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Ursula Kues - One of the best experts on this subject based on the ideXlab platform.
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Paradoxical performance of tryptophan synthase gene trp1 ^+ in transformations of the basidiomycete Coprinopsis cinerea
Applied Microbiology and Biotechnology, 2016Co-Authors: Bastian Dörnte, Ursula KuesAbstract:Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1 , 1-6 which can be complemented by introduction of the trp1 ^+ wild-type gene. Regularly in C. cinerea , single- trp1 ^+-vector transformations yield about half the numbers of clones than Cotransformations with a non- trp1 ^+-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB ^+ gene shown here to function as a selection marker in C. cinerea . Parts of single- trp1 ^ + - or single- trpB ^ + -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single- trp1 ^+-vector transformants and also sets off loss of clones in Cotransformation with a non- trp1 ^+-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in Cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 ^+ copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in Cotransformations due to preferred single trp1 ^+ integration by homologous recombination at its native genomic site.
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Paradoxical performance of tryptophan synthase gene trp1+ in transformations of the basidiomycete Coprinopsis cinerea
Applied microbiology and biotechnology, 2016Co-Authors: Bastian Dörnte, Ursula KuesAbstract:Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1,1-6 which can be complemented by introduction of the trp1 + wild-type gene. Regularly in C. cinerea, single-trp1 +-vector transformations yield about half the numbers of clones than Cotransformations with a non-trp1 +-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB + gene shown here to function as a selection marker in C. cinerea. Parts of single-trp1 + - or single-trpB + -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single-trp1 +-vector transformants and also sets off loss of clones in Cotransformation with a non-trp1 +-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in Cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 + copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in Cotransformations due to preferred single trp1 + integration by homologous recombination at its native genomic site.
Bastian Dörnte - One of the best experts on this subject based on the ideXlab platform.
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Paradoxical performance of tryptophan synthase gene trp1 ^+ in transformations of the basidiomycete Coprinopsis cinerea
Applied Microbiology and Biotechnology, 2016Co-Authors: Bastian Dörnte, Ursula KuesAbstract:Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1 , 1-6 which can be complemented by introduction of the trp1 ^+ wild-type gene. Regularly in C. cinerea , single- trp1 ^+-vector transformations yield about half the numbers of clones than Cotransformations with a non- trp1 ^+-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB ^+ gene shown here to function as a selection marker in C. cinerea . Parts of single- trp1 ^ + - or single- trpB ^ + -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single- trp1 ^+-vector transformants and also sets off loss of clones in Cotransformation with a non- trp1 ^+-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in Cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 ^+ copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in Cotransformations due to preferred single trp1 ^+ integration by homologous recombination at its native genomic site.
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Paradoxical performance of tryptophan synthase gene trp1+ in transformations of the basidiomycete Coprinopsis cinerea
Applied microbiology and biotechnology, 2016Co-Authors: Bastian Dörnte, Ursula KuesAbstract:Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1,1-6 which can be complemented by introduction of the trp1 + wild-type gene. Regularly in C. cinerea, single-trp1 +-vector transformations yield about half the numbers of clones than Cotransformations with a non-trp1 +-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB + gene shown here to function as a selection marker in C. cinerea. Parts of single-trp1 + - or single-trpB + -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single-trp1 +-vector transformants and also sets off loss of clones in Cotransformation with a non-trp1 +-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in Cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 + copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in Cotransformations due to preferred single trp1 + integration by homologous recombination at its native genomic site.
Anna Depicker - One of the best experts on this subject based on the ideXlab platform.
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Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants.
Molecular plant-microbe interactions : MPMI, 2000Co-Authors: S. De Buck, C De Wilde, M. Van Montagu, Anna DepickerAbstract:Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and Cotransformation frequencies of Arabidopsis root cells. Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5%. T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells. A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining. When selection for transformation competence is applied, the picture is completely different. Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome. Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and Cotransformation frequencies and that plant cell competence for transformation is based on these two factors.
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Agrobacterium tumefaciens Transformation and Cotransformation Frequencies of Arabidopsis thaliana Root Explants and Tobacco Protoplasts
Molecular plant-microbe interactions : MPMI, 1998Co-Authors: Sylvie De Buck, Anni Jacobs, Marc Van Montagu, Anna DepickerAbstract:In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and Cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The Cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.
Vadim Zaytsev - One of the best experts on this subject based on the ideXlab platform.
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Cotransforming Grammars with Shared Packed Parse Forests
Electronic Communication of The European Association of Software Science and Technology, 2016Co-Authors: Vadim ZaytsevAbstract:SPPF (shared packed parse forest) is the best known graph representation of a parse forest (family of related parse trees) used in parsing with ambiguous/conjunctive grammars. Systematic general purpose transformations of SPPFs have never been investigated and are considered to be an open problem in software language engineering. In this paper, we motivate the necessity of having a transformation operator suite for SPPFs and extend the state of the art grammar transformation operator suite to metamodel/model (grammar/graph) Cotransformations.
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GCM@ICGT - Coupled Transformations of Shared Packed Parse Forests
2015Co-Authors: Vadim ZaytsevAbstract:SPPF (shared packed parse forest) is the best known graph representation of a parse forest (family of related parse trees) used in parsing with ambiguous/conjunctive grammars. Systematic general purpose transformations of SPPFs have never been investigated and are considered to be an open problem in software language engineering. In this paper, we motivate the necessity of having a transformation operator suite for SPPFs and extend the state of the art grammar transformation operator suite to metamodel/model (grammar/graph) Cotransformations.
Karuppannan Veluthambi - One of the best experts on this subject based on the ideXlab platform.
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Generation of selection marker-free transgenic plants by Cotransformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain
Plant Science, 2002Co-Authors: Subha S. Jacob, Karuppannan VeluthambiAbstract:Cotransformation of Nicotiana tabacum was done using a single Agrobacterium tumefaciens strain harbouring a cointegrate vector (one copy per cell) and a binary vector (ten to 15 copies per cell). The T-DNAs of the cointegrate plasmid, pGV2260::pSSJ1 and the binary plasmid, pGA472 carried hph and nptII as plant selection markers, respectively. When the binary T-DNA marker (nptII) was used for selection, the non-selected cointegrate T-DNA with hph cotransformed at 36% frequency. However, upon using the cointegrate T-DNA with hph for selection, Cotransformation of binary T-DNA with nptII was much higher (56-74%). Segregation of the T-DNA markers hph and nptII was followed in the T 1 generation to screen for the elimination of the selection marker. The elimination of nptII of the multicopy binary vector was found in the progeny of only one of the three T 0 plants and at a low frequency of 3%. However, elimination of hph of the single-copy cointegrate vector was found at 16-18% frequency in the progeny of both the T 0 plants analysed. The use of the T-DNA of low copy number cointegrate vector for initial selection improves the Cotransformation frequency of non-selected T-DNA of the multicopy binary vector. This strategy also increases the frequency of generation of selection marker-free transgenic plants in the T 1 generation.
