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Ahnenforschung unter sozialen Amöben : die morphologische Taxonomie muss umgeschrieben werden
(2007)
Seit fast 150 Jahren forschen Wissenschaftler aus aller Welt über den faszinierenden Wechsel zwischen Einzelligkeit und Vielzelligkeit im Lebenszyklus der »zellulären Schleimpilze«. Diese Forschung war bisher so erfolgreich, dass einem Vertreter der zellulären Schleimpilze, Dictyostelium discoideum, vom US-amerikanischen Gesundheitsministerium National Institutes of Health (NIH) ganz offiziell der Status eines Modellorganismus für biomedizinische Forschung verliehen wurde. Obwohl wir inzwischen glauben, viel über die »sozialen Amöben«, die sich bei Nahrungsmangel von Einzellern zu einem vielzelligen Verband zusammenlagern, gelernt zu haben, basiert unser Wissen doch fast ausschließlich auf Arbeiten mit der einen Art D. discoideum. Man kennt allerdings heute mehr als 100 Arten sozialer Amöben. Alle bilden multizelluläre Fruchtkörper aus, die aus Stielen und Sporenpaketen bestehen. Bisher ging man davon aus, dass die Spezies mit azellulären Stielen in ihren Fruchtkörpern phylogenetische Vorläufer der Vertreter mit zellulären Stielen sind, und dass die Vertreter mit verzweigten Fruchtkörpern näher mit sich selbst als mit den anderen sozialen Amöben verwandt sind. Diese Hypothesen wurden nun durch aktuelle molekulargenetische Analysen widerlegt.
Expeditionen ins Pilzreich Panamas : Pionierarbeit in einer der artenreichsten Regionen unserer Erde
(2007)
Als Bindeglied zwischen Nord- und Südamerika ist Panama ein »Biodiversitäts-Hotspot« – es beherbergt eine außerordentlich hohe Artenvielfalt an Pflanzen, Tieren und Pilzen. Pilze übernehmen in tropischen Ökosystemen wichtige Aufgaben: Sie zersetzen totes organisches Material, helfen den Pflanzen bei der Aufnahme von Wasser und Mineralstoffen aus dem Boden, und sie leisten sogar als Parasiten einen Beitrag zum Erhalt einer großen Artenvielfalt. Aufgrund einzelner Stichproben wissen wir, dass die Anzahl der Pilzarten in den Tropen diejenige der Pflanzen um ein Vielfaches übertrifft. Doch während für Panama zirka 9500 verschiedene Arten von Gefäßpflanzen bekannt sind, zählt eine im Rahmen unserer Arbeit erstellte Checkliste der Pilze nur zirka 1800 Arten. Das zeigt, dass für die Erforschung der Pilze noch umfangreiche Pionierarbeit geleistet werden muss. Zwischen 2003 und 2006 geschah dies im Rahmen einer Universitätspartnerschaft der Universität Frankfurt mit der Universidad Autónoma de Chiriquí, die durch den Deutschen Akademischen Austauschdienst (DAAD) gefördert wurde. Im Zentrum eines Projekts der Deutschen Forschungsgemeinschaft (DFG) steht die Erforschung der Vielfalt und Ökologie pflanzenparasitischer Pilze. Des Weiteren untersucht unsere Arbeitsgruppe Pilze an Insekten sowie an menschlichen Haut- und Nagelläsionen.
Background The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. Results A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 uM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. Conclusions The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6% - 28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.
Tens of thousands of man-made chemicals are in regular use and discharged into the environment. Many of them are known to interfere with the hormonal systems in humans and wildlife. Given the complexity of endocrine systems, there are many ways in which endocrine-disrupting chemicals (EDCs) can affect the body’s signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. A major concern is that some of these EDCs appear to be biologically active at extremely low concentrations. There is growing evidence to indicate that the guiding principle of traditional toxicology that “the dose makes the poison” may not always be the case because some EDCs do not induce the classical dose–response relationships. The European Union project COMPRENDO (Comparative Research on Endocrine Disrupters—Phylogenetic Approach and Common Principles focussing on Androgenic/Antiandrogenic Compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (AACs) to wildlife and humans by focusing on the commonalities and differences in responses to AACs across the animal kingdom (from invertebrates to vertebrates).
Polyploidy is common in higher eukaryotes, especially in plants, but it is generally assumed that most prokaryotes contain a single copy of a circular chromosome and are therefore monoploid. We have used two independent methods to determine the genome copy number in halophilic archaea, 1) cell lysis in agarose blocks and Southern blot analysis, and 2) Real-Time quantitative PCR. Fast growing H. salinarum cells contain on average about 25 copies of the chromosome in exponential phase, and their ploidy is downregulated to 15 copies in early stationary phase. The chromosome copy number is identical in cultures with a twofold lower growth rate, in contrast to the results reported for several other prokaryotic species. Of three additional replicons of H. salinarum, two have a low copy number that is not growth-phase regulated, while one replicon even shows a higher degree of growth phase-dependent regulation than the main replicon. The genome copy number of H. volcanii is similarly high during exponential phase (on average 18 copies/cell), and it is also downregulated (to 10 copies) as the cells enter stationary phase. The variation of genome copy numbers in the population was addressed by fluorescence microscopy and by FACS analysis. These methods allowed us to verify the growth phase-dependent regulation of ploidy in H. salinarum, and they revealed that there is a wide variation in genome copy numbers in individual cells that is much larger in exponential than in stationary phase. Our results indicate that polyploidy might be more widespread in archaea (or even prokaryotes in general) than previously assumed. Moreover, the presence of so many genome copies in a prokaryote raises questions about the evolutionary significance of this strategy.
Background Identification and evaluation of surface binding-pockets and occluded cavities are initial steps in protein structure-based drug design. Characterizing the active site's shape as well as the distribution of surrounding residues plays an important role for a variety of applications such as automated ligand docking or in situ modeling. Comparing the shape similarity of binding site geometries of related proteins provides further insights into the mechanisms of ligand binding. Results We present PocketPicker, an automated grid-based technique for the prediction of protein binding pockets that specifies the shape of a potential binding-site with regard to its buriedness. The method was applied to a representative set of protein-ligand complexes and their corresponding apo-protein structures to evaluate the quality of binding-site predictions. The performance of the pocket detection routine was compared to results achieved with the existing methods CAST, LIGSITE, LIGSITEcs, PASS and SURFNET. Success rates PocketPicker were comparable to those of LIGSITEcs and outperformed the other tools. We introduce a descriptor that translates the arrangement of grid points delineating a detected binding-site into a correlation vector. We show that this shape descriptor is suited for comparative analyses of similar binding-site geometry by examining induced-fit phenomena in aldose reductase. This new method uses information derived from calculations of the buriedness of potential binding-sites. Conclusions The pocket prediction routine of PocketPicker is a useful tool for identification of potential protein binding-pockets. It produces a convenient representation of binding-site shapes including an intuitive description of their accessibility. The shape-descriptor for automated classification of binding-site geometries can be used as an additional tool complementing elaborate manual inspections.
Background The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabdeltaC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E.coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.
Background The Radical Pair model proposes that magnetoreception is a light-dependent process. Under low monochromatic light from the short-wavelength part of the visual spectrum, migratory birds show orientation in their migratory direction. Under monochromatic light of higher intensity, however, they showed unusual preferences in other directions or axial preferences. To determine whether or not these responses are still controlled by the respective light regimes, European robins, Erithacus rubecula, were tested under UV, Blue, Turquoise and Green light at increasing intensities, with orientation in migratory direction serving as a criterion whether or not magnetoreception works in the normal way. Results Under low light with a quantal flux of 8 times 10 to 15 power quanta s-1 m-2, the birds were well oriented in their seasonally appropriate migratory direction under 424 nm Blue, 502 nm Turquoise and 565 nm Green light, indicating unimpaired magnetoreception. Under 373 nm UV of the same quantal flux, they were not oriented in migratory direction, showing a preference of the east-west axis instead, but they showed excellent orientation in migratory direction under UV of lower intensity. Intensities of above 36 times 10 to 15 power quanta s-1 m-2 of Blue, Turquoise and Green light elicited a variety of responses: disorientation, headings along the east-west axis, headings along the north-south axis or 'fixed' direction tendencies. These responses changed as the intensity was increased from 36 times 10 to the 15 power quanta s-1 m-2 to 54 and 72 times 10 to 15 power quanta s-1 m-2. Conclusion The specific manifestation of responses in directions other than migratory direction clearly depends on the ambient light regime. This implies that although mechanisms normally providing magnetic compass information seem disrupted, processes that are activated by light still control the behavior. It suggests complex interactions between different types of receptors, magnetic and visual. The nature of the receptors involved and details of their connections are not yet known; however, a role of the color cones in the processes mediating magnetic input is suggested.
Background: Growth rate is central to the development of cells in all organisms. However, little is known about the impact of changing growth rates. We used continuous cultures to control growth rate and studied the transcriptional program of the model eukaryote Saccharomyces cerevisiae, with generation times varying between 2 and 35 hours.
Results: A total of 5930 transcripts were identified at the different growth rates studied. Consensus clustering of these revealed that half of all yeast genes are affected by the specific growth rate, and that the changes are similar to those found when cells are exposed to different types of stress (>80% overlap). Genes with decreased transcript levels in response to faster growth are largely of unknown function (>50%) whereas genes with increased transcript levels are involved in macromolecular biosynthesis such as those that encode ribosomal proteins. This group also covers most targets of the transcriptional activator RAP1, which is also known to be involved in replication. A positive correlation between the location of replication origins and the location of growth-regulated genes suggests a role for replication in growth rate regulation.
Conclusion: Our data show that the cellular growth rate has great influence on transcriptional regulation. This, in turn, implies that one should be cautious when comparing mutants with different growth rates. Our findings also indicate that much of the regulation is coordinated via the chromosomal location of the affected genes, which may be valuable information for the control of heterologous gene expression in metabolic engineering.
Length variations of repetitive sequences in different AT-rich loop-coding regions of mitochondrial 16S rDNA in two gastropod species were discovered during intraspecific haplotype surveys. Examination of the discrete length variation of the basic repeat unit in a phylogenetic framework led to the conclusion of a microsatellite-like mutational dynamic. The observations suggest that the presence of a repetitive sequence structure alone is sufficient to trigger this dynamic.
I analysed the importance of shell size, shell shape, habitat preferences and availability, experienced climate, active dispersal and influence of Pleistocene glaciations for the range sizes of 37 Western Palaearctic Helicidae s.l. species for which a phylogeny was available. In both cross-species and phylogenetically controlled analyses, the range sizes were positively correlated to climatic tolerance, shell size, active dispersal and influence of Pleistocene glaciations. In addition, range sizes increased significantly with latitude. Multiple regression suggested that, predominantly, the influence of Pleistocene glaciations, tolerance to large annual temperature ranges and shell size influenced the distributional range sizes. Habitat preference, range and availability, active dispersal and shell shape explained no additional variance. The results suggest that the processes influencing species range size of the Helicidae s.l. are mainly related to the climatic shifts after the Pleistocene.
Background Reliable taxonomic identification at the species level is the basis for many biological disciplines. In order to distinguish species, it is necessary that taxonomic characters allow for the separation of individuals into recognisable, homogeneous groups that differ from other such groups in a consistent way. We compared here the suitability and efficacy of traditionally used shell morphology and DNA-based methods to distinguish among species of the freshwater snail genus Radix (Basommatophora, Pulmonata). Results Morphometric analysis showed that shell shape was unsuitable to define homogeneous, recognisable entities, because the variation was continuous. On the other hand, the Molecularly defined Operational Taxonomic Units (MOTU), inferred from mitochondrial COI sequence variation, proved to be congruent with biological species, inferred from geographic distribution patterns, congruence with nuclear markers and crossing experiments. Moreover, it could be shown that the phenotypically plastic shell variation is mostly determined by the environmental conditions experienced. Conclusion Contrary to DNA-taxonomy, shell morphology was not suitable for delimiting and recognising species in Radix. As the situation encountered here seems to be widespread in invertebrates, we propose DNA-taxonomy as a reliable, comparable, and objective means for species identification in biological research.
Background The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome – Ph+) the derivative 9+ encodes either the p40(ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. Methods We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient. Results Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. Conclusion Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.
The radiation-sensitive mutant pso4-1 of Saccharomyces cerevisiae shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have cloned the putative yeast DNA repair gene PSO4 from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for pso4-1. Sequence analysis revealed that gene PSO4 consists of 1512 bp located upstream of UBI4 on chromosome XII and encodes a putative protein of 56.7 kDa. PSO4 is allelic to PRP19, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast genes. Gene disruption with a destroyed reading frame of our PSO4 clone resulted in death of haploid cells, confirming the finding that PSO4/PRP19 is an essential gene. Thus, PSO4 is the third essential DNA repair gene found in the yeast S.cerevisiae.
Background Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. Results We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. Conclusion Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.
Herman P. Schwan [1915–2005] was a distinguished scientist and engineer, and a founding father of the field of biomedical engineering. A man of integrity, Schwan influenced the lives of many, including his wife and children, and his many students and colleagues. Active in science until nearly the end of his life, he will be very much missed by his family and many colleagues.
High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently,~ 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.
Background: Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results: We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions: Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.
The 5'-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3Cpro), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5'-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 A (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel ‘pseudo base pair’ without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschläger, J. Wöhnert, E. Bucci, S. Seitz, S. Häfner, R. Ramachandran, R. Zell and M. Görlach (2004) Structure, 12, 237–248] that the proteinase 3Cpro recognizes structure rather than sequence.