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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 19 (1996), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The sequence and spacing requirements of the archaeal‘distal promoter element’(DPE) were examined by randomizing positions -19 to -32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 414 entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high-activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: -32 RGTWWWWRACYGSY -19 (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)-long transcript, and very resistant clones contained both the 700 nt-long transcript and a second, more abundant, 500 nt-long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3’-untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non-SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non-SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram-positive) bacteria, which we denote ‘segregation and condensation proteins’ (Scps). ScpA genes are localized next to smc genes in nearly all SMC- containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature- sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so-called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two-hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non-SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 171 (1999), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In this study the usage of the halophilic archaeon Haloferax volcanii as a production system for eukaryal G protein-coupled receptors (GPCRs) was characterized. The genes of four GPCRs were fused to the dihydrofolate reductase gene of H. volcanii. In Northern blots both 5′ fragments and full-length fusion transcripts were found. In contrast, only C-terminal fusion protein fragments could be detected in Western blot analyses. Ligand binding experiments revealed that a minor amount of correctly folded human β2 adrenergic receptor was inserted into the membrane. The introduction of different modifications at the 5′ and the 3′ end of the receptor genes did not significantly increase the production level. Determination of the subcellular localization showed that fusion protein fragments containing one or more receptor helices were located in the membrane. The results indicate that neither transcription, translation nor membrane translocation but the activity of one or more proteases limits the level of GPCR production in H. volcanii.
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haloferax volcanii is a moderately halophilic archaeon that can grow aerobically and anaerobically with a variety of substrates. We undertook a novel approach for the characterization of metabolic adaptations, i.e. transcriptome analysis with a onefold-coverage shotgun DNA microarray. A genomic library was constructed and converted into a polymerase chain reaction (PCR) product library, which was used to print two DNA microarrays, a 960-spot test array used for optimization of microarray analysis and a 2880-spot onefold-coverage array. H. volcanii cultures were shifted from casamino acid-based metabolism to glucose-based metabolism, and the transcriptome changes were analysed with the onefold-coverage array at five time points covering the transition phase and the onset of exponential growth with the new carbon source. About 10% of all genes were found to be more than 2.5-fold regulated at at least one time point. The genes fall into five clusters of kinetically co-regulated genes. For members of all five clusters, the results were verified by Northern blot analyses. The identity of the regulated genes was determined by sequencing. Many co-regulated genes encode proteins of common functions. Expected as well as a variety of unexpected findings allowed predictions about the central metabolism, the transport capacity and the cellular composition of H. volcanii growing on casamino acids and on glucose. The microarray analyses are in accordance with the growth rates and ribosome contents of H. volcanii growing on the two carbon sources. Analysis of the results revealed that onefold-coverage shotgun DNA microarrays are well suited to characterize the regulation of metabolic pathways as well as protein complexes in response to changes in environmental conditions.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science, Ltd
    Molecular microbiology 46 (2002), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The genome of Halobacterium salinarum encodes four proteins of the structural maintenance of chromosomes (SMC) protein superfamily. Two proteins form a novel subfamily and are named ‘SMC-like proteins of H. salinarum’ (Sph1 and Sph2). Northern blot analyses revealed that sph1 and hp24, the adjacent gene, are solely transcribed in exponentially growing, but not in stationary phase, cells. A synchronization procedure was developed, which makes use of the DNA polymerase inhibitor aphidicolin and leads to highly synchronous cultures. It allowed us for the first time to study cell cycle-dependent transcription in an archaeon. The sph1 transcript was found to be highly cell cycle regulated, with its maximal accumulation around the time of septum formation. The Sph1 protein level was also elevated at that time, but a basal protein level was found throughout the cell cycle. The hp24 transcript was sharply upregulated about 1 h before sph1 and had already declined at the time of sph1 induction. These and additional transcript patterns revealed that precisely controlled transcriptional regulation is involved in haloarchaeal cell cycle progression. A DNA staining protocol was developed, which opened the possibility of following the dynamic intracellular localization of haloarchaeal nucleoids using synchronized cultures. After an initial dispersed localization, the nucleoid is condensed at mid-cell. Subsequently, DNA is rapidly transported to the 1/4 and 3/4 positions. All staining patterns were also observed in untreated exponentially growing cells, excluding synchronization artifacts. The Sph1 concentration is elevated when segregation of the new chromosomes is nearly complete; therefore, it is proposed to play a role in a late step of replication, e.g. DNA repair, similar to eukaryotic Rad18 proteins.
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 31 (1999), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The basal apparatus for transcription initiation in Archaea is more closely related to the eukaryal than to the bacterial counterpart. The understanding of archaeal transcription initiation has been deepened by recent advances, which include genome sequencing, biochemical approaches and the structure determination of a protein DNA complex. Archaeal promoter elements, transcription factors, RNA polymerase and their interactions are discussed and compared with the eukaryal situation. It is emerging that transcription initiation is not uniform in Archaea. A minimal set of promoter elements and transcription factors is conserved, but the relative importance for transcription initiation can vary. Furthermore, additional basal transcription factors and promoter elements seem to be crucial in subgroups of Archaea. Finally, some aspects of global as well as gene-specific transcriptional regulation are discussed.
    Type of Medium: Electronic Resource
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  • 8
    Publication Date: 2006-12-20
    Electronic ISSN: 1932-6203
    Topics: Medicine , Natural Sciences in General
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  • 9
    Publication Date: 2009-09-01
    Print ISSN: 1386-1425
    Electronic ISSN: 1873-3557
    Topics: Chemistry and Pharmacology , Physics
    Published by Elsevier
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  • 10
    Publication Date: 1993-02-01
    Print ISSN: 0723-2020
    Electronic ISSN: 1618-0984
    Topics: Biology
    Published by Elsevier
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