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Rescue and characterization of episomally replicating DNA from the moss Physcomitrella
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 46, 19444-19449 p.Article in journal (Refereed) Published
Abstract [en]

The moss Physcomitrella is unique among plants in that it permits efficient gene targeting by homologous recombination. Furthermore, transformed DNA can replicate episomally in Physcomitrella. Here we show that episomally replicating DNA can berescued back into E. coli, and use such rescue to study the fate of the transformed DNA. Significantly, plasmids rescued from moss transformed with circular DNA are identical to the original plasmid, whereas plasmids rescued from moss transformed with linearized DNA frequently have deletions created by direct repeat recombination.These events are highly predictable in that they target the longest direct repeat on the plasmid, if this repeat is at least 12 bp. Episomal transformants obtained with linearized DNA show a more than 1000-fold amplification of the DNA whereas transformants obtained with circular DNA have much lower copy numbers. Most episomal transformants quickly lose the plasmid in the absence of selection, but a semi-stable type of transformant that loses the plasmid at a much lower frequency was also observed. The consistent rescue of the original plasmid, or of predictable derivatives thereof, suggests that molecular genetics methods which rely on shuttle plasmids are feasible in Physcomitrella

Place, publisher, year, edition, pages
2009. Vol. 106, no 46, 19444-19449 p.
Keyword [en]
Physcomitrella patens, shuttle plasmid, plasmid rescue, recombination
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-109191DOI: 10.1073/pnas.0908037106ISI: 000271907400041PubMedID: 19892729OAI: oai:DiVA.org:uu-109191DiVA: diva2:272023
Available from: 2009-10-13 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Studies of the Carbon and Energy Metabolism in the Moss Physcomitrella patens
Open this publication in new window or tab >>Studies of the Carbon and Energy Metabolism in the Moss Physcomitrella patens
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Since a proper balance between anabolic and catabolic reactions is essential for all eukaryotes, the basic mechanisms for regulation of the energy and carbon metabolism have been conserved throughout evolution. The moss Physcomitrella patens, which belongs to one of the basal clades among land plants, has many unique properties that make it an excellent plant model system.

We have used a yeast two-hybrid system to identify novel possible regulators or targets of the moss Snf1-related kinases, previously shown to regulate energy homeostasis. The function of the identified interactors PpSki1 and PpSki2 was analyzed in order to better understand the biological role of plant Snf1-related kinases.

The recently completed genome sequence of Physcomitrella was used in a comparative approach to study to what extent key enzyme and gene families involved in transport and metabolism of sugars and in regulation of the energy and carbon metabolism are conserved between mosses and vascular plants.

It has long been known that transformed DNA can replicate episomally in Physcomitrella. We have now shown that such DNA can be rescued back into E. coli. Surprisingly, we found that the original plasmid can be recovered from moss transformants obtained with circular DNA. Plasmids rescued from transformants obtained with linearized DNA had been repaired either by homologous recombination or by cohesive end re-ligation. These findings suggest that methods using shuttle plasmids are feasible in Physcomitrella.

Hexokinase, a key enzyme in the carbon metabolism, catalyzes the first step in hexose metabolism, but is also involved in sugar sensing and signaling. We have now made an initial characterization of the complete hexokinase family in Physcomitrella which is encoded by 11 genes. Two new types of plant hexokinases, types C and D, were found in addition to the previously described types A and B.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 73 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 490
Keyword
Hexokinase, SnRK1, sugar signaling, carbon metabolism, Physcomitrella patens, shuttle vector, subcellular localization, model organism
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-109358 (URN)978-91-554-7631-1 (ISBN)
Public defence
2009-11-27, B41, Uppsala Biomedical Center (BMC), Husargatan 3, 75123 Uppsala, 09:15 (English)
Opponent
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Available from: 2009-11-05 Created: 2009-10-14 Last updated: 2009-11-05Bibliographically approved

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