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The transcriptional regulation of the cellular mechanisms involves many different components and different levels of control which together contribute to fine tune the response of cells to different environmental stimuli. In some responses, diverse signaling pathways can be controlled simultaneously. One of the most important cellular processes that seem to possess multiple levels of regulation is photosynthesis. A model organism for studying photosynthesis-related processes is the unicellular green algae Chlamydomonas reinhardtii, due to advantages related to culturing, genetic manipulation and availability of genome sequence. In the present study, we were interested in understanding the regulatory mechanisms underlying photosynthesis-related processes. To achieve this goal different molecular approaches were followed. In order to indentify protein transcriptional regulators we optimized a method for isolation of nuclei and performed nuclear proteome analysis using shotgun proteomics. This analysis permitted us to improve the genome annotation previously published and to discover conserved and enriched protein motifs among the nuclear proteins. In another approach, a quantitative RT-PCR platform was established for the analysis of gene expression of predicted transcription factor (TF) and other transcriptional regulator (TR) coding genes by transcript profiling. The gene expression profiles for more than one hundred genes were monitored in time series experiments under conditions of changes in light intensity (200 µE m-2 s-1 to 700 µE m-2 s-1), and changes in concentration of carbon dioxide (5% CO2 to 0.04% CO2). The results indicate that many TF and TR genes are regulated in both environmental conditions and groups of co-regulated genes were found. Our findings also suggest that some genes can be common intermediates of light and carbon responsive regulatory pathways. These approaches together gave us new insights about the regulation of photosynthesis and revealed new candidate regulatory genes, helping to decipher the gene regulatory networks in Chlamydomonas. Further experimental studies are necessary to clarify the function of the candidate regulatory genes and to elucidate how cells coordinately regulate the assimilation of carbon and light responses.
The meadow grasshopper, Chorthippus parallelus (Zetterstedt), is common and widespread in Central Europe, with a low dispersal range per generation. A population study in Central Germany (Frankenwald and Thuringer Schiefergebirge) showed strong interpopulation differences in abundance and individual fitness. We examined genetic variability using microsatellite markers within and between 22 populations in a short-to long-distance sampling (19 populations, Frankenwald, Schiefergebirge, as well as a southern transect), and in the Erzgebirge region (three populations), with the latter aiming to check for effects as a result of historical forest cover. Of the 671 C. parallelus captured, none was macropterous (functionally winged). All populations showed a high level of expected and observed heterozygosity (mean 0.80-0.90 and 0.60-0.75, respectively), whereas there was evidence of inbreeding (F(IS) values all positive). Allelic richness for all locus-population combinations was high (mean 9.3-11.2), whereas alleles per locus ranged from 15-62. At a local level, genic and genotypic differences were significant. Pairwise F(ST) values were in the range 0.00-0.04, indicating little interpopulation genetic differentiation. Similarly, the calculated gene flow was very high, based on the respective F(ST) (19.5) and using private alleles (7.7). A Neighbour-joining tree using Nei's D(A) and principal coordinate analysis separated two populations that were collected in the Erzgebirge region. Populations from this region may have escaped the effects of the historical forest cover. The visualization of the spatial arrangement of genotypes revealed one geographical barrier to gene flow in the short-distance sampling.
Microviridins are unique protease inhibitors from bloom-forming cyanobacteria that have both ecological and pharmacological relevance. Their peptide backbones are produced ribosomally, and ATP grasp ligases introduce omega-ester and omega-amide bonds to yield rare cage-like structures. Bioinformatic analysis of the microviridin biosynthesis gene cluster suggests a novel type of processing machinery, which could rationalize the challenging in vivo/in vitro reconstitution of the pathway. In this work, we report the establishment of a minimal expression system for microviridins. Through bioinformatics and mutational analysis of the MdnA leader peptide we identified and characterized a strictly conserved binding motif that is specific for microviridin ligases. Furthermore, we showed that the ABC transporter MdnE is crucial for cyclization and processing of microviridins and demonstrated that MdnE is essential for stability of the microviridin biosynthesis complex.
It is currently controversially discussed if the same freshwater microorganisms occur worldwide wherever their required habitats are realized, i.e., without any adaptation to local conditions below the species level. We performed laboratory experiments with flagellates and ciliates from three acidic mining lakes (AML, pH similar to 2.7) to investigate if similar habitats may affect similar organisms differently. Such man-made lakes provide suitable ecosystem models to test for the significance of strong habitat selection. To this end, we analyzed the growth response of three protist taxa (three strains of the phytoflagellate Chlamydomonas acidophila, two isolates of the phytoflagellate Ochromonas and two species of the ciliate genus Oxytricha) by exposing them to lake water of their origin and from the two other AML in a cross-factorial design. Population growth rates were measured as a proxy for their fitness. Results revealed significant effects of strain, lake (= habitat), and strain X habitat interaction. In the environmentally most adverse AML, all three protist taxa were locally adapted. In conclusion, our study demonstrates that (1) the same habitat may affect strains of the same species differently and that (2) similar habitats may harbor ecophysiologically different strains or species. These results contradict the 'everything is everywhere' paradigm.
The two rhizomatous perennials Solidago canadensis and S. gigantea belong to the most widespread alien plants in Europe. Anecdotal observations suggest that they disperse also by rhizome fragments. For testing their resprouting ability, rhizome fragments of different sizes from both species were buried at four different soil depths (0, 5, 10 and 20 cm, respectively) in a common garden. Rhizome fragments of S. canadensis ranged 3-6 cm in length, those of S. gigantea 5-20 cm in length. Resprouting plants were harvested after 3 months and growth related traits measured to assess their vitality. Resprouting rate in S. gigantea was far higher than in S. canadensis (85 and 19%, respectively). In S. gigantea, fragments of all sizes resprouted from all soil depths whereas none rhizome of S. canadensis emerged from 20 cm burial depth. In S. gigantea, size related traits showed significant interactions between fragment size and burial depth, but not relative shoot growth rate. At all burial depths, vitality of plants emerging from small rhizomes was lower than plants emerging from large rhizomes. Effects of rhizome size became stronger with increasing burial depth. The results show that both species are able to resprout from buried rhizome fragments, and that succesful regeneration is more likely to occur in S. gigantea. Managing these species should avoid any activities promoting rhizome fragmentation and dispersal of fragments.
The production of monoclonal antibodies by hybridoma technology is dependent on lymphocytes taken from vertebrates which have to be immunized against the corresponding antigen. We present here our first experiments which should allow the replacement of this in vivo immunization step by an in vitro immunization procedure. This work provides new possibilities for the specific activation of immune cells in order to use them for the generation of antibodies which are not of murine origin. Bone marrow-derived dendritic cells were loaded with antigen and co-cultured with naive T and B lymphocytes of non-immunized mice. The interaction and activation of the different cell types were investigated by measuring the expression of specific cell surface markers, the release of activation-dependent interleukins and the secretion of antigen-specific antibodies. We could demonstrate that dendritic cells process and present antigen fragments and activate T cells, that T cells proliferate and release activation-induced interleukins, and that B cells maturate under the influence of activated T cells and secrete antigen-specific antibodies.
Preface
(2011)
Background: Soil biota effects are increasingly accepted as an important driver of the abundance and distribution of plants. While biogeographical studies on alien invasive plant species have indicated coevolution with soil biota in their native distribution range, it is unknown whether adaptation to soil biota varies among populations within the native distribution range. The question of local adaptation between plants and their soil biota has important implications for conservation of biodiversity and may justify the use of seed material from local provenances in restoration campaigns.
Methodology/Principal Findings: We studied soil biota effects in ten populations of the steppe grass Stipa capillata from two distinct regions, Europe and Asia. We tested for local adaptation at two different scales, both within (ca. 10-80 km) and between (ca. 3300 km) regions, using a reciprocal inoculation experiment in the greenhouse for nine months. Generally, negative soil biota effects were consistent. However, we did not find evidence for local adaptation: both within and between regions, growth of plants in their 'home soil' was not significantly larger relative to that in soil from other, more distant, populations.
Conclusions/Significance: Our study suggests that negative soil biota effects can prevail in different parts of a plant species' range. Absence of local adaptation points to the possibility of similar rhizosphere biota composition across populations and regions, sufficient gene flow to prevent coevolution, selection in favor of plasticity, or functional redundancy among different soil biota. From the point of view of plant - soil biota interactions, our findings indicate that the current practice of using seeds exclusively from local provenances in ecosystem restoration campaigns may not be justified.
The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.