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Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.
Unlike other eukaryotes, plants possess a complex family of heat stress transcription factors (Hsfs) with usually more than 20 members. Among them, Hsfs A4 and A5 form a group distinguished from other Hsfs by structural features of their oligomerization domains and by a number of conserved signature sequences. We show that A4 Hsfs are potent activators of heat stress gene expression, whereas A5 Hsfs act as specific repressors of HsfA4 activity. The oligomerization domain of HsfA5 alone is necessary and sufficient to exert this effect. Due to the high specificity of the oligomerization domains, other class A Hsfs are not affected. Pull-down assay and yeast two-hybrid interaction tests demonstrate that the tendency to form HsfA4/A5 heterooligomers is stronger than the formation of homooligomers. The specificity of interaction between Hsfs A4 and A5 was confirmed by bimolecular fluorescence complementation experiments. The major role of the representatives of the HsfA4/A5 group, which are not involved in the conventional heat stress response, may reside in cell type-specific functions connected with the control of cell death triggered by pathogen infection and/or reactive oxygen species.
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.
High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.
The genome, antigens of human cytomegalovirus (HCMV) are frequently found in prostatic carcinoma. However, whether this infection is causative or is an epiphenomenon is not clear. We therefore investigated the ability of HCMV to promote metastatic processes, defined by tumor cell adhesion to the endothelium, extracellular matrix proteins. Experiments were based on the human prostate tumor cell line PC3, either infected with the HCMV strain Hi (HCMVHi) or transfected with cDNA encoding the HCMV-specific immediate early protein IEA1 (UL123) or IEA2 (UL122). HCMVHi upregulated PC3 adhesion to the endothelium, to the extracellular matrix proteins collagen, laminin, fibronectin. The process was accompanied by enhancement of β1-integrin surface expression, elevated levels of integrin-linked kinase, phosphorylation of focal adhesion kinase. IEA1 or IEA2 did not modulate PC3 adhesion or β1-integrin expression. Based on this in vitro model, we postulate a direct association between HCMV infection, prostate tumor transmigration, which is not dependent on IEA proteins. Integrin overexpression, combined with the modulation of integrin-dependent signalling, seems to be, at least in part, responsible for a more invasive PC3Hi tumor cell phenotype. Elevated levels of c-myc found in IEA1-transfected or IEA2-transfected PC3 cell populations might promote further carcinogenic processes through accelerated cell proliferation.
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: 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.
Obwohl die reiche Artenvielfalt der westafrikanischen Savannenlandschaften erst in Ansätzen erforscht und dokumentiert ist, geht aus Beobachtungen der ansässigen Bevölkerung hervor, dass viele Pflanzenarten bedroht sind. Dies ist nicht nur ein ökologisches, sondern auch ein soziokulturelles Problem. So werden beispielsweise in Nord-Benin etwa 80 Prozent aller vorkommenden Pflanzen zu medizinischen Zwecken herangezogen und stellen damit die Basisgesundheitsversorgung besonders für die ländliche Bevölkerung dar. Neben der Verwendung der Pflanzen in der traditionellen Medizin kommt ihnen auch in der täglichen Ernährung, als Baumaterial und zur Herstellung von Kosmetika eine entscheidende Rolle zu. Das interdisziplinäre BIOTA-Projekt der Universitäten Frankfurt und Mainz, des Forschungsinstituts Senckenberg und der Universitäten Ouagadougou (Burkina Faso) und Abomey-Calavi (Benin) hat es sich zur Aufgabe gemacht, die biologische Artenvielfalt und das damit verbundene lokale Wissen zu erforschen, zu schützen und zu erhalten. Erste Erfolge konnten bereits durch die Anpflanzung besonders bedrohter Arten und die Einrichtung eines Medizinalpflanzengartens, gemeinsam mit lokalen Heilkundigen in Nord-Benin, erzielt werden.
In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.