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- Arnqvist, L, et al.
(författare)
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Reduction of cholesterol and glycoalkaloid levels in transgenic potato plants by overexpression of a type 1 sterol methyltransferase cDNA
- 2003
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 131:4, s. 1792-1799
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Tidskriftsartikel (refereegranskat)abstract
- Transgenic potato (Solanum tuberosum cv Desiree) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.
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| 4. |
- Ellerström, Mats, 1961, et al.
(författare)
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Ectopic Expression of EFFECTOR OF TRANSCRIPTION Perturbs Gibberellin-Mediated Plant Developmental Processes
- 2005
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Ingår i: Plant Molecular Biology. - : Springer Science and Business Media LLC. - 0167-4412 .- 1573-5028. ; 59:4, s. 663-681
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Tidskriftsartikel (refereegranskat)abstract
- The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific α-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J.␣Biol. Chem. 273: 23313–23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.
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- SITBON, F, et al.
(författare)
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COMPARTMENTATION OF INDOLE-3-ACETIC-ACID METABOLISM IN PROTOPLASTS ISOLATED FROM LEAVES OF WILD-TYPE AND IAA-OVERPRODUCING TRANSGENIC TOBACCO PLANTS
- 1993
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Ingår i: Planta. - 0032-0935 .- 1432-2048. ; 191:2, s. 274-279
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Tidskriftsartikel (refereegranskat)abstract
- Cellular compartmentation of indole-3-acetamide (IAM), indole-3-acetic acid (IAA), and [N-15(1)]IAA synthesised from [N-15(1)]tryptophan was monitored in protoplasts isolated from sterile wild-type tobacco SR1 plants, and in IAA-overproducing plants expressing the Agrobacterium tumefaciens T-DNA IAA genes iaaM and iaaH. Indole-3-acetamide was located exclusively in the cytosol of both iaaM and iaaM/iaaH protoplasts, being 75% lower than in iaaM protoplasts, presumably because of conversion into IAA by action of the iaaH-encoded hydrolase. The free-IAA level, however, was raised only 8% in iaaM/iaaH compared to iaaM protoplasts, whereas the level of IAA-conjugates was increased more than fivefold. For both genotypes, the location of IAA conjugates was restricted to the cytosol, while one-third of the free-IAA pool was present in chloroplasts. Transcription of the iaaM gene was increased by fusion to the strong cautiflower mosaic virus (CaMV) 35S promoter. Compared with the wildtype, this led to an 18-fold higher conversion of [N-15(1)]tryptophan to [N-15(1)]IAA, a three- to fourfold increase in free IAA, and a tenfold higher level of IAA conjugates in 35S-iaaM/iaaH protoplasts. Also in these genotypes, IAA conjugates were exclusively cytosolic. There was no major difference between transgenic and wildtype protoplasts in the proportion of chloroplastic to total cellular IAA, although the chloroplastic IAA and [N-15(1)]IAA pools in the transformant were threefold and eightfold higher, respectively. Since the IAM pool in transgenic plants is exclusively cytosolic, these findings suggest that the increased chloroplastic [N-15(1)]IAA pool in 35S-iaaM/iaaH protoplasts is synthesised in the cytosol but rapidly transported into the chloroplast. Furthermore, the presence of IAA in the chloroplast together with the exclusively cytosolic location of IAA conjugates, suggests the presence of two differentially subcellular pools of IAA. The first is located in the cytosol and mainly regulated by non-decarboxylative catabolism and conjugation (Sandberg et al. 1990, Planta 180, 562-568), whereas the second is located in the chloroplast and is seemingly not directly regulated inside the organelle by either of these two processes. The cytosolic IAA control mechanisms, however, also affect the chloroplastic pool size due to the rapid transport of IAA between the two compartments.
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- Williams, Cecilia, 1969-, et al.
(författare)
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A high frequency of sequence alterations is due to formalin fixation of archival specimens.
- 1999
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Ingår i: American Journal of Pathology. - 0002-9440 .- 1525-2191. ; 155:5
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Tidskriftsartikel (refereegranskat)abstract
- Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.
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- Wycliffe, P., et al.
(författare)
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Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cells
- 2005
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Ingår i: The Plant Journal. - 0960-7412 .- 1365-313X. ; 44:1, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope.
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