title: DNA methylation programming and reprogramming in primate embryonic stem cells

status: Public on Sep 10 2009

city: Rehovot

first: Amos

last: Tanay

address: The Weizmann Insititute

department: Computer Science and Applied Math

country: Israel

institute: The Weizmann Institute

summary: DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem (ES) cells, fibroblasts and ES cells generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper- or hypo- methylated in fibroblasts compared to native ES cells and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ES cells, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.

overall_design: DNA methylation profiles in rhesus monkey native ES cells (ORMES-22), fibroblasts, and ES cells generated by somatic cell nuclear transfer (CRES-2), as well as a distinctly different native ES line (the homozygous parthenote ORMES-9) to control for ES line specific effects were identified via hybridization to a custom array including a set of human ESC bivalent domains combined with additional methylation related domains and control regions.

first: Arnar

last: Palsson

first: Shoukhrat

last: Mitalipov

first: Vikas

last: Dighe

first: Netta

last: Mendelson-Cohen


title: Halobacterium salinarum NRC-1 VNG1451C transcription factor ChIP-chip with and without carbon sources

status: Public on Sep 08 2009

city: Seattle

first: Amy

middle: K

last: Schmid

state: WA

country: USA

institute: Institute for Systemsbiology

type: Genome binding/occupancy profiling by array

summary: Gene regulatory networks play an important role in coordinating biochemical fluxes through diverse metabolic pathways.  The modulation of enzyme levels enables efficient utilization of limited resources as organisms dynamically acclimate to nutritional fluctuations in their environment.  Here we have identified and characterized a novel nutrient-responsive transcription factor from the halophilic archaea, AgmR. Like TrmB, its thermophilic archaeal homolog, AgmR regulates glycolytic and gluconeogenic pathways in response to sugar availability. However, using high throughput genome-scale experiments, we find that AgmR directly governs the transcription of nearly 100 additional genes encoding enzymes in diverse metabolic pathways. Genome-scale in vivo binding site location data reveals that >60% of these are direct targets. Integration of these systems-scale datasets with metabolic reconstruction models suggests that AgmR, a sequence-specific bacterial-like regulator, interacts with the general transcription factor machinery to coordinate nitrogen and carbon metabolism with the de novo synthesis of cognate cofactors and reducing equivalents, achieving system-wide redox and energy balance.

overall_design: Halobacterium salinarum NRC-1 (ATCC700922) ura3 parent and VNG1451C strains were grown in rich medium (CM, 250  NaCl, 20 g/L MgSO4?7H2O, 3 g/L sodium citrate, 2 g/L KCl, 10 g/L peptone) with or without varying concentrations of glucose or glycerol at 37ºC under full-spectrum white light. The cells were cross-linked with 1.2% formaldehyde, then lysed and DNA sheared via sonication. Next cleared cell lysate was subject to immunoprecipitation against an anti-myc antibody. Protein-DNA complexes were purified and DNA was isolated after protease and RNAase treatment. DNA was amplified using universal linkers, labeled directly using Kreatech 547 and 647 dyes. Samples were grown in biological duplicate. Each DNA sample was hybridized against 2 microarrays as dyefilps. At least two experiments (including biological duplicates) were carried out.

first: Schmid

last: Amy

first: Koide

last: Tie

first: Pan

last: Min

first: Baliga

last: Nitin S.


title: The effect of hfq on global gene expression and virulence in Neisseria gonorrhoeae

status: Public on Sep 15 2009

city: Berlin

first: Hans-Joachim

last: Mollenkopf

address: Charitéplatz 1

country: Germany

institute: Max-Planck-Institute for Infection Biology

summary: Hfq is an RNA chaperone, which functions as a pleiotropic regulator for RNA metabolism in bacteria. To characterize the role of Hfq in pathogenicity of Neisseria gonorrhoeae we generated a N. gonorrhoeae hfq mutant, MS11hfq.Transcriptional analysis using a custom-made N. gonorrhoeae microarray revealed that 369 open reading frames were differentially regulated in MS11hfq compared to the wild-type (wt) strain (202 were upregulated, 167 were downregulated).

overall_design:
Arrays were generated as custom 8x15 K microarrays (Agilent Technologies) covering all open reading frames (ORFs) from N. gonorrhoeae strain FA1090 (accession number AE004969) supplemented with all ORFs from the gonoccoccal genetic island of strain MS11 (accession number AY803022) by 6 specific 60-mer oligonucleotides per gene on average.
Microarray analysis was performed as dual-color hybridization with dye-reversal color-swaps as technical replicates for Cy-dye specific effect compensation and three independent biological replicates.
Microarray features were extracted with the FE 9.5.3.1 software from Agilent Technologies using the GE2-v4_95_Feb07 protocol with default settings.
Data were loaded into Resolver (Rosetta Biosoftware) using the MAGE-ML loader and analyzed for differential gene expression. Ratio profiles were combined into ratio experiments and regulated genes were identified with a threshold of 1.75 fold-change and anti-correlation of the color-swapped dye reversals, rendering the analysis highly stringent and robust with P values <0.0001 of the results.

first: Alexandra

last: Friedrich


title: Response of Rhodopirellula baltica to salt shift

status: Public on Sep 09 2009

city: Bremen

first: Patricia

last: Wecker

address: Celsiusstr, 1

department: Molecular Ecology

institute: Max Planck Institut for Marine Microbiology

summary: Expression profiling of the stress response of the marine Planctomycete Rhodopirellula baltica after a salinity up-shift. R. baltica cultures were grown at 28°C in mineral media with 17.5% salinity and up-shifted to 59.9%. Samples were taken after 10, 20, 40, 60 and 300min and compared with cultures before the up-shift.

overall_design:
Reference design, time series experiment
Whole genome array
5 time points, 3 replicates per time point.
In total, 18 samples.

first: Christine

last: Klockow

first: Andreas

last: Ellrott

first: Frank

middle: O

last: Glöckner


title: The type of phagocytic receptor modulates gene expression and phagosome functions in macrophages

city: Rostock

first: Eik

last: Hoffmann

address: Albert-Einstein-Str. 3

department: Institute of Biological Sciences

institute: University of Rostock

summary: The receptors engaged during phagocytic particle uptake determine the signaling events that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here, we used latex beads coupled to single ligands, focusing on IgG, mannan, LPS and avidin, and monitored phagocytic uptake rates, phago-lysosomal fusion events, macrophage gene expression and the proteomic composition of isolated phagosomes. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand altered a distinct pattern of genes and led to a different composition of phagosomes. These data argue that activation of each receptor initiates a specific signature of signaling events that last many hours and influences several phagocytosis functions.

supplemental_files: ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE17nnn/GSE17761/suppl/GSE17761_Hoffmann et al_Microarray data.xls.gz

overall_design:
All samples and controls were carried out in triplicates. J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well plates one day before the experiment. Subsequently, cells were incubated with serum-free DME medium containing 0.01 % latex beads of 1 µm diameter coupled to the following ligands: avidin (Av), the Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation, samples as well as untreated controls were washed twice in PBS and total RNA was extracted using RNeasy kit (Qiagen) following manufacturer's instructions. Each sample was hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages.

CodeLink EXP v4.0-processed data are represented in the Sample tables, GeneSpring-processed data are linked as a supplementary files to the matrix table, additional results available as a supplementary file on the Series record.

http://www.biologie.uni-rostock.de/tierphysiologie/agkuznetsov.html

first: Bibhuti

middle: B

last: Mishra

first: Gareth

last: Griffiths

first: Sergei

last: Kuznetsov


title: Microarray analysis of pig innate immune responses after experimental infection with Actinobacillus pleuropneumoniae

city: Copenhagen

first: Kerstin

last: Skovgaard

address: Bülowsvej 27

department: Immunology and Parasitology

country: Denmark

institute: National Veterinary Institute

summary:
To study hepatic gene expression differences, liver samples from infected pigs (n = 10) were compared with liver samples from the non-infected control group (n = 5). Ten microarrays were performed, such that 5 randomly selected cDNA samples from the infected group were labeled with Oyster 550 (Genisphere Inc., Hatfield, PA, USA) and compared directly to 5 control animals labeled with Oyster 650 (Genisphere) analysing a pair of samples (one from an infected animal and one from a randomly chosen non-infected animal) on each microarray slide. The remaining five cDNA preparations from the infected group were labeled with Oyster 650 and compared in the same way to the five controls labeled with Oyster 550 on the remaining 5 microarray slides. This dye-swap design was applied to reduce variation due to dye effects and to provide as much biological replication as possible.
Keywords: Disease state analysis

overall_design: Two-condition experiment, Disease vs. Control, Biological replicates: 10 disease replicates and 5 control replicates, 10 Microarrays comparing one disease replicate with one control replicate.

first: Shila

last: Mortensen

first: Peter

middle: M

last: Heegaard


title: Genome-wide analysis of toc1-2 transcriptional networks

city: Barcelona

first: Paloma

last: Mas

address: C/ Jordi Girona 18-26

department: Genetica Molecular

country: Spain

institute: Consorcio CSIC-IRTA-UAB (CRAG)

summary: To gain insights into the mechanisms of TOC1 function in the Arabidopsis circadian clock we performed transcriptional profiling of Wild-Type (WT) and and TOC1 mutant  plants (toc1-2) under constant light conditions for two days.

overall_design: Comparisons of WT and toc1-2. Two biological replicates each per array. Two Arabidopsis Oligonucleotide Microarrays (two-color Cy3 and Cy5). synchronized under 12-hour light:12-hour dark (LD) cycles for 10 days followed by two days under constant light conditions. Samples were collected at circadian time 16 (CT16).

first: Tommaso

last: Legnaioli

first: Juan

middle: C

last: Cuevas


title: Effect of Nrg1 on pathogenesis of Cryptococcus neoformans

status: Public on Sep 04 2009

city: Lynchburg

first: Michael

middle: S

last: Price

state: VA

address: 1971 University Blvd.

department: Biology & Chemistry

institute: Liberty University

summary: The Cryptococcus neoformans NRG1 gene was identified using gene microarrays to define putative transcription factor genes regulated by the cyclic AMP (cAMP) signal transduction pathway. Disruption of NRG1 results in delayed capsule formation and mating, two phenotypes that are directly controlled by cAMP signaling. Putative targets of the Nrg1 transcription factor were identified using a second genome microarray to define differences in the transcriptomes of the wild-type and nrg1 mutant strains. These experiments implicate Nrg1 in the transcriptional control of multiple genes involved in carbohydrate metabolism and substrate oxidation, as well as the UGD1 gene encoding a UDP-glucose dehydrogenase required for polysaccharide capsule production and cell wall integrity. In addition to being under transcriptional control of the cAMP pathway, Nrg1 contains a putative protein kinase A phosphorylation site; mutation of this motif results in reduced Nrg1 activity. Consistent with prior studies in hypocapsular mutants, the nrg1 mutant strain is attenuated in an animal model of disseminated cryptococcal disease.

overall_design: WT and nrg1 mutant RNA were each compared to a Reference Pool RNA sample in a modified Reference design.  Three hybridizations were performed for the WT and nrg1 mutant samples, and the dyes were swapped for the third hybridization of both WT and nrg1 mutant.

first: Kari

middle: L

last: Cramer

first: Quincy

middle: D

last: Gerrald

first: Connie

last: Nichols

first: James

middle: A

last: Alspaugh


title: Distribution of macroH2A1 and macroH2A2 at gene promoters in NT2/D1 cells

status: Public on Sep 06 2009

city: Badalona

first: Lauro

last: Sumoy

state: Barcelona

address: Ctra. Can Ruti, Camí de les escoles s/n

department: High Content Genomics and Bioinformatics

institute: IGTP

type: Genome binding/occupancy profiling by genome tiling array

summary:
Chip-on-chip experiment with NT2 wildtype cells and cells treated with shRNA for macroH2A1 and macroH2A2
The goal was to determine the genome-wide occupancy of macroH2A1 and macroH2A2 at gene promoters in undifferentiated cells

overall_design: Biological replicates: Chromatin was prepared from exponentially growing wild-type NT2 cells and cells stably exressing shRNA targetting macroH2A1 and macroH2A2 mRNA. Triplicate of IPs for macroH2A2 and two duplicates with different macroH2A1 antibodies were performed. A triplicate of control IPs was performed with IgGs on chromatin of untreated cells and macroH2A1 specific antibody on shRNA-treated cells.

first: Luciano

last: di Croce

first: Pau

last: Rue

first: Marcus

last: Buschbeck


title: Gene content differences across strains of Streptococcus uberis identified using oligonucleotide microarray CGH

status: Public on Sep 13 2009

city: Ithaca

middle: J

last: Stanhope

state: NY

address: Schurman Hall

department: Population Medicine and Diagnostic Sciences

institute: Cornell University

summary:
Streptococcus uberis is one of the principal causative agents of bovine mastitis. The organism is typically considered an environmental pathogen, however, recent studies also suggest possible host adaptation. In this study, two multilocus sequence typing (MLST) schemes and whole genome DNA microarrays were used to evaluate the degree and nature of genome flexibility between S. uberis strains. The 21 isolates examined in this study arise from a collection of 232 international isolates for which previous epidemiological and preliminary genotyping data existed. The microarray analysis resulted in an estimate of the core genome for S. uberis, consisting of 1530 ORFs, among 1855 tested, representing 82.5% of the S. uberis 0140J genome.  The remaining ORFs were variable in gene content across the 21 tested strains.  A total of 26 regions of difference (RDs), consisting of three or more contiguous ORFs, were identified among the variable genes.  Core genes mainly encoded housekeeping functions, while the variable genes primarily fell within categories such as protection responses, degradation of small molecules, laterally acquired elements, and two component systems.  Recombination detection procedures involving the MLST loci suggested S. uberis is a highly recombinant species, precluding accurate phylogenetic reconstructions involving these data. On the other hand, the microarray data did provide limited support for an association of gene content with strains found in multiple cows and/or multiple herds, suggesting the possibility of genes related to bovine transmissibility or host-adaptation.

Keywords: comparative genomic hybridization

sample_organism:
Streptococcus uberis
Streptococcus equi subsp. zooepidemicus
Streptococcus equi subsp. equi

overall_design:
Combimatrix CustomArray™ 4X2K was used in this study (design1_#4231).  This array is divided into 4 sectors, each of which contains 2,240 in situ synthesized oligonucleotide probes (spots) in 56 columns and 40 rows with the same probe design and layout.   Based on the sequence of S. uberis strain 0140J, oligonucleotide probes were designed to have a similar annealing temperature of 56ºC and a length 35-40 bp.  The first design included 100 negative controls probes as well as 1855 of the 1870 ORFs described in the un-annotated sequence of S. uberis strain 0140J.  Replicate microarrays were hybridized for every 21 strains tested in this study.

Combimatrix CustomArray™ 4X2K was used in this study (design2_#4887).  This array is divided into 4 arrays (sectors), each of which contains 2K in situ synthesized oligonucleotide probes (spots) in 56 columns and 40 rows with the same probe design and layout.  This second design of S. uberis was based on genes that were not clearly present (loci with low intensity or no hybridization for at least one strain) for all 21 strains in the hybridization results involving the first design. Based on the sequence of S. uberis strain 0140J, oligonucleotide probes were designed to have a similar annealing temperature of 56ºC and a length 35-40 bp. The design included four or five probes, separated from one another in order to span the entire gene, for 434 of 451 ambiguous loci, synthesized in situ to occupy the 2,240 independent microarray spots.  Replicate microarrays were hybridized for every 21 strains tested in this study.

first: Ping

last: Lang

first: Wei

last: Wang


title: Functional Genomics Reveals Extended Roles of the Mycobacterium tuberculosis Stress Response Factor sigmaH

city: San Antonio

first: Deepak

last: Kaushal

state: TX

address: 8715 W Military Dr

department: HPI

institute: Texas Biomedical Research Institute

summary:
DNA Microarray based comparison of M. tuberculosis and its isogenic delta-sigmaH mutant for an extended period of time, using a diamide-stress and removal strategy (post-diamide stress or PDS)

Raw data has been requested of, but not provided by, the submitter.

overall_design: Two strains, M. tuberculosis vs delta-sigmaH mutant. At Pre-diamide (control), 0' 30' 60' 90' and 120' post-diamide stress, two biological replicates, six replicates per array

first: Smriti

last: Mehra


title: Response of Rhodopirellula baltica to heat shock

first: Patricia

last: Wecker

summary:
Expression profiling of the stress response of the marine Planctomycete Rhodopirellula baltica after a temperature up-shift. R. baltica cultures were grown at 28°C and then shifted to 37°C. Samples were taken after 10, 20, 40, 60 and 300min and compared with cultures before the up-shift (28°C).

Time series experiment, whole genome array

overall_design:
5 time points, 3 replicates per time point.
Each time point sample was compared with the reference.
3 self-self reference hybridizations.
In total, 18 samples.

first: Andreas

last: Ellrott

first: Christine

last: Klockow

first: Frank

last: Glöckner


title: Mammary transcriptome analysis of lactating dairy cows

city: Puddle Alley

first: Alan

middle: Francis

last: McCulloch

state: Mosgiel

address: Invermay

country: New Zealand

institute: AgResearch NZ

summary:
Growth hormone (GH) has a well established galactopoietic effect
on ruminant lactation, however the molecular mechanisms that mediate these effects are not fully understood. This study aimed to define
the effects of GH on the production of milk protein by the bovine
 mammary gland and to establish the potential molecular
mechanisms mediating these effects. Elevated levels of GH
increased milk yield with corresponding increases in protein,
fat and lactose yield, while milk composition remained unaffected
by treatment. Increased yield (but not concentration) of all
the individual milk proteins was not accompanied by changes in
the levels of the milk protein transcripts, with the exception of
S2-casein, suggesting post-transcriptional regulation of the
milk protein genes is a main factor in the GH-mediated effects on
bovine lactation. There were significant transcriptional changes in
a wide range of pathways including cell signaling, cell death, cell
growth and proliferation, cell cycle regulation and metabolism,
consistent with the broad effects of GH. Differential expression of
genes involved in the PI3K, ERK/MAPK, insulin receptor, JAK/STAT
and IGF-1 signaling pathways were also evident in response to
GH treatment supporting potential cross-talk between the GH and
insulin signaling pathways in the bovine mammary gland. Transcript
 levels of the SHC1 gene were elevated in GH-treated cows.
SHC1 activates many pathways including those associated with
translational regulation and gene transcription and has been implicated
in mediating cross-talk between the insulin and GH signaling
pathways, providing some insights into the potential pathways
mediating the effects of GH on milk protein synthesis in the bovine
mammary gland.

overall_design:
Eight non-pregnant second lactation spring-calved Jersey cows between 178 to 200 days postpartum, were sourced from a commercial herd. Cows were weighed twice weekly for one month prior to treatment, and randomly assigned to two groups of four. On day 0, a slow-release formula of commercially available GH (Lactatropin®, Elanco Animal Health, Bryanston, South Africa) was administered via a single subcutaneous injection to 4 lactating Jersey cows. A further 4 Jersey cows were given a single subcutaneous injection of saline as controls.
18 slides were used in a loop design which requires three aliquots of each test sample labeled alternately with the Cy5 and Cy3 dyes and co-hybridized on arrays with two other test samples.

first: Susan

last: McCoard

first: Amanda

last: Hayashi


title: Functional profiling in yeast with arsenite and monomethylarsonous acid

status: Public on Sep 03 2009

city: Berkeley

first: William

last: Jo

state: CA

address: 119 Morgan Hall

institute: UC Berkeley

summary:
Arsenic is a human toxin and carcinogen commonly found as a contaminant in drinking water. Arsenite (AsIII) is the most toxic inorganic form, but recent evidence indicates that the metabolite monomethylarsonous acid (MMAIII) is even more toxic. We have used a chemical genomics approach to identify the genes that modulate the cellular toxicity of MMAIII and AsIII in the yeast Saccharomyces cerevisiae. Functional profiles provided evidence of the requirement of highly-conserved biological processes in the response against both arsenicals including tubulin folding, DNA double-strand break repair and chromatin modification.
At the equitoxic doses of 150 µM MMAIII and 300 µM AsIII, genes related to glutathione were essential only for resistance to the former, suggesting a higher potency of MMAIII to disrupt glutathione metabolism than AsIII. Treatments with MMAIII induced an increase in glutathione levels, which correlated to the requirement of genes from the sulfur and methionine metabolic pathways. Many of the identified yeast genes have orthologs in humans that could potentially modulate arsenic toxicity in a similar manner as their yeast counterparts.

overall_design:
Pools of homozygous diploid deletion mutants (n = 4,607 strains) were grown in rich media (YPD) at 3 concentrations of arsenite (As3) and monomethylarsonous acid (MMA3) for 5 and 15 generations (5g, 15g).
Each strain has a deletion in a different gene. In each strain, the gene was replaced by a deletion cassette containing a antibiotic resistance gene and two BARCODES, up and down tags (Please see Giaever et al, 2002, Nature).  These tags in the DNA are specific to each strain. We pool all deletion strains and grow them under a selective condition; extract DNA; amplify barcodes using universal primers and hybridize to arrays containing complemetary sequences to the up and down tags. In this way, we can look at growth of each of the strains. Our  strategy is to analyze separately the up and down tags. Therefore, for each array (CEL file), we generate two data files, one for all ups and another one for all downs. In these files, the list of genes are the same but the data come from different set of probes, up or down. The values are log2 averages of replicate probes for the same tag. These pre processed files were used to identify strains with differential growth in arsenic by comparing to the control arrays.

first: Alex

last: Loguinov

first: Chris

last: Vulpe


title: Co-amplification at 8p and 11q in breast tumors and cell lines with 8p11q array Genomic Comparative Hybridization

status: Public on Sep 02 2009

city: San Francsico

first: Serena

last: kwek

address: 2340 Sutter St.

department: Cancer Research Institute

institute: UCSF

type: Genome variation profiling by genome tiling array

summary: Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis.  We used high resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions.  The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site.  Candidate genes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. Funcational analysis for transformation was further carried out with candidate genes to determine candidate oncogenes.

overall_design: Near tiling path coverage of 8p11q array CGH experiments with breast cell lines and ductal invasive and lymph node-negative breast tumors.

first: S

last: Kwek

first: D

middle: G

last: Albertson

first: J

last: Climent

last: Martinez-Climent


title: Analysis of five species of Gossypium allotetraploids and a synthetic F1 hybrid compared to model diploid projenitors

city: Ames

first: Lex

last: Flagel

state: IA

address: 251 Bessey Hall

institute: Iowa State Univ.

summary: We compare gene expression among petals tissues in 5 species of natural allotetraploid and F1 hybrid cottons to  the antecedent conditions existing prior to genome merger and duplication, thus revealing the effects of genome merger and polyploidy on gene expression evolution

sample_organism:
Gossypium
Gossypium barbadense
Gossypium hirsutum
Gossypium arboreum
Gossypium raimondii
Gossypium mustelinum
Gossypium darwinii
Gossypium tomentosum

overall_design: 27 total samples, including 3 reps. each of five natural tetraploid species, a F1 hybrid, two parental species, and a 1:1 RNA mix of the parental species

middle: E

first: Jonathan

middle: F

last: Wendel


city: Durham

first: Jennifer

middle: Lynn

last: McCulley

state: NC

address: 374 CARL Building, Research Drive Box 3054

department: Molecular Genetics and Microbiology

institute: Duke University

summary:
In Saccharomyces cerevisiae, Elevated Levels of Aneuploidy and Chromosome Rearrangements are Separable Genome Instability Events Controlled by the Tel1 and Mec1 Kinases
Cancer cells often have elevated frequencies of chromosomal aberrations, and it is likely that loss of genome stability is one driving force behind tumorigenesis. Deficiencies in DNA replication, DNA repair, or cell cycle checkpoints can all contribute to increased rates of chromosomal duplications, deletions and translocations. The Saccharomyces cerevisiae proteins Tel1 and Mec1 (homologues of the human ATM and ATR proteins, respectively) are known to participate in the DNA damage response, replication checkpoint, and telomere maintenance pathways and are critical to maintain genome stability.  In the absence of induced DNA damage, tel1 mec1 diploid yeast strains exhibit extremely high rates of chromosome aneuploidy. There is a significant bias towards trisomy of chromosomes II, VIII, X, and XII, whereas the smallest chromosomes I and VI are commonly monosomic. tel1 mec1 strains also demonstrate elevated levels of chromosome rearrangements, including translocations as well as interstitial duplications and deletions. Restoring wild-type telomere length with the Cdc13-Est2 fusion protein substantially reduces the amount of chromosome rearrangements in tel1 mec1 strains. This result suggests that most of the rearrangements are initiated by telomere-telomere fusions. However, the telomere defects associated with tel1 mec1 strains do not cause the high rate of aneuploidy, as restoring proper telomere function does not prevent cells from becoming aneuploid. Our data demonstrate that the same mutant genotype can produce both high levels of chromosome rearrangements and high levels of aneuploidy, and these two types of events occur through separate mechanisms.

overall_design: The strains in this series consist of 23 diploid yeast strains homozygous null for TEL1, MEC1, and SML1 as well as four wild-type controls. The experimental strains were subcultured for approximately 100 generations before introducing a wild-type copy of MEC1 on a plasmid to capture the current complement of chromosomal aberrations and limit further genome instability during the course of analysis.  Genomic DNA from each experimental strains was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of alterations to chromosome number and chromosome structure in diploid strains lacking TEL1, MEC1, and SML1 following subculturing.

first: Thomas

last: Petes


title: Expression data comparing human foreskin keratinocytes to epithelial derivatives of human embryonic stem cells (H9)

status: Public on Sep 01 2009

city: Cambridge

first: Christian

last: Metallo

state: MA

address: 77 Mass. Ave.

department: Chemical Engineering

institute: Massachusetts Institute of Technology

summary:
Whole genome expression array comparison of primary keratinocytes to retinoic acid-differentiated hES cell derivatives
Cells were differentiated according to Metallo et al. Stem Cells 2008

overall_design: A 3 chip study was conducted using competitive hybridization comparing pFKs to hEKs obtained in 3 independent differentiation experiments

first: Sean

middle: P

last: Palecek


title: DnaN causes overinitiation of DNA replication

middle: D.

last: Grossman

email: clee2@mit.edu, adg@mit.edu

address: 31 Ames Street, 68-530

department: Biology

institute: MIT

summary: Overexpression of DnaN causes the accumulation of origin of replication proximal DNA.

overall_design: comparing the genome profile of cells before and after DnaN overexpression we see an increase in the amount of origin specific DNA, which is typical of replication overinitiation

first: Alexi

last: Goranov


title: DnaN overexpression does not affect replication in oriN cells

first: Alan

summary: overexpressing DnaN did not affect the genomic DNA pattern in a strain that replicated form the plasmid origin of replication oriN and is deleted for the endogenous origin of replication oriC

overall_design: Comparison of DNA profiles of strains before and after the overexpression of dnaN


title: Effects on expression profile of YabA-null cells after replication arrest

summary: To compare the transcriptional effects of replication arrest that are dependent on YabA we compared the transcriptional profiles of yabA null and yabA+ cells after treatment with the replication inhibitor HPUra. To avoid secondary effects of replication overinitiation that the yabA-null strain exhibits, we did all experiments in oriC-null cells

overall_design: We compare triplicates of treated (HPUra) and untreated samples for each yabA-null (AIG185) and YabA+ (MMB170) strains.

first: Adam

last: Breier


title: Response of Rhodopirellula baltica to cold shock

first: Patricia

last: Wecker

summary: Expression profiling of the stress response of the marine Planctomycete Rhodopirellula baltica after a temperature up-shift. R. baltica cultures were grown at 28°C and then shifted to 6°C. Samples were taken after 10, 20, 40, 60 and 300min and compared with cultures before the up-shift (28°C).

overall_design:
Reference design, time series experiment
Whole Genome Array
5 time points, 3 replicates per time point.
3 self-self reference hybridizations.
In total, 18 samples.

first: Christine

last: Klockow

first: Andreas

last: Ellrott

first: Frank

last: Glöckner


title: FRIK966 and FRIK2000 comparison to pooled EDL933 reference

city: Lubbock

first: Scot

last: Dowd

address: 4321 Marsha Sharp Fwy

department: MBRI

institute: MBRI

summary: The existence of two separate lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that  one of these lineages (lineage I) might be more pathogenic towards human hosts. We have previously shown that the more pathogenic lineage expresses higher levels of Shiga toxin 2 (Stx2) than the non-pathogenic lineage II.  To evaluate why lineage 2 isolates do not express appreciable levels of toxin, two lineage 2 isolates (FRIK966 and FRIK2000) were chosen as representatives of lineage 2 and whole genome microarrays were performed using Agilent microarrays using the E. coli O157:H7 EDL933 lineage I clinical type isolate as a reference.  Microarray results were utilized to evaluate what genes and pathways might be missing or differentially expressed.  Quantitative RT-PCR was utilized to validate the microarray data.

overall_design:
Based upon the transcriptome of Escherichia coli O157:H7 EDL933 an oligonucleotide microarray, made up of 60 mers was designed.  A total of 4873 genes in an 8 x 15K Agilent microarray design.  Designed using a custom script, specifications for gene specific oligos were based upon various design characteristics such as temperature of melting, 3’ location, specificity, lack of repeat nucleotides, etc. (Charbonnier et al., 2005).  Arrays were manufactured using Agilent Sure-print technology.  Each array consisted of duplicate elements for each gene randomly distributed with Agilent control elements included.
All procedures were performed according to respective manufacturer protocols.  Lineage I and lineage II strains were grown overnight as described, a total of 10e7 cells were washed twice in fresh media, normalized based upon optical density,  inoculated into fresh media, incubated at 37oC shaking 120 x g for 3 hours and then suspended in RNAprotect bacteria reagent (Qiagen Inc., Valencia, CA.).  Total RNA was extracted using RNeasy Bacteria Mini Kit (Qiagen Inc., Valencia, CA.) and trace amounts of DNA were removed using RNase-Free DNase Set (Qiagen Inc., Valencia, CA.).  RNA was quantified using Nanodrop system (NanoDrop Technologies, Wilmington, DE) and quality confirmed by electrophoresis on a Bio-rad Experion system (BioRad XXX).  For each sample, 10 ug of RNA were labeled with either CyDye3-dCTP or CyDye5-dCTP (Perkin Elmer) using the LabelStar kit (Qiagen Inc., Valencia, CA.) and Random nonamers (Integrated DNA Technologies ).  Labeled cDNA were hybridized to the microarray using Agilent Hi-RPM hybridization solution in an Agilent Hybridization chamber (Agilent.). A total of 8 arrays, each with duplicate elements for each gene, alternating dye swap for each replicate (4 biological replicates), were analyzed to obtain genes that were consistently and differentially regulated in comparison to EDL933, while limiting false discover rate (FDR) below a stringent 5% (Benjamini and Hochberg, 1995).


title: Yeast W303 SSD1-V cell cycle

city: Seattle

first: lihong

last: li

address: 1100 Fairview Ave N.

department: Basic Science

institute: Fred Hutchinson Cancer Research Center

summary: SSD1 is a polymorphic locus in budding yeast with many pleiotropic effects. Our lab had previously done transcript microarray of W303a in an ssd1-d background, and here we have carried out another transcript microarray across the cell cycle in an isogenic SSD1-V background.  We find that a large fraction of budding yeast transcripts is differentially expressed in these cells.  Ssd1 has recently been shown to bind mRNAs in vivo, but very few of these mRNAs show significant changes in levels in the SSD1-V versus ssd1-d comparison.  About 20% of cell cycle-regulated transcripts are affected by SSD1-V and most of these transcripts show sharper amplitudes of oscillation in SSD1-V cells. Many transcripts whose gene products influence longevity are also affected, the largest class of which are involved in translation.  Ribosomal protein mRNAs are globally down-regulated by SSD1-V.

overall_design: Cells were synchronized with alpha factor and sampled every 10 min across 2 cell cycles. A total of 13 samples were analyzed. No replicates were included. cDNA of the cell cycle samples were labelled with Cy5. For Cy3 labelling, asynchronous yeast population was used.

first: Lihong

last: Li

first: Linda

last: Breeden


title: Expression data of FB2 vs. FB2khd4Delta

city: Marburg

first: Michael

last: Feldbrügge

state: Hessen

address: Karl-von-Frisch-Strasse

department: Organismic Interactions

institute: MPI for Terrestrial Microbiology

summary:
RNA-binding proteins constitute key factors of the posttranscriptional machinery. These regulatory proteins recognise specific elements within target transcripts to promote e.g. maturation, translation or stability of mRNAs. In Ustilago maydis, evidence is accumulating that posttranscriptional processes are important to determine pathogenicity. Deletion of khd4, encoding a predicted RNA-binding protein with five K homology (KH) domains, causes aberrant cell morphology and reduced virulence. Here, we demonstrate that Khd4 recognises the sequence AUACCC in vivo via its tandem KH domains 3 and 4. This sequence functions most likely as regulatory RNA element in U. maydis, since it accumulates in 3’ untranslated regions. Consistently, an independent transcriptional profiling approach revealed that the binding motif is significantly enriched in transcripts differentially regulated in khd4   strains. Since the vast majority of potential Khd4 target mRNAs exhibit increased expression in deletion mutants, Khd4 might promote mRNA instability. Mutants that fail to bind AUACCC resemble deletion mutants exhibiting altered cell morphology, disturbed filamentous growth and severely reduced virulence. Hence, RNA binding is essential for function of Khd4 stressing the importance of posttranscriptional control in regulating morphology and pathogenicity.
keywords: gene deletion, RNA-binding protein, KH domain

overall_design: Strains FB2 (2 replicates, control) and FB2khd4Delta (2 individuals) were grown in minimal medium containing glucose and glutamine.

first: Evelyn

last: Vollmeister


title: Hypoxia effect on embryonic hearts of Tibetan Chicken, Dwarf Recessive White Chicken and Shouguang Chicken

status: Public on Sep 05 2009

city: Beijing

first: mei

address: Haidian district

country: China

institute: China Agricultural University

summary:
Adaptation to hypoxia is a complicated and important physiological course for organisms, but the genetic mechanism underlying the adaptation is not fully understood yet. Tibetan Chicken (T), an indigenous chicken breed in China which inhabit in high areas with an altitude above 2,900 meters. Shouguang Chicken(S) and Dwarf Recessive White Chicken (DRW), two lowland chicken breeds, were used as control groups. The heart was the first functional organ to develop during the embryonic development. Furthermore, the heart is an efficient energy converter utilizing the most appropriate fuel for a given environment. Therefore, GeneChip® Chicken Genome Array was employed to identify the differentially expressed genes in embryonic hearts of Tibetan Chicken and two lowland chicken breeds in both hypoxic and normoxic incubating environments with a genome wide profile.

Keywords: stress response

overall_design: To obtain general expression profiles of embryonic hearts in Tibetan Chicken(T), Dwarf Recessive White Chicken (DRW)and Shouguang Chicken (S)in hypoxia and normoxia, the fertilized full sib eggs of all the three chicken breeds were incubated under two different conditions. The heart was isolated from all the three chicken breeds under the two different conditions for RNA extraction and hybridization on Affymetrix microarrays.

first: C

middle: H

last: Wu

last: Zhao

first: M


title: Sugar sweet springtails: On the transcriptional response of Folsomia candida (Collembola) towards desiccation stress

city: Amsterdam

first: Martijn

middle: Johannes

last: Timmermans

address: De Boelelaan 1085

department: Animal Ecology

country: Netherlands

institute: Vrije Universiteit Amsterdam

summary: Folsomia candida (Collembola) is able to survive dryer conditions by absorbing water vapour from its surroundings. To unravel the genomic responses underlying this intriguing water-absorption mechanism, we exposed the species to 98.2% Relative Humidity (eight, 27, 53 and 174 hours respectively) and subjected it to microarray based transcription profiling.

overall_design:
About twenty-five 25-27 day old animals were placed in plastic containers that had a Relative Humidity of 98.2% (desiccated groups; n=16) or 100% RH (reference groups; n=16). After 8, 27, 53 and 174 hours four reference and four desiccation treated groups were snap-frozen (in liquid nitrogen) and stored at –80 °C until further analysis.

Total RNA was obtained for each of the 32 samples. 500ng total RNA was labelled with the Agilent Low-Input Fluorescent Linear Amplification Kit.

A pair-wise reference design was applied, implying that samples originating from desiccated groups were hybridized onto arrays with samples from reference groups that were exposed for the same duration; 8, 27, 53 or 174 hours). Dyes were swapped over the biological replicates.

first: Dick

last: Roelofs

first: Benjamin

last: Nota

first: Bauke

last: Ylstra

first: Martin

last: Holmstrup


title: Deletion mutants mth1, std1, and mth1+std1 v. wild type yeast grown in galactose

city: Saint Louis

first: Jeffrey

last: Sabina

address: 4444 Forest Park Blvd.

department: Genetics

institute: Washington University in Saint Louis

summary: Transcriptional profiles of S. cerevisiae strains with deletions of mth1, std1, or both were compared to that of a wild-type strain during growth on synthetic complete media with galactose as the carbon source, with the intent to understand each protein's contribution to the cells' response to glucose.

overall_design: Single-condition experiment, mth1 delete, std1 delete or the double mth1/std1 deletion strain vs. a wild type yeast strain. Biological replicates: 2. Rep. 1: Independently isolated RNA preps for each mutant was compared against a single WT sample. Rep. 2: A second set of mutant and WT samples was used to repeat the profiling. Two replicate spots representing each gene are present per array.


title: Plasmodium falciparum Heterochromatin Protein 1 Marks Genomic Loci Linked to Phenotypic Variation of Exported Virulence Factors

city: Nijmegen

first: Richard

last: Bartfai

address: Geert Grooteplein 26-28

department: Molecular Biology

institute: Nijmegen Center of Molecular Life Sciences

summary: Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. PfEMP1 also mediates sequestration of infected erythrocytes in the microvasculature, which is directly linked to severe malaria outcomes. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that the P. falciparum ortholog of heterochromatin protein 1 (PfHP1) binds to H3K9me3 and constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with non-syntenic virulence gene arrays in subtelomeric and chromosome-internal islands. These include not only var genes but the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host-parasite interactions. Over-expression of PfHP1 resulted in decreased  expression of a small number of (virulance) genes and indicated the presence of well-defined heterochromatic boundaries.. In summary, we uncover an unprecedented function of HP1 as a  mayor regulator of virulence gene silencing and phenotypic variation, which will be instrumental for our understanding of this widely used survival strategy of unicellular pathogens.

overall_design: One experimental sample.

first: Christian

last: Flueck

first: Jennifer

last: Volz

first: Igor

last: Niederwieser

first: Adriana

last: Salcedo-Amaya

first: Blaise

middle: T

last: Alako

first: Florian

last: Ehlgen

first: Stuart

last: Ralph

first: Alan

last: Cowman

first: Zbynek

last: Bozdech

first: Hendrik

last: Stunnenberg

first: Till

last: Voss


title: Transcriptome analysis of rice stripe virus-infected rice

city: Sapporo

first: Kouji

last: Satoh

state: Hokkaido

address: Hitsujigaoka 1

country: Japan

institute: NARO/HARC

summary:
Rice stripe virus (RSV) is one of the major virus diseases of rice in East Asia. Rice plants infected with RSV usually show symptoms such as chlorotic leaf stripes, weakness and senescence of leaves, and dwarfism. In order to characterize the host response to RSV infection at the gene expression level, the changes in transcriptome profiles of RSV-infected rice were monitored at three, six, nine, twelve, and fifteen days after inoculation by a rice oligomicroarray. The microarray data indicated that 1. transcription, translation and protein processing machineries were activated, 2. chloroplasts were disintegrated, and mitochondrion function was activated, 3. genes for transporters and cell wall synthesis were suppressed, and 4. the expression levels of pathogenesis-related genes were changed by RSV infection. Concurrent observation of symptom development, virus accumulation and transcriptome profiles in RSV-infected plants indicates that RSV symptoms are caused by unbalanced activities of organelles, suppression of cell elongation, and uncontrolled water transport, while translation activity of host cells may be increased in correlation with RSV propagation.

Keywords: time course, virus infection, disease response

overall_design: Comparison between RSV- and mock-infected rice. Biological replicates: 3 control, 3 infected, independently grown and harvested; 4 time points (3, 6, 9, 12 days after inoculation (DAI)). Biological replicate: 1 control, 1 infected, independently grown and harvested; 1 time point (15 DAI). 1 sample derived from 5 plants grown under the same conditons.

first: Takahide

last: Sasaya

first: Hiroaki

last: Kondoh

first: Setsuko

last: Kimura

first: Toshihiro

last: Omura

first: Shoshi

last: Kikuchi


title: Essential role for Argonaute2 protein in mouse oogenesis

first: Fuchou

last: Tang

state: Cambridgeshire

address: Tennis Court Road

department: Wellcome Trust/Cancer Research UK Gurdon Institute

country: United Kingdom

institute: University of Cambridge

summary: Argonaute2 proteins (Ago2) are key component of RNA-induced gene silencing complexes (RISCs), which is crucial for microRNAs to repress target genes. The function of Ago2 for oogenesis is unknown due to early embryonic lethal of Ago2 knockout mice. Here we show the effect of loss of Ago2 in mouse oogenesis by specific deletion of it in growing oocyte. Although the Ago2-deficient oocytes can develop to mature oocytes, they have abnormal spindle and the chromosomes that can’t cluster together properly, which are very similar to the phenotype of Dicer-deficient oocytes. We checked miRNA expression profile and found that in Ago2-deficient oocyte, the expression of most miRNAs reduced more than 80%. To understand the downstream genes regulated by Ago2, we used microarray on Ago2-deficient oocyte and found that 512 genes were upregulated and 1073 genes were downregulated (FC>2, P<0.05). Our data proved that Ago2 have a key function for mouse oogenesis through globally regulating miRNA stability and function.

overall_design: we run single cell cDNA microarray comparing Ago2-KO oocytes with wild-type GV oocyte controls

first: Kaiqin

last: Lao

first: Azim

middle: M

last: Surani


title: Gene expression profiling of Clostridium Perfringens infection in broilers on medicated and non-medicated diets.

city: College Station

first: Huaijun

last: Zhou

address: Rm 418D Kleberg Center,Texas A&M University

institute: Texas A&M University

summary: Gene expression profiling of clostridium perfringens infection in broilers on medicated and non-medicated diets using chicken 44k agilent microarray. To elucidate molecular and ceelular mechanisms of bacitracin effect on CP infection in chickens by microarray technology.

overall_design: A total number of 600 Ross broilers were reared in 12 pens with each hosting 50 birds.  Each 6 pens of birds were fed bacitracin-medicated (55 ppm) or non-medicated starter diets immediately after the chicks were placed. At day 18, birds were challenged with CP. Spleens were collected from 12 birds of each group at day 18 (before infection), 19, 20, and 22. Total RNA was labeled by Cy3 or Cy5 with dye swap. Gene signal intensity was globally normalized by LOWESS and expressed on natural log scale. A mixed model including treatment, time, array (random effect), dye, and all interactions among treatment, time was used to identify differentially expressed genes between treatments, at the 1% significance level.


title: Identification of androgen-responsive genes in fish testis

city: Rennes

first: Florence

last: Le Gac

address: Campus de Beaulieu

department: Fish Reproduction Group

country: France

institute: Institut National de la Recherche Agronomique

summary: Sexual steroids play an important role in gonad maturation in all vertebrates. However, the molecular mechanisms regulating of spermatogenesis in seasonal breeding species are yet poorly understood. In this study we investigated the effects of testosterone and 11-ketotestosterone (11KT) - a teleost fish specific androgen - on testicular gene expression at the onset of puberty in the rainbow trout, Oncorhynchus mykiss.

overall_design: Rainbow trout 13 months old were used. Immature males (spermatogonia only, stage I-II) were submitted to androgen supplementation by hormone implants for 7 or 14 days. This included : - untreated animal controls (N=14) - animals subjected to testosterone supplementation (0.1mg) for 7 days (N=5) - animals subjected to testosterone supplementation (0.2mg) for 7 days (N=4) - animals subjected to testosterone supplementation (0.2mg) for 14 days (N=5) - animals subjected to 11-ketotestosterone supplementation (0.25mg) for 7 days (N=7). Individual testes were recovered for microarray experiments and histological analyses. Circulating levels of corresponding androgens were also determined.

first: Antoine

last: Rolland

first: Aurélie

last: Lardenois

first: Anne-Sophie

last: Goupil

first: Fréderic

last: Chalmel

first: Rémi

last: Houlgatte

first: Jean-Jacques

last: Lareyre


title: Gene Expression Analysis in Peripheral Blood of Patients with Rheumatoid Arthritis

city: Tokyo

first: TERAO

last: KIMIO

address: 2-1-1,Nihombashimuromachi, Chuo-ku

department: Clinicalpharmacology

institute: Chugai Pharmaceutical co.,ltd.

summary:
Peripheral blood cells in patients with rheumatoid arthritis before/after IL-6 inhibition
The obtained conclusions on the change of each gene expression level before and after MRA medication were as follows.
(1)	In the case of IL-6, the specific time profile that the expression level dropped at day 21 and increased to some extent at day 42 was observed in 2 of 18 subjects. In other subjects, quite small change or small increase after day 7 was observed.
(2)	In the case of IL-6R as well as in IL-6, the specific time profile that the expression level dropped at day 21 and increased to some extent at day 42 was observed in 1 of 18 subjects.  In other subjects, slow and small increase was observed in 6 of 18.
(3)	The ratio of mRNA expression level of membrane and soluble type of IL-6R showed marginal difference after tocilizumab treatment,
(4)	The distinct change of expression level of cytochrome P450 mRNA after tocilizumab treatment was hardly observed in most patients.

overall_design:
The measurement of expression level of several genes shown below in peripheral blood of human subjects (18 patients with rheumatic disease) before and after tocilizumab medication using DNA microarray (Hitachi human oligo chip) was conducted in this test,
1) Interleukin 6 (IL-6), membrane IL-6 receptor (IL-6R) and soluble type of the IL-6R,
2) several cytochrome P450 as follows: CYP3A4, CYP2D6, CYP2C9, CYP2C19.
Number of human subjects was 18 and peripheral blood samples drawn at 4 time points:
day 0 (the first Omeprazole administration was done), day 7  (Tocilizumab administration was done), day 21 and day 42 for each subject were analyzed. On day 0 and day 7, the blood sampling was done before drug administration.

first: Kimio

last: Terao

first: Tomomi

last: Tsuru

first: Midori

last: Suzaki

first: Takahiro

last: Kakehi

first: Norihiro

last: Nishimoto


title: Shg1p associated RNAs

status: Public on Aug 31 2009

city: ETH Hoenggerberg, Zuerich

first: Andre

last: Gerber

address: Wolfgang Pauli Str. 10, HCI H396

department: Institute of Pharmaceutical Science

country: Switzerland

institute: Swiss Federal Institute of Technology

summary: RNA co-immunopurification with TAP-tagged Shg1p (a component of the Set1 histone methyltransferase complex, SET1C) from Saccharomyces cerevisiae. An untagged (BY4741) served as a control (Gerber et al. 2004). Cells were grown to mid-log phase in rich media and harvested by centrifugation. TAP-tagged Shg1 was affinity-purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input) and from the purified protein sample with the RNAeasy Kit (Qiagen). RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast oligo microarrays over night at 42 degrees in formamide-based hybridization buffer. For the method see experimental procedure for RNA IP (Gerber/Dichtl).

overall_design: An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.


title: Rapid Identification of Bacterial Pathogens using a PCR- and Microarray-Based Assay

city: Helsinki

first: Anna-Kaarina

last: Jarvinen

address: Biomedicum Helsinki 2U, Tukholmankatu 8

country: Finland

institute: Mobidiag

summary: A method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level.

sample_organism:
Homo sapiens
unidentified

platform_organism:
Acinetobacter baumannii
Neisseria meningitidis
Klebsiella pneumoniae
Haemophilus influenzae
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus agalactiae
Streptococcus pneumoniae
Streptococcus pyogenes
Enterococcus faecalis
Enterococcus faecium
Listeria monocytogenes

overall_design:
The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.
The processed data are composed of ORGANISM NAME or NEGATIVE answer depending if the pathogen is detected in the sample or not.  See supplementary file linked below.

first: Minna

last: Maki


title: The transcriptional response of Pasteurella multocida to three classes of antibiotics

city: Mississippi state

first: Bindu

last: Nanduri

state: MS

address: Box 6100 Spring Street

department: Basic Sciences

institute: Mississippi State University

summary:
We studied the effects of three classes of antibiotics (amoxicillin, chlortetracycline and enrofloxacin ) on P. multocida transcriptome using custom oligonucleotide microarrays from Nimblegen systems. All the 2015 genes of Pm70 were spotted on the array and hybridizations were carried out with RNA isolated from three independent cultures of Pm70 grown in the presence or absence of sub-minimum inhibitory (sub-MIC) doses of antibiotics.    Differentially expressed genes were identified by ANOVA and Dunnett’s test. Biological modeling of the differentially expressed genes (DE) was carried out based on Clusters of Orthologous (COG) groups and network analysis in Pathway Studio.

Keywords: Response to sub-MIC antibiotics

sample_organism: Pasteurella multocida subsp. multocida str. Pm70

overall_design: The experimental design included three biological replicate cultures of P. multocida grown in the absence or presence of sub-MIC antibiotics. Effects of antibiotics on the transcriptome with each antibiotic were determined by comparing the growth in the presence of antibiotic (treatment) to growth in the absence of antibiotic (control).

first: Shane

middle: C

last: Burgess

first: Mark

last: Lawrence


title: Global analysis of gene expression in MEF cells and villi enterocytes expressing SV40 T antigens

city: Pittsburgh

first: Ulka

last: Sachdev-Ost

address: 300 Halket Street Suite 5414

institute: University of Pittsburgh School of Medicine

summary:
SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.
Keywords: comparative genomic hybridization

overall_design: We used the Agilent Mouse cDNA (G4104A) array for these experiments. For the MEF microarrays, we used a dye-flip reference design (Churchill GA, 2002, Nature Genetics, Vol. 32, p.490-495) to compare a pool of MEF total RNA to three independent T antigen MEF cell lines. Cells were grown to two days post-confluence and harvested. For the mouse small intestine microarrays, we used three different intestinal preparations: Whole, Villi and Vlcm in a replicated dye-flip design (Churchill GA) using three (Vlcm) or four (Whole and Villi) T+ mice and the same number of their wild-type littermates for each intestinal preparation.

first: Paul

last: Cantalupo


title: Gene regulation in Erbb2 transfected NIH3T3 cells using tet-off system

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum München GmbH

summary:
V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2; synonyms HER2, NEU) encodes a transmembrane glycoprotein with tyrosine kinase-specific activity that acts as a major switch in different signal-transduction processes. ERBB2 amplification and overexpression have been found in a number of human cancers, including breast, ovary and kidney carcinoma. Our aim was to detect ERBB2-regulated target genes regulated in Erbb2 transfected murine fibroblast cells after tetracycline treatment to stop the overexpression of ERBB2. Changes in gene expression patterns in these cells after the switch-off of Erbb2 expression was analysed after 8 different periods of time after the treatement (1h-7d). Identification of co-regulated genes and in cluster analysis were performed.
Keywords: Erbb2, cDNA microarrays, murine fibroblasts,

overall_design: gene expression levels of 8 different measurements after tetracycline treatment (1h, 2h, 4h, 12h, 24h, 3d, 5d and 7d) were compared to the non-treated NIH3T3 cell line. As control two different untreated NIH3T3 cell line were ananysed. For all comparisons 4 experiments have been performed including 2 dye swaps.

first: Johannes

last: Beckers

first: Marion

last: Horsch

first: Barbara

last: Seliger


title: Escherichia coli Tiling Array for In vivo Protein Occupancy Display

city: Princeton

first: yirchung

last: Liu

state: NJ

address: 220 Carl Icahn Laboratory

institute: Princeton University

summary: Using a high-density tiling array containing 25-mer oligonucleotides at the resolution of one every four base pairs across the entire genome, we developed In vivo Protein Occupancy Display (IPOD), a technology that reveals protein occupancy across an entire bacterial chromosome at the resolution of individual binding sites.  Application to Escherichia coli reveals thousands of protein occupancy peaks, highly enriched within and in close proximity to non-coding regulatory regions.

overall_design:
We designed an Affymetrix tiling array for the MG1655 E. coli genome, containing probes that cover the entire genome at a resolution of 4 bp between steps; however, the steps alternate strand coverage, so there is an 8 bp step between probes on the same strand. There are a total of 2.47 million probes on the array, of which 2,300,160 directly enter analysis as E. coli probe pairs. The Affymetrix system pairs each 25-mer perfect match genomic sequence with a corresponding 25-mer that has a mismatch in the 13th position. The mismatch probe is intended as a cross-hybridization control. In addition to the E. coli sequences, 33,996 probe pairs are sequence-specific controls against other genomes, including B. subtilis, lambda phage, and A. thaliana. The tiling array (Ecoli_Tab520346F) is a standard-sized 200 μL-volume microarray with 5 micron features.

6 biological replicates of MG1655 DNA, 2 biological replicates of MG1655 DNA isolated from early-phase DNA-protein complexes, 2 biological replicates of MG1655 DNA isolated from late-phase DNA-protein complexes, 1 replicate of MDS42 DNA isolated from early-phase DNA-protein complexes, 2 biological replicates of MG1655 total RNA.

first: Tiffany

last: Vora

first: Alison

middle: K

last: Hottes

first: Saeed

last: Tavazoie


title: Response to the inhibitors of mevalonate pathway in yeast

city: Toulouse

first: Serguei

last: Sokol

address: 135 av de Rangueil

department: Lab. Biotech. and Bioprocess

institute: INSA

summary: In this experiment we performed the transcriptional profiling of the wild type yeast Saccharomyces cerevisiae upon treatment with lovastatin or zaragozic acid. These drugs are known to exert a repressing effect on the sterol branch of the isoprenoid pathway, but their action differs at the level of FPP biosynthesis. While lovastatin decreases FPP availability because it is an inhibitor of HMGR, zaragozic acid increases this level because it inhibits squalene synthase. In this work, we were interested especially in genes, whose expression would be oppositely regulated in the presence of lovastatin or zaragozic acid, since these genes could be affected by variation in FPP level and thus related somehow to the isoprenoid pathway.

overall_design: We measured changes in the gene expresion after exposure of cells to two inhibitors of the mevalonate pathway, Lovastatin and Zaragozic acid. Changes caused by these drugs were followed in time after 10, 30 and 60 minutes. We carried out the experiment, in the presence of 25 µg/mL lovastatin and 10 µg/mL of zaragozic acid, since these concentrations did not inhibit significantly the growth rate of the cells. These low doses permitted to reduce the risk of observing secondary effects due to growth inhibition and cell death. For each point of the experiment, total RNAs from two independent yeast cultures were extracted. For labeling we employed dye swapping (dCTP-Cy3 and dCTP-Cy5) and hybridized cDNA on two independent microarrays. Thus, every gene was represented by four intensities from two independent DNA arrays.

first: Klaudia

last: Kuranda

first: Kariona

last: Grabinska

first: Thierry

last: Berges

first: Veronique

last: Leberre

first: Jean

last: François

first: Grazyna

last: Palamarczyk


title: Murine NALT Asymptomatic Carriage

city: Stanford

first: Elizabeth Ann

last: Joyce

email: ejoyce@stanford.edu, ea.joyce@gmail.com

address: Mail Code: 5124

department: Microbiology and Immunology

institute: Stanford University School of Medicine

summary:
Background: We employed DNA microarray technology to investigate the host response to Streptococcus pneumoniae in a mouse model of asymptomatic carriage.  Over a period of six weeks, we profiled transcript abundance and complexity in the Nasal Associated Lymphoid Tissue (NALT) to identify genes whose expression differed between pneumococcal-colonized and uncolonized states. Results:  Colonization with S. pneumoniae altered the expression of hundreds of genes over the course of the study, demonstrating that carriage is a dynamic process characterized by increased expression of a set of early inflammatory responses, including induction of a Type 1 Interferon response, and the production of several antimicrobial factors.  Subsequent to this initial inflammatory response, we observed increases in transcripts associated with T cell development and activation, as well as wounding, basement membrane remodeling, and cell proliferation.  Our analysis suggests that microbial colonization induced expression of genes encoding components critical for controlling JAK/STAT signaling, including stat1, stat2, socs3, and mapk1, as well as induction of several Type 1 Interferon-inducible genes and other antimicrobial factors at the earliest stages of colonization. Conclusions:  Examining multiple time points over six weeks of colonization demonstrated that asymptomatic carriage stimulates a dynamic host response characterized by temporal waves with distinct biological programs.  Our data suggest that the usual response to the presence of the pneumocccus is an initial controlled inflammatory response followed by activation of host physiological processes such as response to wounding, basement membrane remodeling, and increasing cellular numbers that ultimately allow the host to maintain an intact epithelium and eventually mount a preventive adaptive immune response.
A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied.

first: Elizabeth


title: Tiling arrays from control and flies without Drosha or Pasha

city: Ashburn

first: Ken

last: Sugino

address: 19700 Helix Dr

institute: Janelia Research Campus

type: Expression profiling by genome tiling array

summary:
Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), we knocked down the miRNA biogenesis pathway in  Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify  miRNA–mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs—clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 39 untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 39 UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.
Tilling arrays were hybridized with cDNA from control or flies in which  Drosha or Pasha was downregulated in the timeless-expressing neurons.

overall_design: Control (Tim-gal4) , Tim-Drosha (Tim-gal4; UAS-Drosha IR) or Tim-Pasha (Tim-gal4; UAS-pasha IR) flies were entrained in 12h light:12h dark cycles and heads were collected at ZT3 and ZT15. For each strain, RNA extracted from those heads were mixed and used to prepare labeled cDNA that was hybridized to the tiling arrays. Then, the expression value for the Tim-pasha and Tim-Drosha samples was compared to control samples

first: Sebastian

last: Kadener


title: Global Expression Analysis of Vasculogenesis Induction by Zebrafish Ets1-Related Protein

city: Tampa

first: Saulius

last: Sumanas

state: FL

address: 560 Channelside Dr

department: Department of Pathology and Cell Biology

institute: University of South Florida

summary: Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis.

overall_design: Approximately 50 pg of etsrp RNA was microinjected into zebrafish embryos at the 1-4 cell stages. Three separate batches of approximately 150 microinjected embryos and uninjected controls were frozen on dry ice at the tailbud stage. Total RNA was purified using the RNAquous-4PCR kit (Ambion). Approximately 10-20 µg RNA from each batch was sent to the NimbleGen facility, were it was processed, labeled and hybridized to standard NimbleGen 385K gene expression array. Briefly, RNA was converted into cDNA using the SuperScript II cDNA Conversion Kit (Invitrogen, Carlsbad, CA). Double-stranded cDNA was random-prime labeled with Cy3-nonamers and hybridized to the microarrays for 16 hours at 42°C. The zebrafish microarray was based on Zv6 genome assembly and contained approximately 32,000 genes with approximately 12 probes of 60mer oligonucleotides per each gene. The arrays were washed, dried, and scanned at 5μm resolution using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA). Data were extracted from scanned images using NimbleScan software (Roche NimbleGen). Quantile normalization was performed across replicate arrays, and RMA (Robust Multichip Average) analysis was performed to generate gene expression values. Average ratios of expression values of etsrp-injected vs. control were calculated from three experiments. Results were sorted by the fold change from the highest to the lowest. Genes that displayed values of p>0.05 or showed expression levels less than 2-fold above the background level in both control and experimental samples were eliminated from further analysis.

first: Kuan

middle: S

last: Wong

first: Kira

last: Proulx


title: C. briggsae developmental timecourse

city: Haifa

first: Itai

last: Yanai

address: Technion City

institute: Technion - Israel Institute of Technology

summary: To identify differences in gene expression patterns between C. elegans and C. briggsae we designed whole-genome species-specific microarrays.

overall_design: We designed two organism-specific microarrays which were then manufactured by Agilent as 44k arrays. Probes were designed to target the coding region, preferentially near the 3’ end, as well as in the presumptive 3’ UTR, up to 150bp downstream of the stop codon. 50-60mer probes were determined using OligoWiz (Wernersson and Nielsen 2005) which selects oligos based upon their cross-hybridization to other coding sequences, Tm, position along the transcript, folding potential, and low-complexity in the sequence. The probes were also restricted against spanning splice junctions to avoid missing transcripts due to errors in gene structure predictions. Each gene was assigned between one and five probes, where the probe with the best overall score was selected as the ‘A-probe’, and the remaining ranked ‘B’ through ‘E’.

first: Craig

last: Hunter


title: C. elegans developmental timecourse


title: Mutation Mapping of Streptococcus suis serotype 2 strains

status: Public on Aug 29 2009

city: Ames

first: Zuo-Wei

last: Wu

address: 1113 Veterinary Medicine Bldg

institute: Iowa state university

summary: Streptococcus suis serotype 2 (SS2), an important zoonotic agent, is notorious for causing contagious porcine diseases and human infection. The two outbreaks in China (in 1998 and in 2005) have caused serious economic losses in the pig industry and posed public health for its new toxin shock symptoms (TSS). However, the molecular mechanism of SS2 pathogenicity is still poorly understood. In order to get insights into pathogenecity of SS2, eighteen SS2 strains of different virulence and sources have been subjected to whole genome comparison by NimbleGen CGS arrays

overall_design: Comparative genomic analysis of 18 SS2 strains with 05ZYH33 as reference

first: Zuowei

first: Weilan

last: Wang

first: Baoli

last: Zhu


title: Differential transcriptomic responses of Biomphalaria glabrata to bacteria and metazoan parasites

city: Albuquerque

first: Coen

last: Adema

state: NM

address: 1 University of New Mexico

department: CETI Biology MSC03 2020

institute: University of New Mexico

summary:
Differential transcriptomic responses of Biomphalaria glabrata (Gastropoda, Mollusca) to bacteria and metazoan parasites Schistosoma mansoni and Echinostoma paraensei (Digenea, Platyhelminthes).
Transcriptional profiling of Biomphalaria glabrata comparing control uninfected M-line B. glabrata with five experimental groups.  The experimental groups are: wounded but not infected M-line, Escherichia coli infected, Micrococcus luteus infected, Echinostoma paraensei infected and Schistosoma mansoni infected at 12 hours time point post infection.

overall_design: Seven groups of samples: two controls, wounded, two bacterial- and two trematode-infected B. glabrata were analyzed in triplicate, using universal RNA reference.

first: Eric

last: Loker


title: Serial transcriptome analysis and cross species integration identifies CENPE as novel neuroblastoma target

status: Public on Aug 28 2009

city: Philadelphia

first: Jayanti

last: Jagannathan

state: PA

address: 34th & Civic center blvd

department: Oncology

institute: Childrens Hospital of Philadelphia

summary: Cancer genomic studies that rely on analysis of diagnostic biopsies from primary tumors may not fully identify the molecular events associated with tumor progression.  We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic neuroblastoma model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in 9 hyperplastic ganglia harvested at 3 time points, and 4 tumor cohorts of progressively larger size (n=6 each group) in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We manually curated the 24 candidates and prioritized 3 targets (Cenpe, Gpr49, Impdh2) with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell line models we further prioritized CENPE due to potent inhibition of cellular proliferation. Targeting of CENPE with the selective small molecular inhibitor GSK923295 showed potent inhibition of in vitro proliferation of 19 neuroblastoma derived cell lines (median IC50=41 nM; range 27-266 nM), and significantly delayed tumor growth kinetics in 3 xenograft models (p-values ranged from p<0.0001 to p=0.018). Here we provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets, and identify CENPE as a novel therapeutic candidate in neuroblastoma.

overall_design:
set 1: 24 tumor samples analyzed
set 2: 9 hyperplastic ganglia harvested at 3 time points (day 0, day 7 and day 14) and 2 tumors "small" size were analysed

first: Naomi

last: Balamuth

first: Andrew

last: Wood

first: Qun

first: Zhe

last: Zhang

first: Zhongxue

last: Chen

first: Patrick

last: Mayes

last: Rappaport

first: Bruce

last: Pawel

first: Barbara

last: Weber

first: Richard

last: Wooster

last: Sekyere

first: Glenn

last: Marshall

first: John

last: Maris


title: Gene expression-based assessment of the effect of PQQ

status: Public on Aug 26 2009

city: davis

first: eskouhi

last: Tchaparian

address: one shields avenue

institute: UC-Davis

summary: Pyrroloquinoline quinone (PQQ) is a naturally occurring compound and known to improve growth and reproductive performance when added to diets of PQQ-deprived rodents. To understand its mechanism of action, changes in hepatic gene expression were measured in rats fed diets with or without added PQQ.  Of the ~10,000 genes and ESTs analyzed, 4.7% of the transcripts were sensitive to changes in PQQ dietary status.  PQQ deprivation generally caused downregulation of genes associated with mitochondriogenesis, cell differentiation, and immune function.  These gene expression changes provide the basis for most of the previously published functional observations associated with PQQ deficiency and PQQ administered in pharmacological amounts. To assess PQQ’s potential functions, we used gene expression profiling through microarray technology as part of a comprehensive approach to identify potential pathways and mechanisms.  Given that the systemic effects of PQQ deprivation are influenced at levels of dietary intake in the micromolar range, highly purified diets were used to reduce expression from other bioactive factors and xenobiotics, such as those found in typical rodent chow diets. Dietary conditions were also chosen to clarify the response to PQQ deprivation.  A goal was to determine if specific changes in dietary protocol or patterns could be used to identify genes important to the function of PQQ.  Because it has been observed that mitochondrial-related functions are influenced by PQQ, we hypothesized that changes in genes important to fatty acid and amino acid metabolism and mitochondrial function would be likewise affected by dietary levels of PQQ. The overall study was carried out using two experiments. In this specific experiment, PQQ depletion and repletion was studied to understand the effect of PQQ on gene expression.

overall_design: In this experiment, a CodeLink® UniSet Rat I Bioarray was used. Unpooled RNA samples were hybridized to the UniSet arrays, hence expression profiles were obtained from individual rats for each of the 5 groups.

first: Eskouhie

first: Lisa

first: Gene

last: Cutler

first: Kathryn

last: Bauerly

first: Winyoo

last: Chowanadisai

first: Calliandra

last: Harris

first: Robert

middle: B

last: Rucker


title: Cse4 ChIP-chip

city: Kansas City

first: Raymond

last: Camahort

state: MO

institute: Stowers Institute

summary: In order to take an unbiased approach and discover all the locations of Cse4 in the genome, we utilized formaldehyde crosslinking and immunoprecipitation followed by hybridization to DNA microarrays. We analyzed the location of Cse4 in three different strains; one in which the endogenous Cse4 is tagged with 12Myc epitopes (Cse4-12Myc), and one which contained both the endogenous Cse4 (untagged) and an ectopic copy of Cse4-12Myc expressed from the GAL1-10 promoter (pGAL1-10-Cse4-12Myc), and one in which Cse4 is tagged with 3HA epitopes (Cse4-3HA).

overall_design: ChIP-chip was performed using custom microarrays, to look at the genome-wide localization of the centromeric histone variant Cse4.  Biological replicates were performed for each Cse4 epitope-tagged strain

first: Jennifer

last: Gerton


title: Sub1 occupancy in yeast cells in exponential growth condition

status: Public on Aug 27 2009

city: Gif-sur-Yvette cedex

first: Christine

last: Conesa

address: CEA Saclay

department: iBiTeC-S

institute: CEA

summary:
A genome-wide location analysis by ChIP on chip of the gene occupancy by Sub1 was undertaken to define the gene targets of Sub1 in vivo. Chromatin immunoprecipitation (ChIP) assays were performed on epitope-tagged Sub1-3HA cross-linked chromatin from exponentially growing cells. Immunopurified DNA and DNA from whole-cell extracts were fluorescently labelled and competitively hybridized to DNA microarrays harbouring ORFs and intergenic regions. The ratio of fluorescence intensities at each site in the microarray provided a measure of the extent of Sub1 binding to a specific genomic locus. Data from three independent experiments were compiled. Many loci were found to be significantly enriched in active growth conditions where Sub1 is not essential for cell viability. Approximately one-fourth of the enriched loci were located within ORF and the others corresponded to intergenic regions and to genes encoding non-translated RNAs. The ACT1, PMA1, PYK1, ADH1 and snoRNA genes previously identified as DNA targets of Sub1 were indeed enriched in our data. Sub1 was also preferentially bound to a subset of Pol II-transcribed genes encoding constituents of the cell wall, the nucleosome and the ribosome. Remarkably, these Pol II genes represented only two-third of the enriched loci. The other ones corresponded to Pol III-transcribed genes present on the arrays or to the intergenic regions and ORFs adjacent to these genes, suggesting the association of Sub1 to all Pol III-transcribed genes in conditions of active growth. The arrays used had a poor coverage of the rDNA gene locus. Nevertheless, we found a significant enrichment of the different loci corresponding to that region suggesting that Sub1 associates at many locations throughout the rDNA gene. Altogether, the results suggested that Sub1 could be a regulator of all three transcription systems.
Keywords: ChIP-Chip

overall_design:
Chromatin immunoprecipitation

Chromatin immunoprecipitations were performed essentially as described by Ren et al. (2000) with some modifications described below. Briefly, overnight yeast cultures were used to inoculate fresh YPD medium at 0.1 OD600. The cells were grown in 100 mL of YPD to 1 OD600 and fixed with formaldehyde at a final concentration of 1% for 15 min at room temperature. Fixation was stopped by the addition of glycine (final concentration 340 mM) and incubation for 5 min. The fixed cells were harvested by centrifugation and washed twice with cold TBS. The cell pellets were frozen and stored at –80°C for further use.
The cell pellets were thawed, resuspended in 700 µL of lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM PMSF, O Complete protease inhibitor (Roche)) and transferred to 1.5 mL Eppendorf tubes. 0.5 mL glass beads (425-600 µm, Sigma) were then added to each tube, and the yeast cells were lysed at 4°C for 2 hours using an Eppendorf Thermomixer Comfort at 1400 rpm. Each tube was pierced at the bottom and the cell lysate was collected by centrifugation in a fresh tube. The lysate was then sonicated 4 times for 10 s with 20 s cooling on ice between each sonication. The power of the Vibra Cell™ sonicator (Sonics & Materials Inc., Danbury, Connecticut, USA) was set to 4 with 60% duty cycle. Sonication resulted in chromatin fragments of 0.25–1 kb in size with a mean size of 600 base pairs as determined by gel electrophoresis. After 5 min of centrifugation in 1.5 mL Eppendorf tubes at 15 000 rpm, the supernatant (named whole cell extract, WCE) was transferred to another tube on ice. The preparation of magnetic beads, immunoprecipitation (with anti-HA 12CA5 or anti-myc 9E10), elution from beads and reversal of crosslinking, DNA precipitation, DNA blunting and ligation of blunted DNA to linkers were done exactly as described in Ren et al. (2000).

Construction of yeast microarrays

The S. cerevisiae ORF and intergenic regions were amplified from S288C genomic DNA with oligonucleotides from ResGen. The primers allow the amplification of the sequence located on either side of elements such as open reading frames, tRNAs, small nuclear RNAs, Ty elements, solo δ, etc. A complete description of the primers can be obtained on the web site (ftp://ftp.resgen.com/pub/genepairs/yeast_intergenic). The PCR products were purified by ethanol precipitation and their size and concentration were measured by agarose gel electrophoresis. 95 % of the regions were correctly amplified (as evidenced by gel electrophoresis) and purified at a mean plate concentration of 70 ng/µL. The purified DNAs were spotted onto amino-silane coated glass slides (GAPS II, Corning) using an automated arrayer (MicroGrid II, BioRobotics) with a spotting success percentage higher than 96 %. The final success rate is 93-94 %.
LM-PCR, probe labeling and hybridization
Ligation-mediated PCR was done as described by Ren et al. (2000) except that amino-allyl conjugated dUTP (150 µM final) was used instead of Cy3-dUTP or Cy5-dUTP and only 30 cycles of PCR were performed. After PCR amplification, 5 μl of the reaction mixtures was run on 1.5% agarose gel to measure the amount of amplified DNA. The PCR product size ranged from 200 to 1000 bp with an average size of 300 bp. The PCR products were purified using a Microcon YM-30 filter (Amicon/Millipore). Amino-allyl modified DNA was recovered with 20 µL of H2O and the DNA was lyophilized.
The DNA pellet was resuspended in 9 µL of 100 mM sodium bicarbonate, pH 9.0. This sample DNA was used to dissolve a dry pellet of monofunctional NHS-ester Cy3 or Cy5 (1/16e of the quantity delivered from the mono-reactive dye pack PA23001 or PA25001 respectively from Amersham) and the mixture was incubated for 1 hour at room temperature in the dark. The coupling reaction of the Cy dyes to the amino-allyl dUTP was stopped by quenching with the addition of 4.5 µL of 4M hydroxylamine (Sigma) and the solution was further incubated for 15 min in the dark. Cy3-immunoprecipitated DNA and Cy5-WCE control DNA were mixed and unincorporated/quenched Cy dyes were removed using a Microcon YM-30 filter (Amicon/Millipore). DNA was recovered with 60 µL of TE and ethanol precipitated.
Prehybridization and hybridization conditions were those described on P. Brown's web site (http://brownlab.stanford.edu/protocols.html) with few minor modifications. The labeled precipitated DNA samples were recovered in hybridization buffer (50% formamide, 5X Denhardt’s, 0.5% SDS, 7X SSPE, 10 µg herring sperm DNA) and hybridized to the DNA microarray in a Corning slide chamber at 42°C overnight. Arrays were washed at room temperature once with 0.1X SSC + 0.1% SDS, twice with 0.1X SSC, then dried by centrifugation.

Data analysis

Hybridized arrays were scanned using a GenePix 4000A scanner (Axon Instruments, Inc.) and fluorescence ratio measurements were determined with the GenePix Pro 6.0 software (Axon Instruments, Inc.). Array analyses were undertaken using the Limma package (Smyth, 2005) from the R/Bioconductor software (R-Development-Core-Team, 2007). Data from three independent experiments were compiled. ChIP-chip spot intensities have been normalized using the weighted median as implemented in the normalizeWithinArrays function of the Limma package. Normalized measures served to compute the log2-ratio for each probe. To exhibit enriched probes, we have used a moderated t-test. The moderated t test applied here was based on an empirical Bayes analysis and was equivalent to shrinkage (or expansion) of the estimated sample variances towards a pooled estimate, resulting in a more stable inference, but we have considered a one-sided alternative hypothesis, since we expect higher intensity levels in the IP-enriched hybridizations than in the negative control hybridizations.

first: C

last: Ducrot

first: A

last: Suleau

first: M

last: Michaut

first: JC

last: Aude


title: Identification of Vaccinia Virus HLA Class I Peptides Presented by the Immune System

city: Rochester

first: Kenneth

middle: L.

last: Johnson

state: MN

address: 200 First Street S.W.

institute: Mayo Clinic

summary:
Seeking to identify HLA class I peptides that originate from vaccinia virus proteins to understand the mechanism of immune protection.  Note that vaccinia-infected B cells will still continue to present (primarily) a wide variety of peptides originating from endogenous proteins; this data set contains evidence for more than 5000 such peptides.  The objective and challenge is to detect and identify the peptides that originate from the pathogen (vaccinia virus) in the presence (background) of this large number of endogensous 'self' peptides.
Keywords: Peptide search results from multiple injections of multiple strong cation exchange fractions combined into one set of results.

sample_organism:
Homo sapiens
Bos taurus
Orthopoxvirus vaccinia

overall_design: Immortalized B-cells (Priess cells) were infected with live vaccinia virus and incubated 24 hours; HLA proteins binding and presenting peptide fragments of cellular proteins (HLA class I molecules/peptide complexes) were immunopurified and the peptides were identified by 2-dimensional liquid chromatography and tandem mass spectrometry.

middle: L


title: Identification of proteins from vacuoles of red algae

city: Nishiikebukuro

first: Fumi

last: Yagisawa

state: Tokyo

department: Research Information Center for Extremophiles

institute: Rikkyo (St. Paul's) University

summary:
Vacuoles and lysosomes are single-membrane-bound organelles involved in diverse functions such as intracellular digestion and storage or secretion of metabolites. To understand their origin, evolution and fundamental features, the identification of proteins comprising these compartments in missing links would be invaluable. So, we isolated the vacuoles from Cyanidioschyzon merolae, which is considered to be one of the most primitive photosynthetic eukaryotes, and identified 46 proteins by matrix-assisted laser desorption/ionization time of flight-mass spectrometry.

Keywords: peptide mass fingerprinting, MALDI-TOF

overall_design: one sample


title: Transcriptional response of ethanol-stressed vs. non-stressed culture

first: Laurent

last: Benbadis

address: 135 Av de Rangueil

institute: LISBP INSA CNRS UMR5504

summary:
The yeast Saccharomyces cerevisiae is well known for its high ethanol production performances. An original fermentation process that allows the yeast S. cerevisiae to produce in less than 45 h more than 150 g/l ethanol (i.e. 18.9°GL) was set up in our laboratory [1]. Under this condition, the yeast cells induce a dynamic process to adapt to increased ethanol concentration by a mechanism that is likely different to the stress response triggered by sudden ethanol addition to exponentially growing cells [2]. Kinetic analysis of the growth curve identified two main phases: a growth phase that ended up at 90 g/l ethanol and then an uncoupling phase during which non-growing cells kept producing ethanol. This latter phase is also characterized with an increased loss of viability.
In order to investigate on a genome scale the expression changes occurring during this process, gene expression was quantified using DNA chips technology at six different time-points during fed-batch fermentation.
[1] Alfenore et al, Appl. Microbiol. Biotechnol. 60 : 67-72, 2002.
[2] Alexandre H. et al., FEBS Lett. 498(1) : 98-103, 2001.

overall_design:
We measure the changes in the gene expression of ethanol stressed culture at five different time-points during fed-batch fermentation compared to a common reference consisting of exponentially growing yeast cells ( sample number 1 : growing cells ; low ethanol concentration of  17 g/l ; specific growth rate of 0.3 h-1) . The sets corresponded to sample number 2 : growing cells/ethanol concentration of 60 g/l ; sample number 3 : before growth arrest/ethanol concentration of 90 g/l ; sample number 4 : growth arrest/ethanol concentration of 95 g/l ; sample number 5 : 1 hour after growth arrest/ethanol concentration of 100 g/l and sample number 6 : uncoupling phase/ethanol concentration of 125 g/l.
For each sample, total RNAs from one yeast culture (no biological replicate) were extracted three times (technical replicates -extract). For labelling, we labelled the common reference with dCTP-Cy5 and labelled the sample with dCTP-Cy3 and hybridized cDNA on three independent microarrays, given rise to six data value per gene (each gene is duplicate in the slide).

first: Marlène

last: Cot

first: Jean

last: François


title: Control HepG2 vs miR-24 HepG2 cells

city: Boston

first: Ashish

last: Lal

address: 260 WAB, 200 Longwood Ave

institute: Harvard University

summary: miR-24 targets were identified in this study using mRNA microarrays. Combined with Bioinformatics, our results show that miR-24 regulates cell cycle and DNA repair.

overall_design: Total RNA was isolated from C or miR-24 over-expressing HepG2 cells after 48 hr of transfection and microarray analysis was performed


title: The Expression Profiles of Zebra Mussel Byssus Genes along Byssogenesis

city: Chicago

first: Wei

last: Xu

state: IL

address: 303 E Chicago Ave, Tarry 4-714

department: Surgery

institute: Northwestern University, Feinberg School of Medicine

summary: To identify the expression profiles of zebra mussel byssus unique genes with the regeneration of the byssal threads, a time-course study was performed. The RNA samples from the feet of fully attached zebra mussels (maintained attachment for more than 4 weeks) were extracted as common reference. The detached mussels were allowed to re-attach on underwater surface. Then the RNA samples from feet of the mussels at different time points post detachment were taken, namely, 12 hours, 1 day, 2 days, 3 days, 4 days, 7 days, and 21 days post detachment. The comparisons of the genes expression profiles were made between different time points and reference, as well as the any two connected time points (expect for 7 days and 21 days).

overall_design: The dual-channel microarray hybridizations were performed as following: 12h→R, 12h←R, 1D→R, 1D←R, 2D→R, 2D←R, 3D→R, 3D←R, 4D→R 4D←R, 7D→R, 7D←R, 21D→R, 21D←R, 12h→1D, 12h←1D, 1D→2D, 1D←2D, 2D→3D, 2D←3D, 3D→4D, 3D←4D, 4D→7D, 4D←7D, 7D→21D, 7D←21D. The direction of the arrows stand for the way of labeling: Alexa555→Alexa647. Six biological replicates of 1D, 2D, 3D, 4D, and 7D were used; four biological repllicates of 12h and 21D were taken; fourteen biological replicates of reference were applied. The whole experiment includes 26 microarray slide hybridizations.

first: Mohamed

last: Faisal