Gene stability in transgenic aspen (Populus). I. Flanking DNA sequences and T-DNA structure.
Identifieur interne : 004935 ( Main/Curation ); précédent : 004934; suivant : 004936Gene stability in transgenic aspen (Populus). I. Flanking DNA sequences and T-DNA structure.
Auteurs : M. Fladung [Allemagne]Source :
- Molecular & general genetics : MGG [ 0026-8925 ] ; 1999.
Descripteurs français
- KwdFr :
- ADN bactérien (isolement et purification), ADN des plantes (isolement et purification), Amplification de gène (MeSH), Arbres (génétique), Fragilité des chromosomes (MeSH), Phénotype (MeSH), Protéines bactériennes (génétique), Régulation de l'expression des gènes (MeSH), Technique de Northern (MeSH), Technique de Southern (MeSH), Végétaux génétiquement modifiés (génétique), bêta-Glucosidase (MeSH).
- MESH :
- génétique : Arbres, Protéines bactériennes, Végétaux génétiquement modifiés.
- isolement et purification : ADN bactérien, ADN des plantes.
- Amplification de gène, Fragilité des chromosomes, Phénotype, Régulation de l'expression des gènes, Technique de Northern, Technique de Southern, bêta-Glucosidase.
English descriptors
- KwdEn :
- Bacterial Proteins (genetics), Blotting, Northern (MeSH), Blotting, Southern (MeSH), Chromosome Fragility (MeSH), DNA, Bacterial (isolation & purification), DNA, Plant (isolation & purification), Gene Amplification (MeSH), Gene Expression Regulation (MeSH), Phenotype (MeSH), Plants, Genetically Modified (genetics), Trees (genetics), beta-Glucosidase (MeSH).
- MESH :
- chemical , genetics : Bacterial Proteins.
- chemical , isolation & purification : DNA, Bacterial, DNA, Plant.
- genetics : Plants, Genetically Modified, Trees.
- Blotting, Northern, Blotting, Southern, Chromosome Fragility, Gene Amplification, Gene Expression Regulation, Phenotype, beta-Glucosidase.
Abstract
The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events. In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. x tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one of these transgenic lines (Esch5:35S-rolC-#1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders, and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of single-stranded DNA during mitotic cell divisions.
DOI: 10.1007/s004380050931
PubMed: 9928937
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pubmed:9928937Le document en format XML
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<author><name sortKey="Fladung, M" sort="Fladung, M" uniqKey="Fladung M" first="M" last="Fladung">M. Fladung</name>
<affiliation wicri:level="1"><nlm:affiliation>Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics and Forest Tree Breeding, Grosshansdorf, Germany. mfladung@rrz.uni-hamburg.de</nlm:affiliation>
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<wicri:regionArea>Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics and Forest Tree Breeding, Grosshansdorf</wicri:regionArea>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Bacterial Proteins (genetics)</term>
<term>Blotting, Northern (MeSH)</term>
<term>Blotting, Southern (MeSH)</term>
<term>Chromosome Fragility (MeSH)</term>
<term>DNA, Bacterial (isolation & purification)</term>
<term>DNA, Plant (isolation & purification)</term>
<term>Gene Amplification (MeSH)</term>
<term>Gene Expression Regulation (MeSH)</term>
<term>Phenotype (MeSH)</term>
<term>Plants, Genetically Modified (genetics)</term>
<term>Trees (genetics)</term>
<term>beta-Glucosidase (MeSH)</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>ADN bactérien (isolement et purification)</term>
<term>ADN des plantes (isolement et purification)</term>
<term>Amplification de gène (MeSH)</term>
<term>Arbres (génétique)</term>
<term>Fragilité des chromosomes (MeSH)</term>
<term>Phénotype (MeSH)</term>
<term>Protéines bactériennes (génétique)</term>
<term>Régulation de l'expression des gènes (MeSH)</term>
<term>Technique de Northern (MeSH)</term>
<term>Technique de Southern (MeSH)</term>
<term>Végétaux génétiquement modifiés (génétique)</term>
<term>bêta-Glucosidase (MeSH)</term>
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<term>DNA, Plant</term>
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<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Plants, Genetically Modified</term>
<term>Trees</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Arbres</term>
<term>Protéines bactériennes</term>
<term>Végétaux génétiquement modifiés</term>
</keywords>
<keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>ADN bactérien</term>
<term>ADN des plantes</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Blotting, Northern</term>
<term>Blotting, Southern</term>
<term>Chromosome Fragility</term>
<term>Gene Amplification</term>
<term>Gene Expression Regulation</term>
<term>Phenotype</term>
<term>beta-Glucosidase</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Amplification de gène</term>
<term>Fragilité des chromosomes</term>
<term>Phénotype</term>
<term>Régulation de l'expression des gènes</term>
<term>Technique de Northern</term>
<term>Technique de Southern</term>
<term>bêta-Glucosidase</term>
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<front><div type="abstract" xml:lang="en">The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events. In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. x tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one of these transgenic lines (Esch5:35S-rolC-#1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders, and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of single-stranded DNA during mitotic cell divisions.</div>
</front>
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<Abstract><AbstractText>The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events. In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. x tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one of these transgenic lines (Esch5:35S-rolC-#1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders, and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of single-stranded DNA during mitotic cell divisions.</AbstractText>
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