TY  - THES
A1  - Gonzalez Duran, Enrique
T1  - Genetic control of intracellular gene transfer by DNA repair in N. tabacum
N2  - 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.
N2  - Mitochondrien und Plastiden sind beides Organellen endosymbiotischen Ursprungs. Im Laufe der Evolution gehen viele Gene aus den Organellengenomen verloren und werden in das Kerngenom integriert, was als intrazellulärer/endosymbiotischer Gentransfer (IGT/EGT) bezeichnet wird. IGT konnte experimentell in Nicotiana tabacum mit einer Gentransferrate (GTR) von einem Ereignis in fünf Millionen Zellen nachgestellt werden, aber trotz seiner zentralen Bedeutung für die eukaryotische Evolution sind keine genetischen Faktoren bekannt, die die Häufigkeit von IGT in höheren Eukaryoten beeinflussen. Der Schwerpunkt dieser Arbeit lag auf der Bestimmung der Rolle verschiedener DNA-Reparaturwege der Doppelstrangbruchreparatur (DSBR) bei der Integration von Organellen-DNA in das Kerngenom während des IGT. Dazu wurde in N. tabacum eine CRISPR/Cas9-basierte Mutagenesestrategie angewandt, mit dem Ziel, Mutanten in Kerngenen zu erzeugen, für die keine sichtbaren Phänotypen zu erwarten sind. Diese Strategie führte zur Erzeugung einer Reihe unabhängiger Mutanten im LIG4-Gen (notwendig für „non-homologous end joining“, die nicht-homologe Verbindung von Enden, NHEJ) und POLQ (notwendig für „microhomology mediated end joining“, die Mikrohomologie-vermittelte Verbindung von Enden, MMEJ). Die gezielte Beeinflussung anderer DSBR-Gene (KU70, KU80, RPA1C) führte zu Mutanten mit unerwartet starken Entwicklungsphänotypen. Diese Gene spielen eine Rolle beim Erhalt der Telomere, was auf einen möglichen Zusammenhang zwischen der Telomerlänge und der Stärke der Entwicklungsstörung bei Verlust der Telomerstruktur in Pflanzen hindeutet. Die Mutanten wurden in einem genetischen Hintergrund erzeugt, der über einen in den Plastiden lokalisierten IGT-Reporter verfügt, der nach Übertragung in den Zellkern Kanamycin-Resistenz vermittelt. In groß angelegten unabhängigen Experimenten wurde in lig4-Mutanten sowie in Linien, die für ein POLQ-Gen mit einer defekten Polymerase-Domäne (polqΔPol) kodieren, eine erhöhte GTR vom Chloroplasten zum Zellkern beobachtet. Dies zeigt, dass NHEJ oder MMEJ eine zweischneidige Beziehung zum IGT haben: Während übertragene Gene folglich über jeden der beiden Mechanismen integriert werden können, unterdrückt das gleichzeitige Vorhandensein beider Wege IGT in somatischen Wildtyp-Zellen, wodurch zum ersten Mal gezeigt wird, in welchem Ausmaß Kerngene die IGT-Häufigkeit in Pflanzen kontrollieren. Die erhöhte Verfügbarkeit von Doppelstrangbrüchen für die Integration könnte für die erhöhte IGT-Häufigkeit in den Mutanten verantwortlich sein. Darüber hinaus zeigt die Analyse des Zeitverlaufs, dass Gentransferereignisse (GT) in Abhängigkeit von der Zeit, die im Experiment unter Antibiotikaselektion verbracht wurde, linear akkumulieren, was beweist, dass es, anders als bisher angenommen, in somatischen IGT-Experimenten keine statische GTR gibt. Darüber hinaus scheint IGT in Gewebekulturexperimenten das Ergebnis eines Wettlaufs mit der Zeit um die Integration in das Kerngenom zu sein, der beginnt, wenn die Organellen-DNA in den Zellkern gelangt und eine vorübergehende Antibiotikaresistenz gewährt. Echte GT-Ereignisse und „Escapes“ (scheinbare Resistenz, ein vorläufiges Ausweichen vor der Kanamycin-Selektion) können zwei mögliche Ergebnisse dieses Wettlaufs sein: Die Fälle, in denen die Organellen-DNA integriert wird, werden als GT-Ereignisse gewertet, und in den Fällen, in denen die rechtzeitige Integration scheitert, kann die Antibiotikaresistenz nicht aufrechterhalten werden und sie werden als „Escape“ betrachtet. In den Mutanten verändern sich die Möglichkeiten zur Integration in das Kerngenom, wodurch sich das Gesamtverhältnis zwischen IGT- und „Escape“-Ereignissen ändert. Die hier erzeugten Ressourcen sind vielversprechende Ausgangspunkte für künftige Forschungen: (1) die Mutantensammlung, für die Untersuchung von weiteren Prozessen, die von der DNA-Reparatur in Pflanzen abhängen, (2) die Sammlung von GT-Linien, die aus den hier beschriebenen Experimenten gewonnen wurden, für die Untersuchung der Auswirkungen von DSBR-Mechanismen auf Integrationsmuster und Stabilität übertragener Gene und (3) der hier entwickelte Arbeitsablauf für die Mutantenerzeugung mittels CRISPR/Cas9, damit N. tabacum sein Potenzial als attraktives Modell für die Beantwortung komplexer biologischer Fragestellungen erfüllen kann.
T2  - Genetische Kontrolle des intrazellulären Gentransfers durch DNA-Reparatur in N. tabacum
KW  - endosymbiosis
KW  - organelles
KW  - gene
KW  - transfer
KW  - DNA
KW  - repair
KW  - genome
KW  - editing
KW  - evolution
KW  - plant
Y1  - 2023
ER  - 
TY  - JOUR
A1  - Van Hout, Cristopher V.
A1  - Tachmazidou, Ioanna
A1  - Backman, Joshua D.
A1  - Hoffman, Joshua D.
A1  - Liu, Daren
A1  - Pandey, Ashutosh K.
A1  - Gonzaga-Jauregui, Claudia
A1  - Khalid, Shareef
A1  - Ye, Bin
A1  - Banerjee, Nilanjana
A1  - Li, Alexander H.
A1  - O'Dushlaine, Colm
A1  - Marcketta, Anthony
A1  - Staples, Jeffrey
A1  - Schurmann, Claudia
A1  - Hawes, Alicia
A1  - Maxwell, Evan
A1  - Barnard, Leland
A1  - Lopez, Alexander
A1  - Penn, John
A1  - Habegger, Lukas
A1  - Blumenfeld, Andrew L.
A1  - Bai, Xiaodong
A1  - O'Keeffe, Sean
A1  - Yadav, Ashish
A1  - Praveen, Kavita
A1  - Jones, Marcus
A1  - Salerno, William J.
A1  - Chung, Wendy K.
A1  - Surakka, Ida
A1  - Willer, Cristen J.
A1  - Hveem, Kristian
A1  - Leader, Joseph B.
A1  - Carey, David J.
A1  - Ledbetter, David H.
A1  - Cardon, Lon
A1  - Yancopoulos, George D.
A1  - Economides, Aris
A1  - Coppola, Giovanni
A1  - Shuldiner, Alan R.
A1  - Balasubramanian, Suganthi
A1  - Cantor, Michael
A1  - Nelson, Matthew R.
A1  - Whittaker, John
A1  - Reid, Jeffrey G.
A1  - Marchini, Jonathan
A1  - Overton, John D.
A1  - Scott, Robert A.
A1  - Abecasis, Goncalo R.
A1  - Yerges-Armstrong, Laura M.
A1  - Baras, Aris
T1  - Exome sequencing and characterization of 49,960 individuals in the UK Biobank
JF  - Nature : the international weekly journal of science
N2  - The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world(1). Here we describe the release of exome-sequence data for the first 49,960 study participants, revealing approximately 4 million coding variants (of which around 98.6% have a frequency of less than 1%). The data include 198,269 autosomal predicted loss-of-function (LOF) variants, a more than 14-fold increase compared to the imputed sequence. Nearly all genes (more than 97%) had at least one carrier with a LOF variant, and most genes (more than 69%) had at least ten carriers with a LOF variant. We illustrate the power of characterizing LOF variants in this population through association analyses across 1,730 phenotypes. In addition to replicating established associations, we found novel LOF variants with large effects on disease traits, includingPIEZO1on varicose veins,COL6A1on corneal resistance,MEPEon bone density, andIQGAP2andGMPRon blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical importance, and show that 2% of this population has a medically actionable variant. Furthermore, we characterize the penetrance of cancer in carriers of pathogenicBRCA1andBRCA2variants. Exome sequences from the first 49,960 participants highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community. <br /> Exome sequences from the first 49,960 participants in the UK Biobank highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.
KW  - clinical exome
KW  - breast-cancer
KW  - mutations
KW  - recommendations
KW  - gene
KW  - metaanalysis
KW  - variants,
KW  - BRCA1
KW  - risk
KW  - susceptibility
Y1  - 2020
U6  - https://doi.org/10.1038/s41586-020-2853-0
SN  - 0028-0836
SN  - 1476-4687
VL  - 586
IS  - 7831
SP  - 749
EP  - 756
PB  - Macmillan Publishers Limited
CY  - London
ER  - 
TY  - JOUR
A1  - Perscheid, Cindy
T1  - Integrative biomarker detection on high-dimensional gene expression data sets
BT  - a survey on prior knowledge approaches
JF  - Briefings in bioinformatics
N2  - Gene expression data provide the expression levels of tens of thousands of genes from several hundred samples. These data are analyzed to detect biomarkers that can be of prognostic or diagnostic use. Traditionally, biomarker detection for gene expression data is the task of gene selection. The vast number of genes is reduced to a few relevant ones that achieve the best performance for the respective use case. Traditional approaches select genes based on their statistical significance in the data set. This results in issues of robustness, redundancy and true biological relevance of the selected genes. Integrative analyses typically address these shortcomings by integrating multiple data artifacts from the same objects, e.g. gene expression and methylation data. When only gene expression data are available, integrative analyses instead use curated information on biological processes from public knowledge bases. With knowledge bases providing an ever-increasing amount of curated biological knowledge, such prior knowledge approaches become more powerful. This paper provides a thorough overview on the status quo of biomarker detection on gene expression data with prior biological knowledge. We discuss current shortcomings of traditional approaches, review recent external knowledge bases, provide a classification and qualitative comparison of existing prior knowledge approaches and discuss open challenges for this kind of gene selection.
KW  - gene selection
KW  - external knowledge bases
KW  - biomarker detection
KW  - gene
KW  - expression
KW  - prior knowledge
Y1  - 2021
U6  - https://doi.org/10.1093/bib/bbaa151
SN  - 1467-5463
SN  - 1477-4054
VL  - 22
IS  - 3
PB  - Oxford Univ. Press
CY  - Oxford
ER  - 
TY  - JOUR
A1  - Choi, Youngeun
A1  - Schmidt, Carsten
A1  - Tinnefeld, Philip
A1  - Bald, Ilko
A1  - Rödiger, Stefan
T1  - A new reporter design based on DNA origami nanostructures for quantification of short oligonucleotides using microbeads
JF  - Scientific Reports
N2  - The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters.
KW  - nucleic-acids
KW  - hybridization
KW  - microrna
KW  - flourescence
KW  - biomarkers
KW  - platform
KW  - particle
KW  - binding
KW  - array
KW  - gene
Y1  - 2019
U6  - https://doi.org/10.1038/s41598-019-41136-x
SN  - 2045-2322
IS  - 9
PB  - Macmillan Publishers Limited
CY  - London
ER  -