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In dieser Arbeit wurde mit verschiedenen Methoden die Funktion des Ribosomen-BiogeneseFaktors Nep1p in dem Modellorganismus S. cerevisiae untersucht. Die bedeutendste Entdeckung war dabei die Identifizierung einer spezifischen Wechselwirkung dieses Proteins mit den Nukleotiden 1564-1569 der 18S rRNA unter Verwendung eines Hefe-Drei-HybridSystems. Dieser kurze Bereich stellt sehr wahrscheinlich die Nep1p-Bindestelle in einem rRNA-Vorläufermolekül während der Reifung der Ribosomen-Untereinheiten im Nukleolus dar, obwohl diese Interaktion für das humane Protein nicht nachgewiesen werden konnte. Da das 18S rRNA-Nukleotid GB 1573B eine Basenmethylierung aufweist, wurde zunächst vermutet, dass Nep1p an dieser Modifikation beteiligt ist. Diese Hypothese konnte aber durch den biochemischen Nachweis dieser Methylierung in Zellen ohne Nep1p widerlegt werden. Zur weiteren Aufklärung der Funktion dieses Proteins im Prozess der Ribosomen-Biogenese wurden unter Verwendung verschiedener Methoden Mutationen isoliert, die den Wachstumsdefekt von nep1-1P tsP bzw. ∆nep1 partiell kompensieren können. So wurde gezeigt, dass Funktionsverlust-Mutanten der beiden nukleolären Faktoren Nop20p und Nop6p nep1- Suppressoren darstellen. Bei diesen handelt es sich um sehr basische nichtessentielle Proteine, die nur bei Pilzen vorkommen. Auch der Ausfall der snoRNA snR57 zeigte einen nep1- Suppressionseffekt, der sehr wahrscheinlich durch die in dieser Mutante ausbleibende RiboseMethylierung von GB 1570B verursacht wird. Die Entdeckung von RPS19B als MulticopySuppressor von nep1-Mutanten spricht ebenfalls für die in den Drei-Hybrid-Experimenten ermittelte 18S rRNA-Region als Wirkort von Nep1p, da die rRNA-Bindestelle von Rps19p sich nach den in dieser Arbeit zusammengefassten Protein-Protein-Crosslink-Daten höchstwahrscheinlich auch in diesem Bereich befindet. Weitere Analysen lassen sogar vermuten, dass Nep1p für die effiziente Assoziation dieses ribosomalen Proteins mit der rRNA verantwortlich ist. Mit Hilfe eines genetischen Arrays wurden außerdem Mutanten identifiziert, die synthetische Wechselwirkungen mit einem ∆nep1 nop6-1-Stamm, der zudem RPS19B überexprimiert, zeigen. Unter diesen befinden sich neben einigen für verschiedene Ribosomen-BiogeneseFaktoren kodierenden Genen auch die Isogene des ribosomalen Proteins Rps18p. Dessen rRNA-Binderegion ist bekannt und liegt in direkter Nachbarschaft der für Nep1p und Rps19p postulierten Bindestellen, was diese somit bestätigt. Mit den Ubiquitin-spezifischen Proteasen Ubp3p und Ubp6p bzw. dem Ubp3p-Cofaktor Bre5p sind in dem genetischen Array außerdem Faktoren isoliert worden, die eine Verbindung von Nep1p zum Ubiquitin-Stoffwechsel aufzeigen. Neben den Befunden zur Funktion von Nep1p haben die in dieser Arbeit ermittelten Daten auch Hinweise auf weitere Faktoren gegeben, die bei Menschen an der Pathogenese der Diamond-Blackfan-Anämie mitwirken. Die Ubiquitin-spezifische Protease DUB-3 ist dabei ein interessanter Kandidat für den bisher unbekannten Lokus auf Chromosom VIII, dessen Defekt in einigen DBA-Patienten an der Entstehung der Krankheit beteiligt ist.
In the present studies, phylogenetic methodologies were used to study patterns and processes of diversification among freshwater pulmonate gastropods, the Basommatophora, which are important organisms of virtually all aquatic ecosystems. It was anticipated to identify monophyla of different hierarchical rank and investigate datasets of morphological and anatomical characters and their utility in phylogenetic reconstructions. Global biogeographic patterns of the limnic Basommatophora are studied using the new phylogenetic hypotheses as basic assumptions of relationships among the taxa of concern. Finally, a case study deals with microevolutionary patterns and processes from an ancient lake system on the island of Sulawesi, Indonesia. First, the composition and position of basommatophoran gastropods among the Euthyneura was investigated using multi-locus DNA sequencing and employing maximum parsimony, maximum likelihood, and Bayesian inference methodologies. According to these analyses, Pulmonata are a monophyletic unit. Within pulmonates, Stylommatophora and Archaeopulmonata are monophyletic, whereas Basommatophora s.l. are paraphyletic and branch into several differently supported groups. Siphonaria is independent of the archaeopulmonate clade. Hygrophila, a clade comprising the superfamilies Lymnaeoidea, Planorboidea, Physoidea, Acroloxoidea, and Latia is monophyletic and the superfamilies themselves are well supported. Parsimonious and maximum likelihood reconstructions of ancestral habitat types occupied by the taxa studied suggest that starting from the ancestral marine habitat, the invasion of the land happened two times independently in the monophyletic Stylommatophora and within the archaeopulmonates. However, the freshwater was invaded only ones by the freshwater pulmonates (Hygrophila). Contrary to previous assumptions, this pathway was entirely aquatic with no trace of terrestrial intermediate stages. The phylogeny of Basommatophora s.str. was studied using a larger taxon set. Besides the previously recognized superfamilies of Hygrophila, the families Acroloxidae, Physidae, Bulinidae, and Lancidae are monophyletic and supported as well. The Ancylidae and Planorbidae, however, were not monophyletic and cluster within the Planorboidea, a clade that is well supported. A sister-group relationship of Physoidea and Lymnaeoidea was well supported in both maximum likelihood and Bayesian inference analyses. Within Acroloxidae, Acroloxus lacustris is basal to the clade comprising lake Baikal endemic genera. Physidae consist of two clearly distinct well supported groups that do not correspond to the subfamilies commonly accepted. Although in Lymnaeoidea, relationships between groups are not well resolved, Lancidae appear as sister-group of Lymnaeidae. A rapid, possibly simultaneous, radiation event (hard polytomy) of the splits between Acroloxoidea, Planorboidea, Physoidea plus Lymnaeoidea is suggested. The diversification patterns of lineages through time was analyzed with two molecular clock approaches. A fossil calibrated tree and the constant rate molecular clock tree were almost identical in their temporal branching pattern. Two phases of accelerated lineage splitting could be recognized in both approaches. Fitting a geological time scale to the molecular trees showed that accelerated lineage splitting periods coincide with recovery phases in the aftermath of the well known pulse mass extinction event at the Cretaceous-Tertiary (K-T) boundary 65 MYA and the biotic crisis at the boundary from Cenomanian-Turonian (C-T) 93.5 MYA, an anoxic event. It appears as if impacts on the evolutionary history of the Basommatophora due to mass extinctions were world-wide and not restricted to a particular biogeographic region. While recovery from mass extinctions and subsequent radiations have been repeatedly demonstrated for marine and terrestrial taxa, the mass extinction events also triggered evolutionary bursts in this major freshwater taxon, supporting the general importance of these global bioevents for the evolution of freshwater ecosystems. A comprehensive phylogenetic hypothesis was inferred for the most diverse taxon of freshwater basommatophorans, the Planorboidea. Besides the distinct Burnupia clade, two major clades were recovered that correspond to subfamily level taxa. All ancylid taxa (except Burnupia) cluster within Bulininae. This ancylid clade is most advanced within Bulininae and consists of two subclades. Sister to this clade is a monophyletic group comprising highspired (buliniform) Australian planorbids and the patelliform endemic Protancylus from Sulawesi. Planorbinae fell into two major groups. Clades recognized by current classification that are supported by the recent analysis are Planorbinae, Bulinini, Planorbini, Segmentinini. The present study found that Ancylidae, as traditionally understood, is paraphyletic. Monophyly of Planorboidea was demonstrated. Previous hypotheses of shell shape evolution were not supported by this study; limpet-shaped taxa are most basal within Planorboidea. Although many taxa still remain to be studied, these results, have strengthened our understanding of planorbid evolutionary history. The utility of morphological and anatomical characters in phylogenetic reconstructions of the Basommatophora was tested. A maximum parsimony analysis of 47 shell characters demonstrated monophyly for Physidae and Ancylidae, whereas Bulinidae, Lymnaeidae and Planorbidae are non-monophyletic according to this dataset. The analysis of 81 morphological and anatomical characters resulted in monophyly for the Lymnaeoidea, Physidae with good supported, whereas the monophylum Acroloxidae was only weakly supported. 128 combined characters were analyzed and showed Acroloxidae, Lymnaeoidea, Ancylidae, and Physidae to be monophyletic. However, considerable bootstrap support was obtained for Physidae and Ancylidae only. Apomorphies inferred for the supported monophyletic taxa are discussed. The inclusion of more taxa and new characters did improve the phylogenetic inferences as compared to previous studies based on morphological and anatomical datasets. The low resolution and absence of support in large parts of the obtained trees must be attributed to an extraordinarily high degree of homoplasy among the taxa of concern. Comparing all the morphological trees and particularly speaking about the absence of resolution and/or support, it could well be that the mosaic-pattern found among many of the characters is an indication of a rapid initial radiation event of lineages leading to the major superfamily taxa that are extant. “Hotspots” or centers of diversity among world-wide Basommatophora are identified as eastern North America, the European–Mediterranean region and the Australian region. There is a general trend of more generic diversity in the northern hemisphere. The lowest diversity was found in South America, Sahara Africa and Madagascar. A parsimony analysis of endemicity (PAE) with taxa-as-characters (generic dataset) yielded a tree characterized by a fairly broad basal polytomy. From this analysis, the relationship between certain geographic superregions can not be resolved by the recent taxa occurring there. Within the superregions, many neighboring biogeographic (sub-) regions cluster together. Brooks Parsimony Analysis (BPA) and Dispersal-Vicariance Analysis (DIVA) yielded complex, partly conflicting reconstruction of the biogeographic history of the world-wide Basommatophora s.str. involving multiple vicariance and dispersal events. An initial vicariance event separated the Acroloxidae (basal lineage to lake Baikal endemics), while in the Palaearctic region both the Physidae and Lymnaeidae and the widespread genus Acroloxus arose independently. The Palaearctic region became separated from eastern and western North America, South Asia, Africa, the Malagassian region, the Guayana-Brazilian subregion, the Australian region, and Indo-westpacific region. Patterns and processes of dispersal and vicariance are discussed for the taxa studied and the geographic subregions involved. It is apparent from this study that no clear-cut pattern can be drawn of either vicariance or dispersal as predominant components in the long history of word-wide distribution of Basommatophora s.str. Rather, the picture is very complex with both processes and additionally strong extinction events at times being in force probably throughout the taxons existence. Comparatively recent and repeated longdistance (cross-continent) dispersals in post-drift times obviously represents a major contribution to the present wide range of several genera and species, particularly in Lymnaeidae and Planorbidae. Thus, the results of this study point to dispersal as a valid counterpart and addition to vicariance theory in explaining patterns found in biogeography. Freshwater molluscs and the Basommatophora s.str. in particular would provide a prime model group for a new methodological framework that places dispersal into the place it deserves. Macroecological rules were tested using the global dataset of distribution patterns of Basommatophora. Bergmann’s rule could not be confirmed for basommatophoran gastropods on a global scale. These findings are in concert with the growing evidence that body size in ectotherms is not at all related to latitude or even follow the converse to Bergmann’s rule. The analysis of generic ranges revealed no indication of a Rapoport effect, which states that a species’ geographical extent decreases from higher to lower latitudes, on a global scale either. Therefore, this is another example of the growing number of studies that raise doubts on the generality of a latitudinal gradient. Among the potential factors limiting the species ranges of freshwater pulmonates on a worldwide scale, two explaining variables were retained in multiple regressions. A climate variable was composed of climatic factors related to the water regime a given species experiences and, therefore, to species’ intrinsic factors and generally to physiological ecology of freshwater pulmonates. Differential adaptations may explain differences in tolerances to temporally changing environmental conditions. These adaptations involve temperature tolerance and life history characteristics. Other factors responsible for obtaining larger range sizes are resistence of a given species to anoxia and desiccation tolerance. The second variable found in this study explaining range size variation was the potential area of different biogeographic regions that were actually occupied by a species. Once again, dispersal capabilities were identified to be crucial in range size evolution. Current ranges are not an adequate reflection of historical ranges in Basommatophora. It can indirectly be concluded that pronounced dispersal by passive means is crucial in obtaining a certain range size. Additionally, it becomes clear from this study, that large scale environmental variation figures prominently in the possession of a particular range size by a particular taxon. Microevolutionary patterns and processes among Basommatophora were studied using a model of endemic limpet-like gastropods from the ancient lakes on Sulawesi (Indonesia), which exhibit an interesting case of diversification in a generally species-poor group of freshwater gastropods. Within these lakes, at least three species of Protancylus evolved allopatrically. These species are characterized by low morphological variation and a specific life history living as epizoa on the caenogastropod Tylomelania spp. They possess an unique reproduction strategy involving the only known case of brood care among Basommatophora. Phylogenetic relationships, phylogeographic pattern, morphological differentiation, species delimitation, life history parameters, and ecology are discussed in the context of evolutionary history. Among the potential modes of speciation, drift-based speciation in the lakes of the Malili Lake system and lake Poso was identified as most likely. Altogether, the studies summarized in this thesis contribute to the understanding of patterns and processes of diversification, and thus evolution, of the Basommatophora. Hopefully, they can provide the basis and trigger more interest in this fascinating model group of pulmonate gastropods.
Regulatory RNA elements fulfill functions such as translational regulation, control of transcript levels, and regulation of viral genome replication. Trans-acting factors (i.e., RNA-binding proteins) bind the so-called cis elements and confer functionality to the complex. The specificity during protein-RNA complex (RNP) formation often exploits the structural plasticity of RNA. Functional integrity of cis-trans pairs depends on the availability of properly folded RNA elements, and RNA conformational transitions can cause diseases. Knowledge of RNA structure and the conformational space is needed for understanding complex formation and deducing functional effects. However, structure determination of RNAs under in vivo conditions remains challenging. This review provides an overview of structured eukaryotic and viral RNA cis elements and discusses the effect of RNA structural equilibria on RNP formation. We showcase implications of RNA structural changes for diseases, outline strategies for RNA structure-based drug targeting, and summarize the methodological toolbox for deciphering RNA structures.
Background The degree of gene and sequence preservation across species provides valuable insights into the relative necessity of genes from the perspective of natural selection. Here, we developed novel interspecies metrics across 462 mammalian species, GISMO (Gene identity score of mammalian orthologs) and GISMO-mis (GISMO-missense), to quantify gene loss traversing millions of years of evolution. GISMO is a measure of gene loss across mammals weighed by evolutionary distance relative to humans, whereas GISMO-mis quantifies the ratio of missense to synonymous variants across mammalian species for a given gene.
Rationale Despite large sample sizes, current human constraint metrics are still not well calibrated for short genes. Traversing over 100 million years of evolution across hundreds of mammals can identify the most essential genes and improve gene-disease association. Beyond human genetics, these metrics provide measures of gene constraint to further enable mammalian genetics research.
Results Our analyses showed that both metrics are strongly correlated with measures of human gene constraint for loss-of-function, missense, and copy number dosage derived from upwards of a million human samples, which highlight the power of interspecies constraint. Importantly, neither GISMO nor GISMO-mis are strongly correlated with coding sequence length. Therefore both metrics can identify novel constrained genes that were too small for existing human constraint metrics to capture. We also found that GISMO scores capture rare variant association signals across a range of phenotypes associated with decreased fecundity, such as schizophrenia, autism, and neurodevelopmental disorders. Moreover, common variant heritability of disease traits are highly enriched in the most constrained deciles of both metrics, further underscoring the biological relevance of these metrics in identifying functionally important genes. We further showed that both scores have the lowest duplication and deletion rate in the most constrained deciles for copy number variants in the UK Biobank, suggesting that it may be an important metric for dosage sensitivity. We additionally demonstrate that GISMO can improve prioritization of recessive disorder genes and captures homozygous selection.
Conclusions Overall, we demonstrate that the most constrained genes for gene loss and missense variation capture the largest fraction of heritability, GISMO can help prioritize recessive disorder genes, and identify the most conserved genes across the mammalian tree.
Heat stress transcription factors (HSFs) are the core regulators of the heat stress (HS) response in plants. HSFs are considered as a molecular rheostat: their activities define the response intensity, incorporating information about the environmental temperature through a network of partner proteins. A prompted activation of HSFs is required for survival, for example the de novo synthesis of heat shock proteins. Furthermore, a timely attenuation of the stress response is necessary for the restoration of cellular functions and recovery from stress. In an ever-changing environment, the balance between thermotolerance and developmental processes such as reproductive fitness highlights the importance of a tightly tuned response. In many cases, the response is described as an ON/OFF mode, while in reality, it is very dynamic. This review compiles recent findings to update existing models about the HSF-regulated HS response and address two timely questions: How do plants adjust the intensity of cellular HS response corresponding to the temperature they experience? How does this adjustment contribute to the fine-tuning of the HS and developmental networks? Understanding these processes is crucial not only for enhancing our basic understanding of plant biology but also for developing strategies to improve crop resilience and productivity under stressful conditions.
Highlights
• Histone modifications alter chromatin structure and gene accessibility, allowing timely stress response, and enhancing tomato's ability to cope with environmental challenges.
• miRNAs and lncRNAs fine-tune gene expression, playing essential roles in stress tolerance, particularly in heat and drought stress responses.
• Leveraging epigenetic modifications can develop tomato varieties that maintain high productivity and quality under adverse environmental conditions.
• Detailed mapping of the tomato epigenome under various stress conditions can identify key regulatory regions and guide targeted breeding programs
Abstract
Climate change poses a major challenge to agriculture, affecting crop production through shifting weather patterns and an increase in extreme conditions such as heat waves, droughts, and floods, all of which are further compounded by biotic stress factors. Tomatoes, a vital dietary staple and significant agricultural product worldwide, are particularly susceptible to these changes. The need for developing climate-resilient tomato varieties is more urgent than ever to ensure food security. Epigenetic modifications, such as DNA methylation and histone modifications, play essential roles in gene expression regulation. These modifications can affect plant traits and responses to environmental stresses, enabling tomatoes to maintain productivity despite variable climates or disease pressures. Tomato, as a model plant, offers valuable insights into the epigenetic mechanisms underlying fruit development and responses to stress. This review provides an overview of key discoveries regarding to tomato response and resilience mechanisms related to epigenetics, highlighting their potential in breeding strategies to enhance tomato resilience against both abiotic and biotic challenges, thereby promoting sustainable agricultural practices in the context of global climate change.
Background: Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work.
Results: In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.
Conclusions: The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.
Highlights
• Different NADPH supply strategies are compared in Saccharomyces cerevisiae.
• Example products are d-xylitol and l-galactonate.
• ZWF1 overexpression is the most robust strategy in the diauxic batch fermentation.
• Carbon source dependencies and interferences of different strategies are explored.
Abstract
Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L−1 xylitol from 20 g L−1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.
Highlights
• Determination of styrene-butadiene rubber as tire constituent using TED-GC/MS.
• Determination of zinc content as tire constituent using ICP-OES.
• Representative sampling strategy with large-volume mixed samples.
• Tire wear content is decreasing with increasing sampling depth and distance to road.
• Deposited tire wear particles are mainly present in soil fraction <100 μm.
Abstract
Tire wear (TW) constitutes a significant source of microplastic in terrestrial ecosystems. It is known that particles emitted by roads can have an effect up to 100 m into adjacent areas. Here, we apply for the first-time thermal extraction desorption gas chromatography-mass spectrometry (TED-GC/MS) to determine TW in soil samples by detection of thermal decomposition products of styrene-butadiene rubber (SBR), without additional enrichment. Additionally, zinc contents were determined as an elemental marker for TW. Mixed soil samples were taken along three transects along a German motorway in 0.3, 2.0, and 5.0 m distance from the road. Sampling depths were 0–2, 2–5, 5–10, and 10–20 cm. Four fine fractions, 1 000–500, 500–100, 100–50, and <50 μm, were analyzed.
TW contents based on SBR ranged from 155 to 15 898 mg kg−1. TW contents based on zinc were between 413 and 44 812 mg kg−1. Comparison of individual values of SBR and zinc reveals SBR as a more specific marker. Results confirm that most TW ends up in the topsoil within a 2 m distance.
The sampling strategy resulted in representative data for a larger area. Standard deviations of quadruple TED-GC/MS determination of SBR were <10% for all grain size fractions. TED-GC/MS is a suitable analytical tool for determining TW in soil samples without the use of toxic chemicals, enrichment, or special sample preparation.