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Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD+ (~40 µM) but variable affinities for the substrate UDP-glucose (120–335 µM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes.
Summary The basal transcription apparatus of archaea is well characterized. However, much less is known about the mechanisms of transcription termination and translation initation. Recently, experimental determination of the 5´-ends of ten transcripts from Pyrobaculum aerophilum revealed that these are devoid of a 5´-UTR. Bioinformatic analysis indicated that many transcripts of other archaeal species might also be leaderless. The´-ends and 3´-ends of 40 transcripts of two haloarchaeal species, Halobacterium salinarum and Haloferax volcanii, have been determined. They were used to characterize the lengths of 5´-UTRs and 3´-UTRs and to deduce consensus sequence-elements for transcription and translation. The experimental approach was complemented with a bioinformatics analysis of the H. salinarum genome sequence. Furthermore, the influence of selected 5´-UTRs and 3´-UTRs on transcript stability and translational efficiency in vivo was characterized using a newly established reporter gene system, gene fusions, and real-time PCR. Consensus sequences for basal promoter elements could be refined and a novel element was discovered. A consensus motif probably important for transcriptional termination was established. All 40 haloarchaeal transcripts analyzed had a 3´-UTR (average size 57 nt), and their 3´-ends were not posttranscriptionally modified. Experimental data and genome analyses revealed that the majority of haloarchaeal transcripts are leaderless, indicating that this is the predominant mode for translation initiation in haloarchaea. Surprisingly, the 5´-UTRs of most leadered transcripts did not contain a Shine-Dalgarno (SD) sequence. A genome analysis indicated that less than 10% of all genes are preceded by a SD sequence and even most proximal genes in operons lack a SD sequence. Seven different leadered transcripts devoid of a SD sequence were efficiently translated in vivo, including artificial 5´-UTRs of random sequences. Thus, an interaction of the 5´-UTRs of these leadered transcripts with the 16S rRNA could be excluded. Taken together, either a scanning mechanism similar to the mechanism of translation initiation operating in eukaryotes or a novel mechanism must operate on most leadered haloarchaeal transcripts. Author Summary Expression of the information encoded in the genome of an organism into its phenotype involves transcription of the DNA into messenger RNAs and translation of mRNAs into proteins. The textbook view is that an mRNA consists of an untranslated region (5´-UTR), an open reading frame encoding the protein, and another untranslated region (3´-UTR). We have determined the 5´-ends and the 3´-ends of 40 mRNAs of two haloarchaeal species and used this dataset to gain information about nucleotide elements important for transcription and translation. Two thirds of the mRNAs were devoid of a 5´-UTR, and therefore the major pathway for translation initiation in haloarchaea involves so-called leaderless transcripts. Very unexpectedly, most leadered mRNAs were found to be devoid of a sequence motif believed to be essential for translation initiation in bacteria and archaea (Shine-Dalgarno sequence). A bioinformatic genome analysis revealed that less than 10% of the genes contain a Shine-Dalgarno sequence. mRNAs lacking this motif were efficiently translated in vivo, including mRNAs with artificial 5´-UTRs of total random sequence. Thus, translation initiation on these mRNAs either involves a scanning mechanism similar to the mechanism operating in eukaryotes or a totally novel mechanism operating at least in haloarchaea.
Ob das Alter ein Segen oder ein Fluch ist, darüber gehen seit der Antike die Meinungen auseinander, und es hat nicht an Versuchen gefehlt, für die doch unleugbaren Gebrechen und Gebresten die Gegenrechnung aufzumachen. Auf der einen Seite also Verfall des Körpers, Krankheit, Nachlassen oder Absterben der Sinnesvermögen und des fleischlichen Begehrens, auf der anderen Seite dafür aber Weisheit, Gelassenheit, Gemütsruhe, Abgeklärtheit, Milde, vielleicht Heiterkeit, da nichts mehr erreicht werden will. Prudentia – Klugheit – und Sophrosyne – Beherrschung der Begierden durch Vernunft und Besonnenheit – heißen die altersgemäßen Stichwörter, die vielleicht sogar Handlungsspielräume eröffnen, die den früheren Lebensaltern fehlten. ...
In den hoch entwickelten Industriestaaten wird seit längerem eine dramatische Veränderung der Bevölkerungsstruktur beobachtet. Bei einer Erhöhung der Lebenserwartung und einer gleichzeitigen Abnahme der Geburtenrate verschiebt sich das Verhältnis von jungen zu alten Individuen immer mehr hin zu den Älteren. Längst wird von einem »Ergrauen« oder gar einer »Vergreisung« Europas gesprochen. Hieraus ergeben sich bereits heute schwerwiegende Probleme für die bestehenden Sozial- und Gesundheitssysteme. Diese drohen sich in der Zukunft dramatisch zu verschärfen. Eine Entlastung wird sicher nur dann erreicht werden können, wenn es gelingt, das Auftreten gesundheitlicher Beeinträchtigungen und Erkrankungen nachhaltig zu verhindern oder zumindest zu verzögern und damit eine Verbesserung der Lebensqualität in fortgeschrittenen Lebensabschnitten zu gewährleisten. Entscheidende Voraussetzung zum Erreichen dieser Ziele ist ein grundlegendes Verständnis der Mechanismen biologischen Alterns.
Was passiert auf molekularer Ebene, wenn der Körper altert? Eine Antwort darauf lautet: Es häufen sich irreparable Schäden an Zellen, an Zellbestandteilen wie den Organellen, der DNA oder Eiweißen und anderen Molekülen. DassFehler passieren, ist unvermeidlich, denn jeder Stoffwechselvorgang birgt eine gewisse Störanfälligkeit in sich. Ein junger Organismus ist dank ausgefeilter Reparatursysteme in der Lage, Fehler zu korrigieren. Nimmt diese Fähigkeit mit dem Altern ab, so treten zwei Arten von Problemen mit besonders weitreichenden Folgen auf: Fehler bei der Replikation (dem Kopieren) der DNA und molekulare Schäden, die freie Radikale anrichten. So können Defekte der DNA einerseits die Entstehung von Tumoren verursachen, andererseits aber auch Alterungsprozesse beschleunigen.
Enzymes involved in tRNA maturation are essential for cytosolic, mitochondrial, and plastid protein synthesis and are therefore localized to these different compartments of the cell. Interestingly, only one isoform of tRNA nucleotidyltransferase (responsible for adding the 3′-terminal cytidine–cytidine–adenosine to tRNAs) has been identified in plants. The present study therefore explored how signals contained on this enzyme allow it to be distributed among the different cell compartments. It is demonstrated that the N-terminal portion of the protein acts as an organellar targeting signal and that differential use of multiple in-frame start codons alters the localization of the protein. Moreover, it is shown that the mature domain has a major impact on the distribution of the protein within the cell. These data indicate that regulation of dual localization involves not only specific N-terminal signals, but also additional factors within the protein or the cell.
Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5′splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5′SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA–ligand interaction for regulation of gene expression.
Background: In sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila.
Results: Whole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished.
Conclusion: Our experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly.
Background: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. Methodology/Principal Findings: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. Conclusion/Significance: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
(2007)
Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.