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Search Results: 1 - 10 of 223266 matches for " Stephan C. Schuster "
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Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA
Felix Oehme, Stephan C Schuster
BMC Biochemistry , 2001, DOI: 10.1186/1471-2091-2-2
Abstract: We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [32P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction.A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases.Two-component systems are central elements of the bacterial signaling circuitry [1]. Signal transduction by these systems usually involves autophosphorylation of a histidine kinase on a conserved histidine residue and subsequent transfer of the phosphoryl group to a conserved aspartate on a receiver domain. Until recently, two-component systems had only been found in bacteria. In the past few years, genes coding for histidine kinase homologues and their corresponding receivers have also been discovered in eukaryotic organisms [for a review see 2]. Most of the corresponding eukaryotic gene products are part of a phosphoryl relay, which consists of a hybrid histidine kinase with a kinase and a receiver domain on the same polypeptide, a histidine phosphotransfer protein and a second receiver as part of a response regulator [3]. The function of eukaryotic two-component systems as histidine kinases was questio
Hisactophilin is involved in osmoprotection in Dictyostelium
Tanja Pintsch, Hans Zischka, Stephan C Schuster
BMC Biochemistry , 2002, DOI: 10.1186/1471-2091-3-10
Abstract: We identified hisactophilin as one of the proteins that are enriched in the cytoskeletal fraction during osmotic shock. In mutants lacking hisactophilin, viability is reduced under hyperosmotic stress conditions. In wild type cells, serine phosphorylation of hisactophilin was specifically induced by hypertonicity, but not when other stress conditions were imposed on cells. The phosphorylation kinetics reveals a slow accumulation of phosphorylated hisactophilin from 20–60 min after onset of the hyperosmotic shock condition.In the present study, we identified hisactophilin as an essential protein for the osmoprotection of Dictyostelium cells. The observed phosphorylation kinetics suggest that hisactophilin regulation is involved in long-term osmoprotection and that phosphorylation occurs in parallel with inactivation of the dynamic actin cytoskeleton.Cells steadily face changes of the external osmolarity, to which they have to adapt. To withstand a steep increase in osmolarity, eukaryotic cells activate responses like "regulatory volume increase", accumulation of compatible osmolytes and stimulated expression of stress proteins [1-4]. Recently, an exception from this scheme has been identified: Dictyostelium cells protect themselves against hyperosmolarity by largely rearranging cellular proteins, whereas no "regulatory volume increase", no accumulation of compatible osmolytes and no change of the expression pattern of the most abundant proteins were observed [5]. Among the translocated proteins identified, cytoskeletal proteins appear to be predominant. In particular, the rearrangement of actin and myosin II to the cell cortex beneath the plasma membrane [6] was shown to constitute a pivotal element of osmoprotection in Dictyostelium. These two proteins form the core of a rigid network resembling a shell-like structure [7]. Conversely, DdLIM, a cytoskeletal protein involved in the formation of protrusions [8], is depleted from the cytoskeletal fraction under hyperton
Poor Man’s 1000 Genome Project: Recent Human Population Expansion Confounds the Detection of Disease Alleles in 7,098 Complete Mitochondrial Genomes
Hie Lim Kim,Stephan C. Schuster
Frontiers in Genetics , 2013, DOI: 10.3389/fgene.2013.00013
Abstract: Rapid growth of the human population has caused the accumulation of rare genetic variants that may play a role in the origin of genetic diseases. However, it is challenging to identify those rare variants responsible for specific diseases without genetic data from an extraordinarily large population sample. Here we focused on the accumulated data from the human mitochondrial (mt) genome sequences because this data provided 7,098 whole genomes for analysis. In this dataset we identified 6,110 single nucleotide variants (SNVs) and their frequency and determined that the best-fit demographic model for the 7,098 genomes included severe population bottlenecks and exponential expansions of the non-African population. Using this model, we simulated the evolution of mt genomes in order to ascertain the behavior of deleterious mutations. We found that such deleterious mutations barely survived during population expansion. We derived the threshold frequency of a deleterious mutation in separate African, Asian, and European populations and used it to identify pathogenic mutations in our dataset. Although threshold frequency was very low, the proportion of variants showing a lower frequency than that threshold was 82, 83, and 91% of the total variants for the African, Asian, and European populations, respectively. Within these variants, only 18 known pathogenic mutations were detected in the 7,098 genomes. This result showed the difficulty of detecting a pathogenic mutation within an abundance of rare variants in the human population, even with a large number of genomes available for study.
Abstract not submitted for online publication
Stephan Schuster
Genome Biology , 2010, DOI: 10.1186/gb-2010-11-s1-i16
Interpretation of custom designed Illumina genotype cluster plots for targeted association studies and next-generation sequence validation
Elizabeth A Tindall, Desiree C Petersen, Stina Nikolaysen, Webb Miller, Stephan C Schuster, Vanessa M Hayes
BMC Research Notes , 2010, DOI: 10.1186/1756-0500-3-39
Abstract: We illustrate the dramatic effect of outliers in genotype calling and data interpretation, as well as suggest simple means to avoid genotyping errors. Furthermore we present this platform as a successful method for two-cluster rare or non-autosomal variant calling. The success of high-throughput technologies to accurately call rare variants will become an essential feature for future association studies. Finally, we highlight additional advantages of the Illumina GoldenGate chemistry in generating unusually segregated cluster plots that identify potential NGS generated sequencing error resulting from minimal coverage.We demonstrate the importance of visually inspecting genotype cluster plots generated by the Illumina software and issue warnings regarding commonly accepted quality control parameters. In addition to suggesting applications to minimise data exclusion, we propose that the Illumina cluster plots may be helpful in identifying potential in-put sequence errors, particularly important for studies to validate NGS generated variation.Commercially available genome-wide single nucleotide polymorphism (SNP) arrays and high-throughput "custom designed" genotyping of targeted variants are desirable for biologically focused research. The generation of next-generation sequencing (NGS) de novo and resequencing data is contributing to the increased desire for custom, high-throughput arrays for variant validation and determination of allele frequencies [1,2]. For custom designed genotyping, assay reliability and productivity is considered more crucial than perhaps for genome-wide association studies (GWAS). Variants are chosen to answer a specific question, and therefore variant selection is more thoughtful and less redundant.The Illumina platform (Illumina Inc., San Diego, CA, USA) has proven reliable and efficient for a number of high-throughput genotyping applications using DNA extracted from several sources, [3-9]. VeraCode and BeadArray technologies are used with t
Simultaneous Assessment of Soil Microbial Community Structure and Function through Analysis of the Meta-Transcriptome
Tim Urich, Anders Lanzén, Ji Qi, Daniel H. Huson, Christa Schleper, Stephan C. Schuster
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002527
Abstract: Background Soil ecosystems harbor the most complex prokaryotic and eukaryotic microbial communities on Earth. Experimental approaches studying these systems usually focus on either the soil community's taxonomic structure or its functional characteristics. Many methods target DNA as marker molecule and use PCR for amplification. Methodology/Principal Findings Here we apply an RNA-centered meta-transcriptomic approach to simultaneously obtain information on both structure and function of a soil community. Total community RNA is random reversely transcribed into cDNA without any PCR or cloning step. Direct pyrosequencing produces large numbers of cDNA rRNA-tags; these are taxonomically profiled in a binning approach using the MEGAN software and two specifically compiled rRNA reference databases containing small and large subunit rRNA sequences. The pyrosequencing also produces mRNA-tags; these provide a sequence-based transcriptome of the community. One soil dataset of 258,411 RNA-tags of ~98 bp length contained 193,219 rRNA-tags with valid taxonomic information, together with 21,133 mRNA-tags. Quantitative information about the relative abundance of organisms from all three domains of life and from different trophic levels was obtained in a single experiment. Less frequent taxa, such as soil Crenarchaeota, were well represented in the data set. These were identified by more than 2,000 rRNA-tags; furthermore, their activity in situ was revealed through the presence of mRNA-tags specific for enzymes involved in ammonia oxidation and CO2 fixation. Conclusions/Significance This approach could be widely applied in microbial ecology by efficiently linking community structure and function in a single experiment while avoiding biases inherent in other methods.
Calling SNPs without a reference sequence
Aakrosh Ratan, Yu Zhang, Vanessa M Hayes, Stephan C Schuster, Webb Miller
BMC Bioinformatics , 2010, DOI: 10.1186/1471-2105-11-130
Abstract: We describe a computational pipeline, called DIAL (De novo Identification of Alleles), for identifying single-base substitutions between two closely related genomes without the help of a reference genome. The method works even when the depth of coverage is insufficient for de novo assembly, and it can be extended to determine small insertions/deletions. We evaluate the software's effectiveness using published Roche/454 sequence data from the genome of Dr. James Watson (to detect heterozygous positions) and recent Illumina data from orangutan, in each case comparing our results to those from computational analysis that uses a reference genome assembly. We also illustrate the use of DIAL to identify nucleotide differences among transcriptome sequences.DIAL can be used for identification of nucleotide differences in species for which no reference sequence is available. Our main motivation is to use this tool to survey the genetic diversity of endangered species as the identified sequence differences can be used to design genotyping arrays to assist in the species' management. The DIAL source code is freely available at http://www.bx.psu.edu/miller_lab/ webcite.Next-generation sequencing technologies have revolutionized genomics, leading to a tremendous increase in the amount of available sequence data, while bringing down the cost per base. However, de novo assembling of mammalian genomes using these short reads has met with limited success [1], despite recent strides in assembling smaller microbial genomes [1-3]. The market for these short-read technologies has largely been driven by resequencing efforts, where reads are mapped to a genome sequence that was typically assembled using some other sequencing technology such as Sanger sequencing. The deduced genomic differences are then studied to characterize and understand the genetic diversity among individuals of the species. The last few years have seen tremendous progress in the development of algorithms and software
Cytosolic acidification as a signal mediating hyperosmotic stress responses in Dictyostelium discoideum
Tanja Pintsch, Michel Satre, Gérard Klein, Jean-Baptiste Martin, Stephan C Schuster
BMC Cell Biology , 2001, DOI: 10.1186/1471-2121-2-9
Abstract: We determined pH changes in response to hyperosmotic stress using FACS or 31P-NMR. Hyperosmolarity was found to acidify the cytosol from pH 7.5 to 6.8 within 5 minutes, whereas the pH of the endo-lysosomal compartment remained constant. Fluid-phase endocytosis was identified as a possible target of cytosolic acidification, as the inhibition of endocytosis observed under hypertonic conditions can be fully attributed to cytosolic acidification. In addition, a deceleration of vesicle mobility and a decrease in the NTP pool was observed.Together, these results indicate that hyperosmotic stress triggers pleiotropic effects, which are partially mediated by a pH signal and which all contribute to the downregulation of cellular activity. The comparison of our results with the effect of hyperosmolarity and intracellular acidification on receptor-mediated endocytosis in mammalian cells reveals striking similarities, suggesting the hypothesis of the same mechanism of inhibition by low internal pH.Cells steadily face fluctuations of the external osmolarity due to dehydration. Occasionally, dramatic changes in osmolarity can occur, resulting in a stress condition [1]. Hyperosmolarity of the external medium leads to the extrusion of water and the concomitant shrinkage of cells [2]. Within a few minutes, the cells activate mechanisms, termed "regulatory volume increase" (RVI), to regain their volume [3]. Under prolonged hyperosmotic conditions, compatible osmolytes, e.g. polyols or amines are accumulated inside the cells [3]. These osmolytes exhibit a stabilizing effect on proteins and thereby avoid the deleterious effect of protein aggregation. In addition, the expression of stress proteins, as chaperones and DNA repair proteins was observed in various organisms in response to hypertonicity [4,5,6].Recently it could be shown, that the amoeba Dictyostelium discoideum exhibits an unusual response to hypertonic stress which is distinct from the response observed in other organisms [
Characterization of meiotic crossovers and gene conversion by whole-genome sequencing in Saccharomyces cerevisiae
Ji Qi, Asela J Wijeratne, Lynn P Tomsho, Yi Hu, Stephan C Schuster, Hong Ma
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-475
Abstract: We used high throughput sequencing to uncover over 46 thousand single nucleotide polymorphisms (SNPs) between two budding yeast strains and investigated meiotic recombinational events. We provided a detailed analysis of CO and NCO events, including number, size range, and distribution on chromosomes. We have detected 91 COs, very close to the average number from previous genetic studies, as well as 21 NCO events and mapped the positions of these events with high resolution. We have obtained DNA sequence-level evidence for a wide range of sizes of chromosomal regions involved in CO and NCO events. We show that a large fraction of the COs are accompanied by gene conversion (GC), indicating that meiotic recombination changes allelic frequencies, in addition to redistributing existing genetic variations.This work is the first reported study of meiotic recombination using high throughput sequencing technologies. Our results show that high-throughput sequencing is a sensitive method to uncover at single-base resolution details of CO and NCO events, including some complex patterns, providing new clues about the mechanism of this fundamental process.Meiosis is essential for eukaryotic sexual reproduction and reduces the number of chromosomes in half to generate haploid cells [1-3]. To ensure the proper meiotic homolog segregation, the homologs must recognize and pair with each other in early prophase I [1-3]. It is thought that a key pairing mechanism is via DNA heteroduplex formation, which is intimately coupled with the initiation of meiotic recombination [2]. One major type of outcome of meiotic recombination is crossover (CO), which involves the exchange of flanking markers, as well as possible gene conversion (GC) [4,5]. Another result of recombination is GC without exchange of flanking markers (Non-CO, or NCO) [4,5]. Meiosis is also the process that re-distributes the genetic variations in a eukaryotic population. The extent of meiotic recombination directly impacts t
Comparison of Sequencing Platforms for Single Nucleotide Variant Calls in a Human Sample
Aakrosh Ratan, Webb Miller, Joseph Guillory, Jeremy Stinson, Somasekar Seshagiri, Stephan C. Schuster
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055089
Abstract: Next-generation sequencings platforms coupled with advanced bioinformatic tools enable re-sequencing of the human genome at high-speed and large cost savings. We compare sequencing platforms from Roche/454(GS FLX), Illumina/HiSeq (HiSeq 2000), and Life Technologies/SOLiD (SOLiD 3 ECC) for their ability to identify single nucleotide substitutions in whole genome sequences from the same human sample. We report on significant GC-related bias observed in the data sequenced on Illumina and SOLiD platforms. The differences in the variant calls were investigated with regards to coverage, and sequencing error. Some of the variants called by only one or two of the platforms were experimentally tested using mass spectrometry; a method that is independent of DNA sequencing. We establish several causes why variants remained unreported, specific to each platform. We report the indel called using the three sequencing technologies and from the obtained results we conclude that sequencing human genomes with more than a single platform and multiple libraries is beneficial when high level of accuracy is required.
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