TY - JOUR A1 - Hermanussen, Michael A1 - Scheffler, Christiane T1 - Evidence of chronic undernutrition in late 19th century German infants of all social classes JF - Human biology and public health N2 - 125 years ago, European infants grew differently from modern infants. We show weight gains of 20 healthy children weighed longitudinally from birth to age 1 year, published by Camerer in 1882. The data illustrate the historically prevalent concepts of infant nutrition practiced by German civil servants, lawyers, merchants, university professors, physicians, foresters and farmers. Breastfeeding by the mother was not truly appreciated in those days; children were often breastfed by wet nurses or received bottled milk. Bottle feeding mainly used diluted cow’s milk with some added carbohydrates, without evidence that appropriate amounts of oil, butter or other fatty components were added. French children from 1914 showed similar weight gain patterns suggesting similar feeding practices. The historical data suggest that energy deficient infant formula was fed regularly in the late 19th and early 20th century Europe, regardless of wealth and social class. The data question current concerns that temporarily feeding energy deficient infant formula may warrant serious anxieties regarding long-term cognitive, social and emotional behavioral development. KW - chronic undernutrition KW - breastfeeding KW - historical growth KW - social class KW - translation Y1 - 2022 U6 - https://doi.org/10.52905/hbph2022.2.42 SN - 2748-9957 VL - 2022 IS - 2 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - JOUR A1 - Haueis, Lisa A1 - Stech, Marlitt A1 - Kubick, Stefan T1 - A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies JF - Frontiers in Bioengineering and Biotechnology N2 - Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (VHH, similar to 15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the "on demand" synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation. KW - cell-free protein synthesis KW - In vitro transcription KW - translation KW - nanobody KW - VHH KW - camelid KW - CHO cell lysate Y1 - 2022 U6 - https://doi.org/10.3389/fbioe.2022.896763 SN - 2296-4185 VL - 10 PB - Frontiers Media CY - Lausanne ER - TY - THES A1 - Schuster, Maja T1 - High resolution decoding of the tobacco chloroplast translatome and its dynamics during light-intensity acclimation N2 - Chloroplasts are the photosynthetic organelles in plant and algae cells that enable photoautotrophic growth. Due to their prokaryotic origin, modern-day chloroplast genomes harbor 100 to 200 genes. These genes encode for core components of the photosynthetic complexes and the chloroplast gene expression machinery, making most of them essential for the viability of the organism. The regulation of those genes is predominated by translational adjustments. The powerful technique of ribosome profiling was successfully used to generate highly resolved pictures of the translational landscape of Arabidopsis thaliana cytosol, identifying translation of upstream open reading frames and long non-coding transcripts. In addition, differences in plastidial translation and ribosomal pausing sites were addressed with this method. However, a highly resolved picture of the chloroplast translatome is missing. Here, with the use of chloroplast isolation and targeted ribosome affinity purification, I generated highly enriched ribosome profiling datasets of the chloroplasts translatome for Nicotiana tabacum in the dark and light. Chloroplast isolation was found unsuitable for the unbiased analysis of translation in the chloroplast but adequate to identify potential co-translational import. Affinity purification was performed for the small and large ribosomal subunit independently. The enriched datasets mirrored the results obtained from whole-cell ribosome profiling. Enhanced translational activity was detected for psbA in the light. An alternative translation initiation mechanism was not identified by selective enrichment of small ribosomal subunit footprints. In sum, this is the first study that used enrichment strategies to obtain high-depth ribosome profiling datasets of chloroplasts to study ribosome subunit distribution and chloroplast associated translation. Ever-changing light intensities are challenging the photosynthetic capacity of photosynthetic organism. Increased light intensities may lead to over-excitation of photosynthetic reaction centers resulting in damage of the photosystem core subunits. Additional to an expensive repair mechanism for the photosystem II core protein D1, photosynthetic organisms developed various features to reduce or prevent photodamage. In the long-term, photosynthetic complex contents are adjusted for the efficient use of experienced irradiation. However, the contribution of chloroplastic gene expression in the acclimation process remained largely unknown. Here, comparative transcriptome and ribosome profiling was performed for the early time points of high-light acclimation in Nicotiana tabacum chloroplasts in a genome-wide scale. The time- course data revealed stable transcript level and only minor changes in translational activity of specific chloroplast genes during high-light acclimation. Yet, psbA translation was increased by two-fold in the high light from shortly after the shift until the end of the experiment. A stress-inducing shift from low- to high light exhibited increased translation only of psbA. This study indicate that acclimation fails to start in the observed time frame and only short-term responses to reduce photoinhibition were observed. N2 - Chloroplasten sind die photosynthetischen Organellen in Pflanzen- und Algenzellen, die photoautotrophes Wachstum ermöglichen. Aufgrund ihrer prokaryotischen Herkunft besitzen moderne Chloroplasten ein Genom mit 100 bis 200 Gene. Diese kodieren für zentrale Komponenten der Photosynthesekomplexe und des Genexpressionsapparates, was sie für die Lebensfähigkeit des gesamten Organismus essenziell macht. Die leistungsstarke Methode Ribosome Profiling wurde bereits erfolgreich eingesetzt, um hochaufgelöste Bilder der zytosolischen Translationslandschaft von Arabidopsis thaliana zu erstellen, wobei Translation von der Hauptsequenz vorgelagerten, kodierenden Sequenzen und langen, nicht-kodierenden Transkripten identifiziert wurde. Ferner wurden mit dieser Technik Regulationen der Plastidentranslation und spezifische Regionen mit unterschiedlicher Elongationsgeschwindigkeit aufgedeckt. Es fehlen jedoch hochaufgelöste Datensätze des Chloroplasten-Translatoms. Chloroplastenisolation und Affinitätsaufreinigung chloroplastidiärer Ribosomen wurde verwendet, um hochangereicherte Ribosome Profiling-Datensätze des Chloroplastentranslatoms für Nicotiana tabacum im Dunkeln und unter Licht zu erzeugen. Wenngleich sich die Chloroplastenisolation als ungeeignet für eine unverfälschte Analyse der Translation im Chloroplast erwies, ermöglichte sie die Identifizierung von potentiellem co-translationalen Proteinimport. Die entsprechenden Datensätze spiegelten die Ergebnisse des zellulären Ribosome Profilings wider. Für psbA wurde im Licht erhöhte Translationsaktivität festgestellt. Alternative Initiationsmechanismen konnten durch spezifische Anreicherung der kleinen ribosomalen Untereinheit nicht verifiziert werden. Zusammenfassend, dies ist die erste Studie, die mittels Anreicherungsstrategien hochaufgelöste Ribosome Profiling-Datensätze zur Analyse von Ribosomuntereinheitsverteilungen und Chloroplast-assoziierter Translation nutzte. Ständig wechselnde Lichtintensitäten stellen die Photosynthesekapazität von photosynthetischen Organismen auf die Probe. Erhöhte Lichtintensitäten können zu einer Überreizung der photosynthetischen Reaktionszentren führen, was Beschädigungen von zentralen Komplexeinheiten der Photosysteme verursacht. Neben einem aufwändigen Reparaturmechanismus für das Photosystem II-Protein D1 entwickelte der photosynthetische Organismus verschiedene Mechanismen um lichtinduzierte Schäden zu reduzieren oder zu verhindern. Langfristig kommt es zu einer Anreicherung spezifischer Photosynthesekomplexen um eine effiziente Ausnutzung der erhöhten Strahlung zu gewährleisten. Der Beitrag der chloroplastidiäeren Genexpressionsregulation zum Akklimatisierungsprozess ist jedoch weitgehend unbekannt. Hier wurde ein vergleichendes Transkript- und Ribosomen Profiling für die frühen Zeitpunkte der Akklimatisierung unter Starklicht in Tabakchloroplasten in einem genomweiten Maßstab durchgeführt. Die Zeitverlaufsdaten zeigten ein unverändertes Transkriptniveau und nur geringe Änderungen der translationalen Aktivität von chloroplastidiären Genen im Hochlicht im Vergleich zu Kontrollproben. Die psbA-Translation war jedoch unter Hochlicht schon kurz nach Beginn bis zum Ende des Experiments um etwa das Zweifache erhöht. Der stressinduzierende Wechsel von Schwach- zu Hochlicht bewirkte ebenfalls eine auf psbA-beschränkt, erhöhte Translation. Die Ergebnisse zeigen, dass die Akklimatisierung im beobachteten Zeitrahmen nicht begonnen hatte und nur kurzfristige Reaktionen zur Verringerung der Photoinhibition wirksam gewesen sein konnten. KW - translation KW - chloroplast KW - high light KW - ribosome profiling Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-512680 ER - TY - THES A1 - Janowski, Marcin Andrzej T1 - Investigating role of the essential GTPase - AtRsgA in the assembly of the small ribosomal subunit in Arabidopsis thaliana chloroplast N2 - Plastid protein biosynthesis occurs on bacterial-type 70S ribosomes consisting of a large (50S) and a small (30S) subunit. However, since many steps of ribosome biogenesis are not thermodynamically favorable at biological conditions, it requires many assembly factors. One group of assembly factors, circularly permuted GTPases, was implicated in 30S subunit maturation in E. coli, by a protein RsgA. RsgA orthologues are present in bacteria and plastid-containing species and in silico analysis revealed presence of a RsgA-like protein in Arabidopsis thaliana. To functionally characterize the Arabidopsis orthologue, two AtRsgA T-DNA insertion lines were analyzed in this study. The exon line (rsgA-e) led to embryo lethality, while the intron line (rsgA-i) caused severe dwarf, pale green phenotype. Further investigation of rsgA-i mutant line revealed defects in chloroplast biogenesis which led to increased number of chloroplasts, decreased chloroplast size, decreased air space between mesophyll cells and smaller shoot apical meristems, which showed unusual proplastid accumulation. Moreover, rsgA-i plants showed reduction in chlorophyll A and B content, decreased electron transport rate and photosynthetic efficiency. Further analyses revealed that the protein is involved in chloroplast 30S subunit maturation. Interestingly, we observed that while chloroplast-targeted and chloroplast-encoded proteins are generally downregulated in the mutant, a contrasting upregulation of the corresponding transcripts is observed, indicating an elaborate compensatory mechanism. To conclude, the study presented here reveals a ribosome assembly factor and a compensatory mechanism activated during impaired chloroplast function. KW - ribosome assembly KW - GTPase KW - chloro-ribosome KW - translation Y1 - 2017 ER - TY - GEN A1 - Lukoszek, Radoslaw A1 - Feist, Peter A1 - Ignatova, Zoya T1 - Insights into the adaptive response of Arabidopsis thaliana to prolonged thermal stress by ribosomal profiling and RNA-Seq T2 - BMC plant biology N2 - Background: Environmental stress puts organisms at risk and requires specific stress-tailored responses to maximize survival. Long-term exposure to stress necessitates a global reprogramming of the cellular activities at different levels of gene expression. Results: Here, we use ribosome profiling and RNA sequencing to globally profile the adaptive response of Arabidopsis thaliana to prolonged heat stress. To adapt to long heat exposure, the expression of many genes is modulated in a coordinated manner at a transcriptional and translational level. However, a significant group of genes opposes this trend and shows mainly translational regulation. Different secondary structure elements are likely candidates to play a role in regulating translation of those genes. Conclusions: Our data also uncover on how the subunit stoichiometry of multimeric protein complexes in plastids is maintained upon heat exposure. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 438 KW - translation KW - ribosome profiling KW - transcription KW - RNA-Seq KW - secondary structure KW - G-quadruplexes, KW - heat stress response Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-407262 ER - TY - JOUR A1 - Saffert, Paul A1 - Adamla, Frauke A1 - Schieweck, Rico A1 - Atkins, John F. A1 - Ignatova, Zoya T1 - An Expanded CAG Repeat in Huntingtin Causes+1 Frameshifting JF - The journal of biological chemistry N2 - Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the -1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded repeats with more than 35 consecutive CAG codons. An atypical +1 shift site, UUC C at the 5 end of CAG repeats, which has some resemblance to the influenza A virus shift site, triggers the +1 frameshifting and is enhanced by the increased propensity of the expanded CAG repeats to form a stem-loop structure. The +1 trans-frame-encoded product can directly influence the aggregation of the parental Htt exon 1. KW - aggregation KW - Huntington disease KW - translation KW - translation regulation KW - trinucleotide repeat disease KW - frameshifting KW - seeding Y1 - 2016 U6 - https://doi.org/10.1074/jbc.M116.744326 SN - 0021-9258 SN - 1083-351X VL - 291 SP - 18505 EP - 18513 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Gorochowski, Thomas E. A1 - Aycilar-Kucukgoze, Irem A1 - Bovenberg, Roel A. L. A1 - Roubos, Johannes A. A1 - Ignatova, Zoya T1 - A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes JF - ACS synthetic biology N2 - Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources in executing orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exposure to additional ambient stress. Our results demonstrate that a simple model of resource allocation faithfully captures the redistribution of protein synthesis resources when faced with the burden of synthetic gene expression and environmental stress. The tractable nature of the model makes it a versatile tool for exploring the guiding principles of efficient heterologous expression and the indirect interactions that can arise between synthetic circuits and their host chassis because of competition for shared translational resources. KW - protein biosynthesis KW - translation KW - synthetic biology KW - systems biology Y1 - 2016 U6 - https://doi.org/10.1021/acssynbio.6b00040 SN - 2161-5063 VL - 5 SP - 710 EP - 720 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Bartholomäus, Alexander A1 - Fedyunin, Ivan A1 - Feist, Peter A1 - Sin, Celine A1 - Zhang, Gong A1 - Valleriani, Angelo A1 - Ignatova, Zoya T1 - Bacteria differently regulate mRNA abundance to specifically respond to various stresses JF - Geology N2 - Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up-and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs. KW - transcription KW - translation KW - deep sequencing KW - Escherichia coli KW - copy numbers Y1 - 2016 U6 - https://doi.org/10.1098/rsta.2015.0069 SN - 1364-503X SN - 1471-2962 VL - 374 PB - Royal Society CY - London ER - TY - JOUR A1 - Benina, Maria A1 - Ribeiro, Dimas Mendes A1 - Gechev, Tsanko S. A1 - Müller-Röber, Bernd A1 - Schippers, Jos H. M. T1 - A cell type-specific view on the translation of mRNAs from ROS-responsive genes upon paraquat treatment of Arabidopsis thaliana leaves JF - Plant, cell & environment : cell physiology, whole-plant physiology, community physiology N2 - Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5-untranslated region (5-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress. The study illustrates the response of different Arabidopsis thaliana leaf cells and tissues to oxidative stress at the translational level, an aspect of reactive oxygen species (ROS) biology that has been little studied in the past. Our data reveal insights into how translational regulation of ROS-responsive genes is fine-tuned at the cellular level, a phenomenon contributing to the integrated physiological response of leaves to stresses involving changes in ROS levels. KW - Arabidopsis KW - gene regulation KW - oxidative stress KW - tissue-specific KW - translation Y1 - 2015 U6 - https://doi.org/10.1111/pce.12355 SN - 0140-7791 SN - 1365-3040 VL - 38 IS - 2 SP - 349 EP - 363 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Kannan, Krishna A1 - Kanabar, Pinal A1 - Schryer, David A1 - Florin, Tanja A1 - Oh, Eugene A1 - Bahroos, Neil A1 - Tenson, Tanel A1 - Weissman, Jonathan S. A1 - Mankin, Alexander S. T1 - The general mode of translation inhibition by macrolide antibiotics JF - Proceedings of the National Academy of Sciences of the United States of America KW - ribosome KW - antibiotics KW - macrolides KW - translation KW - peptidyl transferase Y1 - 2014 U6 - https://doi.org/10.1073/pnas.1417334111 SN - 0027-8424 VL - 111 IS - 45 SP - 15958 EP - 15963 PB - National Acad. of Sciences CY - Washington ER -