@phdthesis{GonzalezDuran2023,
  author    = {Gonzalez Duran, Enrique},
  title     = {Genetic control of intracellular gene transfer by DNA repair in N. tabacum},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XII, 127, XLI},
  year      = {2023},
  abstract  = {Mitochondria and plastids are organelles with an endosymbiotic origin. During evolution, many genes are lost from the organellar genomes and get integrated in the nuclear genome, in what is known as intracellular/endosymbiotic gene transfer (IGT/EGT). IGT has been reproduced experimentally in Nicotiana tabacum at a gene transfer rate (GTR) of 1 event in 5 million cells, but, despite its centrality to eukaryotic evolution, there are no genetic factors known to influence the frequency of IGT in higher eukaryotes. The focus of this work was to determine the role of different DNA repair pathways of double strand break repair (DSBR) in the integration step of organellar DNA in the nuclear genome during IGT. Here, a CRISPR/Cas9 mutagenesis strategy was implemented in N. tabacum, with the aim of generating mutants in nuclear genes without expected visible phenotypes. This strategy led to the generation of a collection of independent mutants in the LIG4 (necessary for non-homologous end joining, NHEJ) and POLQ genes (necessary for microhomology mediated end joining, MMEJ). Targeting of other DSBR genes (KU70, KU80, RPA1C) generated mutants with unexpectedly strong developmental phenotypes.. These factors have telomeric roles, hinting towards a possible relationship between telomere length, and strength of developmental disruption upon loss of telomere structure in plants. The mutants were made in a genetic background encoding a plastid-encoded IGT reporter, that confers kanamycin resistance upon transfer to the nucleus. Through large scale independent experiments, increased IGT from the chloroplast to the nucleus was observed in lig4 mutants, as well as lines encoding a POLQ gene with a defective polymerase domain (polqΔPol). This shows that NHEJ or MMEJ have a double-sided relationship with IGT: while transferred genes may integrate using either pathway, the presence of both pathways suppresses IGT in wild-type somatic cells, thus demonstrating for the first time the extent on which nuclear genes control IGT frequency in plants. The IGT frequency increases in the mutants are likely mediated by increased availability of double strand breaks for integration. Additionally, kinetic analysis reveals that gene transfer (GT) events accumulate linearly as a function of time spent under antibiotic selection in the experiment, demonstrating that, contrary to what was previously thought, there is no such thing as a single GTR in somatic IGT experiments. Furthermore, IGT in tissue culture experiments appears to be the result of a "race against the clock" for integration in the nuclear genome, that starts when the organellar DNA arrives to the nucleus granting transient antibiotic resistance. GT events and escapes of kanamycin selection may be two possible outcomes from this race: those instances where the organellar DNA gets to integrate are recovered as GT events, and in those cases where timely integration fails, antibiotic resistance cannot be sustained, and end up considered as escapes. In the mutants, increased opportunities for integration in the nuclear genome change the overall ratio between IGT and escape events. The resources generated here are promising starting points for future research: (1) the mutant collection, for the further study of processes that depend on DNA repair in plants (2) the collection of GT lines obtained from these experiments, for the study of the effect of DSBR pathways over integration patterns and stability of transferred genes and (3) the developed CRISPR/Cas9 workflow for mutant generation, to make N. tabacum meet its potential as an attractive model for answering complex biological questions.},
  language  = {en}
}
@article{LauxBierHoelzel2018,
  author    = {Laux, Eva-Maria and Bier, Frank Fabian and H{\"o}lzel, Ralph},
  title     = {Dielectrophoretic Stretching of DNA},
  series = {DNA Nanotechnology},
  journal   = {DNA Nanotechnology},
  edition   = {2},
  publisher = {Humana Press Inc.},
  address   = {New York},
  isbn      = {978-1-4939-8582-1},
  issn      = {1064-3745},
  doi       = {10.1007/978-1-4939-8582-1_14},
  pages     = {199 -- 208},
  year      = {2018},
  abstract  = {The spatial control of DNA and of self-assembled DNA constructs is a prerequisite for the preparation of DNA-based nanostructures and microstructures and a useful tool for studies on single DNA molecules. Here we describe a protocol for the accumulation of dissolved lambda-DNA molecules between planar microelectrodes by the action of inhomogeneous radiofrequency electric fields. The resulting AC electrokinetic forces stretch the DNA molecules and align them parallel to the electric field. The electrode preparation from off-the-shelf electronic components is explained, and a detailed description of the electronic setup is given. The experimental procedure is controlled in real-time by fluorescence microscopy.},
  language  = {en}
}
@article{LauxErmilovaPannwitzetal.2018,
  author    = {Laux, Eva-Maria and Ermilova, Elena and Pannwitz, Daniel and Gibbons, Jessica and H{\"o}lzel, Ralph and Bier, Frank Fabian},
  title     = {Dielectric Spectroscopy of Biomolecules up to 110 GHz},
  series = {Frequenz},
  volume    = {72},
  journal   = {Frequenz},
  number    = {3-4},
  publisher = {De Gruyter},
  address   = {Berlin},
  issn      = {0016-1136},
  doi       = {10.1515/freq-2018-0010},
  pages     = {135 -- 140},
  year      = {2018},
  abstract  = {Radio-frequency fields in the GHz range are increasingly applied in biotechnology and medicine. In order to fully exploit both their potential and their risks detailed information about the dielectric properties of biological material is needed. For this purpose a measuring system is presented that allows the acquisition of complex dielectric spectra over 4 frequency decade up to 110 GHz. Routines for calibration and for data evaluation according to physicochemical interaction models have been developed. The frequency dependent permittivity and dielectric loss of some proteins and nucleic acids, the main classes of biomolecules, and of their sub-units have been determined. Dielectric spectra are presented for the amino acid alanine, the proteins lysozyme and haemoglobin, the nucleotides AMP and ATP, and for the plasmid pET-21, which has been produced by bacterial culture. Characterisation of a variety of biomolecules is envisaged, as is the application to studies on protein structure and function.},
  language  = {en}
}
@article{LiCorriganYangetal.2015,
  author    = {Li, Chenhong and Corrigan, Shannon and Yang, Lei and Straube, Nicolas and Harris, Mark and Hofreiter, Michael and White, William T. and Naylor, Gavin J. P.},
  title     = {DNA capture reveals transoceanic gene flow in endangered river sharks},
  series = {Proceedings of the National Academy of Sciences of the United States of America},
  volume    = {112},
  journal   = {Proceedings of the National Academy of Sciences of the United States of America},
  number    = {43},
  publisher = {National Acad. of Sciences},
  address   = {Washington},
  issn      = {0027-8424},
  doi       = {10.1073/pnas.1508735112},
  pages     = {13302 -- 13307},
  year      = {2015},
  abstract  = {For over a hundred years, the "river sharks" of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks.},
  language  = {en}
}