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Search Results: 1 - 10 of 90768 matches for " Ronald W. Davis "
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A Genome-Wide Screen for Regulators of TORC1 in Response to Amino Acid Starvation Reveals a Conserved Npr2/3 Complex
Taavi K. Neklesa ,Ronald W. Davis
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000515
Abstract: TORC1 is a central regulator of cell growth in response to amino acid availability, yet little is known about how it is regulated. Here, we performed a reverse genetic screen in yeast for genes necessary to inactivate TORC1. The screen consisted of monitoring the expression of a TORC1 sensitive GFP-based transcriptional reporter in all yeast deletion strains using flow cytometry. We find that in response to amino acid starvation, but not to carbon starvation or rapamycin treatment, cells lacking NPR2 and NPR3 fail to fully (1) activate transcription factors Gln3/Gat1, (2) dephosphorylate TORC1 effector Npr1, and (3) repress ribosomal protein gene expression. Both mutants show proliferation defects only in media containing a low quality nitrogen source, such as proline or ammonia, whereas no defects are evident when cells are grown in the presence of glutamine or peptone mixture. Proliferation defects in npr2Δ and npr3Δ cells can be completely rescued by artificially inhibiting TORC1 by rapamycin, demonstrating that overactive TORC1 in both strains prevents their ability to adapt to an environment containing a low quality nitrogen source. A biochemical purification of each demonstrates that Npr2 and Npr3 form a heterodimer, and this interaction is evolutionarily conserved since the human homologs of NPR2 and NPR3 (NPRL2 and NPRL3, respectively) also co-immunoprecipitate. We conclude that, in yeast, the Npr2/3 complex mediates an amino acid starvation signal to TORC1.
A direct comparison of the KB? Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry
Richard W Hyman, Hui Jiang, Marilyn Fukushima, Ronald W Davis
BMC Research Notes , 2010, DOI: 10.1186/1756-0500-3-257
Abstract: The high quality sequence segment of reads derived from the KB? Basecaller were, on average, 30-to-50 bases longer than reads derived from phred. However, microbe identification appeared to have been unaffected by the change in software.We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB? Basecaller compared to phred. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.DNA sequencing by DNA polymerase chain termination was introduced by Sanger et al. [1] in 1977. In this technology, sequence is determined from the lengths of the terminated DNA chains. Electrophoresis is employed to separate the chains based upon length. A different fluorescent dye is covalently attached to each of the four dideoxy chain terminators. The presence of the dyes significantly affects the electrophoretic mobility of the chains. Therefore, sophisticated software must be employed to deconvolute the fluorescent signals into bases.For some years, the suite of software of choice for DNA sequencing was introduced by Green and associates in 1998: phred for calling the bases in sequence reads, phrap for assembling the reads into contigs, and consed for displaying the contigs for editing [2-4]. Relatively recently, the manufacturer of the sequencing equipment, Applied Biosystems (ABI, Foster City, CA), introduced its own base calling software, the KB? Basecaller, to replace phred http:/ / www3.appliedbiosystems.com/ cms/ groups/ mcb_marketing/ documents/ generaldocuments/ cms_040412.pdf webcite.In our published study [5], we identified the microbes in the healthy adult human vagina by PCR amplifying the 16S ribosomal RNA genes, sequencing the genes with dideoxy chemistry, and identifying the microbes by comparison of the sequence to the data in the Ribosomal Database Project (RDP) [6]. We were concerned that the change in base-calling software would change the microbe
A Rapid, Cost-Effective Method of Assembly and Purification of Synthetic DNA Probes >100 bp
Michael A. Jensen, Lauren Jauregui, Ronald W. Davis
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034373
Abstract: Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.
DMSO and Betaine Greatly Improve Amplification of GC-Rich Constructs in De Novo Synthesis
Michael A. Jensen,Marilyn Fukushima,Ronald W. Davis
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0011024
Abstract: In Synthetic Biology, de novo synthesis of GC-rich constructs poses a major challenge because of secondary structure formation and mispriming. While there are many web-based tools for codon optimizing difficult regions, no method currently exists that allows for potentially phenotypically important sequence conservation. Therefore, to overcome these limitations in researching GC-rich genes and their non-coding elements, we explored the use of DMSO and betaine in two conventional methods of assembly and amplification. For this study, we compared the polymerase (PCA) and ligase-based (LCR) methods for construction of two GC-rich gene fragments implicated in tumorigenesis, IGF2R and BRAF. Though we found no benefit in employing either DMSO or betaine during the assembly steps, both additives greatly improved target product specificity and yield during PCR amplification. Of the methods tested, LCR assembly proved far superior to PCA, generating a much more stable template to amplify from. We further report that DMSO and betaine are highly compatible with all other reaction components of gene synthesis and do not require any additional protocol modifications. Furthermore, we believe either additive will allow for the production of a wide variety of GC-rich gene constructs without the need for expensive and time-consuming sample extraction and purification prior to downstream application.
Pervasive and Persistent Redundancy among Duplicated Genes in Yeast
E. Jedediah Dean ,Jerel C. Davis,Ronald W. Davis,Dmitri A. Petrov
PLOS Genetics , 2008, DOI: 10.1371/journal.pgen.1000113
Abstract: The loss of functional redundancy is the key process in the evolution of duplicated genes. Here we systematically assess the extent of functional redundancy among a large set of duplicated genes in Saccharomyces cerevisiae. We quantify growth rate in rich medium for a large number of S. cerevisiae strains that carry single and double deletions of duplicated and singleton genes. We demonstrate that duplicated genes can maintain substantial redundancy for extensive periods of time following duplication (~100 million years). We find high levels of redundancy among genes duplicated both via the whole genome duplication and via smaller scale duplications. Further, we see no evidence that two duplicated genes together contribute to fitness in rich medium substantially beyond that of their ancestral progenitor gene. We argue that duplicate genes do not often evolve to behave like singleton genes even after very long periods of time.
High-Resolution, In Vivo Magnetic Resonance Imaging of Drosophila at 18.8 Tesla
Brian Null, Corey W. Liu, Maj Hedehus, Steven Conolly, Ronald W. Davis
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002817
Abstract: High resolution MRI of live Drosophila was performed at 18.8 Tesla, with a field of view less than 5 mm, and administration of manganese or gadolinium-based contrast agents. This study demonstrates the feasibility of MR methods for imaging the fruit fly Drosophila with an NMR spectrometer, at a resolution relevant for undertaking future studies of the Drosophila brain and other organs. The fruit fly has long been a principal model organism for elucidating biology and disease, but without capabilities like those of MRI. This feasibility marks progress toward the development of new in vivo research approaches in Drosophila without the requirement for light transparency or destructive assays.
A dynamic network of transcription in LPS-treated human subjects
Junhee Seok, Wenzhong Xiao, Lyle L Moldawer, Ronald W Davis, Markus W Covert
BMC Systems Biology , 2009, DOI: 10.1186/1752-0509-3-78
Abstract: In this study, we analyzed a gene expression data set in blood leukocytes from human subjects administered with lipopolysaccharide (LPS), a prototypical inflammatory challenge, in the context of a reconstructed regulatory network including 10 transcription factors, 99 target genes and 149 regulatory interactions. We found that the computationally estimated activities were well correlated to their coordinated action. Furthermore, we found that clustering the genes in the context of regulatory influences greatly facilitated interpretation of the expression data, as clusters of gene expression corresponded to the activity of specific factors or more interestingly, factor combinations which suggest coordinated regulation of gene expression. The resulting clusters were therefore more biologically meaningful, and also led to identification of additional genes under the same regulation.Using NCA, we were able to build a network that accounted for between 8–11% genes in the known transcriptional response to LPS in humans. The dynamic network illustrated changes of transcription factor activities and gene expressions as well as interactions of signaling proteins, transcription factors and target genes.An achievement that would have a major impact on our understanding of transcriptional regulatory networks would be to map out the coordinated dynamic responses of signaling proteins, transcription factors and target genes over time. The primary challenges to such an effort are development of high-throughput technologies to measure transcription factor activities at the genome-scale, and computational tools to interpret the data and predict the structure and dynamics of the underlying networks.Recent development of high-throughput technologies has enabled large-scale measurements of biological signals related to transcription, such as the expression of target genes and the activities of transcription factors. For target gene expression, microarrays measure the expression levels
Cationic Amphiphilic Drugs Are Potent Inhibitors of Yeast Sporulation
Ulrich Schlecht, Robert P. St. Onge, Thomas Walther, Jean-Marie Fran?ois, Ronald W. Davis
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042853
Abstract: Meiosis is a highly regulated developmental process that occurs in all eukaryotes that engage in sexual reproduction. Previous epidemiological work shows that male and female infertility is rising and environmental factors, including pollutants such as organic solvents, are thought to play a role in this phenomenon. To better understand how organic compounds interfere with meiotic development, the model organism Saccharomyces cerevisiae was exposed to 446 bioactive molecules while undergoing meiotic development, and sporulation efficiency was quantified employing two different high-throughput assays. 12 chemicals were identified that strongly inhibited spore formation but did not interfere with vegetative growth. Many of these chemicals are known to bind to monoamine-receptors in higher eukaryotes and are cationic amphiphilic drugs. A detailed analysis of one of these drugs, tripelennamine, revealed that it induces sporulation-specific cytotoxicity and a strong inhibition of meiotic M phase. The drug, however, only mildly interfered with pre-meiotic DNA synthesis and the early meiotic transcriptional program. Chemical-genomic screening identified genes involved in autophagy as hypersensitive to tripelennamine. In addition, we found that growing and sporulating yeast cells heterozygous for the aminophospholipid translocase, NEO1, are haploinsufficient in the presence of the drug.
Knowledge-Based Reconstruction of mRNA Transcripts with Short Sequencing Reads for Transcriptome Research
Junhee Seok, Weihong Xu, Hui Jiang, Ronald W. Davis, Wenzhong Xiao
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031440
Abstract: While most transcriptome analyses in high-throughput clinical studies focus on gene level expression, the existence of alternative isoforms of gene transcripts is a major source of the diversity in the biological functionalities of the human genome. It is, therefore, essential to annotate isoforms of gene transcripts for genome-wide transcriptome studies. Recently developed mRNA sequencing technology presents an unprecedented opportunity to discover new forms of transcripts, and at the same time brings bioinformatic challenges due to its short read length and incomplete coverage for the transcripts. In this work, we proposed a computational approach to reconstruct new mRNA transcripts from short sequencing reads with reference information of known transcripts in existing databases. The prior knowledge helped to define exon boundaries and fill in the transcript regions not covered by sequencing data. This approach was demonstrated using a deep sequencing data set of human muscle tissue with transcript annotations in RefSeq as prior knowledge. We identified 2,973 junctions, 7,471 exons, and 7,571 transcripts not previously annotated in RefSeq. 73% of these new transcripts found supports from UCSC Known Genes, Ensembl or EST transcript annotations. In addition, the reconstructed transcripts were much longer than those from de novo approaches that assume no prior knowledge. These previously un-annotated transcripts can be integrated with known transcript annotations to improve both the design of microarrays and the follow-up analyses of isoform expression. The overall results demonstrated that incorporating transcript annotations from genomic databases significantly helps the reconstruction of novel transcripts from short sequencing reads for transcriptome research.
Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action
Maureen E Hillenmeyer, Elke Ericson, Ronald W Davis, Corey Nislow, Daphne Koller, Guri Giaever
Genome Biology , 2010, DOI: 10.1186/gb-2010-11-3-r30
Abstract: Yeast competitive fitness data constitute a unique, genome-wide assay of the cellular response to environmental and chemical perturbations [1-8]. Here, we systematically analyzed the largest fitness dataset available, comprising measurements of the growth rates of barcoded, pooled deletion strains in the presence of over 400 unique perturbations [1] and show that the dataset reveals novel aspects of cellular physiology and provides a valuable resource for systems biology. In the haploinsufficiency profiling (HIP) assay consisting of all 6,000 heterozygous deletions (where one copy of each gene is deleted), most strains (97%) grow at the rate of wild type [9] when assayed in parallel. In the presence of a drug, the strain deleted for the drug target is specifically sensitized (as measured by a decrease in growth rate) as a result of a further decrease in 'functional' gene dosage by the drug binding to the target protein. In this way, fitness data allow identification of the potential drug target [3,4,10]. In the homozygous profiling (HOP) assay (applied to non-essential genes), both copies of the gene are deleted in a diploid strain to produce a complete loss-of-function allele. This assay identifies genes required for growth in the presence of compound, often identifying functions that buffer the drug target pathway [5-8].The field of functional genomics aims to predict gene functions using high-throughput datasets that interrogate functional genetic relationships. To address the value of fitness data as a resource for functional genomics, we asked how well co-fitness (correlated growth of gene deletion strains in compounds) predicts gene function compared to other large-scale datasets, including co-expression, protein-protein interactions, and synthetic lethality [11-13]. Interestingly, co-fitness predicts cellular functions not evident in these other datasets. We also investigated the theory that genes are essential because they belong to essential complexes [14,1
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