title: Sex-dimorphic gene expression and ineffective dosage compensation of Z-linked genes in gastrulating chicken embryos

status: Public on May 13 2009

city: kansas city

first: Sachin

last: Mathur

state: MO

address: 5100 Rockhill Drive

department: School of Computing & Engineering

institute: University of Missouri Kansas City

summary: Sex determination is still poorly understood in birds and no key determinants have so far been identified. In contrast to most other species, dosage compensation of bird sex chromosomal genes appears rather ineffective. By comparing microarrays of microdissected primitive streak from single chicken embryos, we identified a large number of genes differentially expressed between male and female embryos at a very early stage (Hamburger and Hamilton stage 4), long before any sexual differentiation occurs. Most of these genes are located on the Z chromosome, which indicates that dosage compensation is ineffective in early chicken embryos. Gene ontology analyses using an enhanced annotation tool for Affymetrix probesets of the chicken genome shows that among the male-biased genes found on the Z chromosome, more than 20 genes have a role in sex differentiation.

overall_design: Primitive streak tissues were dissected out of individual Hamburger and Hamilton (HH) stage 4 chicken embryos and extracted for total RNA. Total RNA was amplified 2-cycles, biotin-labeled and hybridized to Affymetrix chicken GeneChip. Gene expression profiles of female and male samples were analyzed for sex-dimorphic expression.

first: Shaobing

middle: O

last: Zhang

first: Gaye

last: Hattem

first: Olivier

last: Tassy

last: Pourquié


title: Fathead minnow ovaries exposed in vivo or in vitro to fadrozole

city: Vicksburg

first: Natalia

last: Vinas

state: MS

address: 3909 Halls Ferry Rd

country: USA

institute: Mississippi State University

summary: Interpreting proteomic and genomic data is a major challenge in predictive ecotoxicology that can be addressed by a systems biology approach.  Mathematical modeling provides an organizational platform to consolidate protein dynamics with possible genomic regulation. Here, a model of ovarian steroidogenesis in the fathead minnow (Pimephales promelas) is developed to evaluate possible transcriptional regulation of the steroid production observed in microarray studies. Steroidogenesis, the production of sex steroids, is a highly conserved pathway whose regulation is critical to sexual reproduction. The model was developed from literature sources, integrating key signaling components (G-protein and PKA activation) with their ensuing effect on steroid production. The model properly predicted steroid behavior when fish were exposed to fadrozole, a specific aromatase inhibitor, but failed to predict testosterone and estradiol behavior during depuration. In vivo microarray data implicated three modes of regulation which may account for over-production of steroids during depuration: P450 enzyme up-regulation, inhibin down-regulation, or luteinizing hormone up-regulation. Simulation studies and sensitivity analysis were used to evaluate each of the three modes as possible sources of compensation.

overall_design: FHM 22,000 gene arrays (4 X 44k format) were manufactured by Agilent and were purchased from EcoArray (Alachua, FL). Array hybridizations were performed using a reference design, where each sample was compared to a common reference sample. The reference consisted of equal amounts of RNA from female and male FHM tissues (liver, brain and gonad). Four replicates consisting of four different individuals were analyzed for each of the treatments (24hr FAD in vivo and 12hr FAD in vitro along with corresponding controls). cDNA synthesis, cRNA labeling and amplification and hybridization were performed following the manufacturer’s kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA). Briefly, a primer containing poly dT and a T7 polymerase promoter was added to 500 ng of total RNA. Reverse transcriptase was added to the reaction to synthesize the first and second strands of cDNA. Next, cRNA was synthesized from the double-stranded cDNA using T7 RNA polymerase, which simultaneously incorporates cyanine 3- (Cy3) or cyanine 5- (Cy5) labeled CTP (Perkin-Elmer, Boston, MA). The ovary samples were labeled with Cy5 while the reference sample was labeled with Cy3. Once the labeling was complete, samples were hybridized to the microarray for 17 hours. The microarrays were washed and scanned with a laser-based detection system (Agilent, Palo Alto, CA).

first: Jason

middle: E

last: Shoemaker

first: Kalyen

last: Gayen

first: Edward

middle: J

last: Perkins

first: Daniel

middle: L

last: Villeneuve

first: Li

last: Liu

first: Francis

last: Doyle III


title: Genome-wide binding profiles of Drosophila melanogaster insulator proteins in Kc and Mbn2 cells

status: Public on May 15 2009

city: Atlanta

first: Ashley

middle: M

last: Bushey

state: GA

address: 1510 Clifton Rd. RRC 1071B

department: Biology

institute: Emory University

type: Genome binding/occupancy profiling by genome tiling array

summary: ChIP-chip experiments with NimbleGen whole-genome tiling arrays to compare Su(Hw), dCTCF, BEAF, and CP190 localization on DNA in Kc and Mbn2 cells revealed that BEAF is a third subclass of CP190-containing insulators.  The DNA binding proteins, Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization.  Notably, cell line specific localization sites for all three DNA binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression.

overall_design: Three replicates of Su(Hw), dCTCF, and CP190 ChIP-chip experiments in both Kc and Mbn2 cells as well as two replicates of BEAF ChIP-chip experiments in both Kc and Mbn2 cells were compared.

first: Victor

last: Corces

last: Ramos


title: Transcriptome analysis of Leishmania infantum PNA+ and PNA- promastigotes by complete shotgun DNA microarrays

status: Public on May 14 2009

city: MADRID

first: PEDRO

middle: JOSÉ

last: ALCOLEA ALCOLEA

address: Ramiro de Maeztu, 9

department: CENTRO DE INVESTIGACIONES BIOLÓGICAS

country: Spain

institute: CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS

summary:
Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in Mediterranean areas and also acts as an opportunistic parasite in HIV patients. Metacyclic promastigotes are transmitted during bloodmeals of the sand-fly host after development. Metacyclogenesis can be micmiked in axenic cultures and peanut lectin (PNA) agglutination followed by two-step centrifugation allows the separation of procyclic and metacyclic promastigotes in L. major. The purpose of this study is to isolate both fractions simultaneously from the same population of L. infantum in stationary phase of axenic culture and compare their expression profiles through DNA microarrays, specially focusing on metacyclic promastigotes.
Whole-genome shotgun DNA microarrays were constructed and used to analyse the stationary-phase procyclic and metacyclic expression profiles. Four biological replicates of the experiment were performed and analysed, so that 322 clones with meaningful values of stage-specific regulation were selected. We found several genes dealing with primary metabolism, differentiation in procyclic promastigotes and with development of infectivity in metacyclic promastigotes.
The differences we have found between the procyclic (PNA+) and metacyclic (PNA-) transcriptomes demonstrate that negative selection of metacyclic promastigotes through PNA agglutination is suitable in L. infantum and both fractions can be isolated. In addition, up-regulation of genes implied in lipophosphoglycan (LPG), proteophosphoglycan (PPG) and glycoprotein biosynthesis indicate that metacyclic promastigotes are related with infectivity.
Keywords: comparative hybridization between cDNAs from procyclic PNA+ and metacyclic PNA- promastigotes of L.infantum

overall_design:
-CONSTRUCTION OF SHOTGUN DNA GENOME LIBRARY IN PUC18 VECTOR
-PCR AMPLIFICATION WITH EXPAND LONG TEMPLATE. 29952 AMPLIFICATIONS
-CONTROL ITEMS: positive control genes from Leishmania sp., genomic DNA from L.infantum, herring sperm DNA; negative control genes (from the nif regulon) from Leptospirillum ferrooxidans (BACTERIA), buffer 1xSSC
-SPOTTING OF 30576 PCR PRODUCTS/CONTROL ITEMS: ARRAY CONSTRUCTION
-LEISHMANIA GROWTH CURVE AND PNA+/- SEPARATION BY CENTRIFUGATION AFTER AGGLUTINATION
-TOTAL RNA EXTRACTION, PURIFICATION, AMPLIFICATION AND CDNA SYNTHESIS/INDIRECT LABELLING
-HYBRIDIZATION AND SCANNING (DUAL CHANNEL)
-ANALYSIS: RAW DATA (GENEPIX 6.0)
-NORMALIZATION: LOWESS PER PIN (ALMAZEN SOFTWARE)
-COMPARATIVE EXPERIMENT OF THREE BIOLOGICAL REPLICATES BY T-TEST
-FILTERING OF INTERESTING SPOTS (THAT MAY CONTAIN DIFFERENTIALLY REGULATED GENES)
-CULTURES OF THE SELECTED CLONES, MINIPREPS AND SEQUENCING REACTIONS.
-ASSEMBLY OF SEQUENCES, BLAST, PROCESSING WITH A CUSTOM PERL SCRIPT AND MAPPING AGAINST THE GENOME OF L.INFANTUM SEQUENCED BY THE SANGER CENTER WITH A GBROWSE APPLICATION.
-CLUSTERING: GENE ONTOLOGY MOLECULAR FUNCTIONS

last: ALCOLEA

first: ANA

last: ALONSO

first: ALICIA

last: SÁNCHEZ-GOROSTIAGA

first: MERCEDES

last: MORENO-PAZ

first: MANUEL

last: GÓMEZ

first: IRENE

last: RAMOS

first: VÍCTOR

last: PARRO

first: VICENTE

last: LARRAGA


title: Gene Expression analysis of Kc and Mbn2 cell lines from Drosophila melanogaster.

summary: Comparison of gene expression between Kc and Mbn2 cell lines derived from Drosophila melanogaster.

overall_design: Microarray analysis was performed with two biological replicates for each cell line.  Each array contains 15,634 Drosophila genes in duplicate with 12, 60 mer probes per gene.


title: Expression in myocardial tissues of Great Dane dogs with cardiomyopathy

status: Public on May 12 2009

city: Rensselaer

first: Sridar

middle: V

last: Chittur

state: NY

address: One Discovery Drive, CRC 342G

department: Center for Functional Genomics

institute: SUNY-University at Albany

summary:
Oligonucleotide microarray analysis of left ventricular tissues from 3 Great dane dogs with dilated cardiomyopathy and 3 control dogs was performed.  Three hundred and twenty three differentially expressed transcripts were detected.  The transcript demonstrating the highest degree of upregulation was FKBP12.6 (calstabin2) and the transcript with the greatest amount of down regulation was triadin.  Both of these transcripts are critical to proper functioning of the cardiac ryanodine receptor.  Abnormalities of both calstabin2 and triadin have been reported in humans with heart disease.  Further investigation into the role of calstabin2 and triadin in canine cardiomyopathy is warranted, and the canine disease may serve as a model of human disease.

Keywords: control vs diseased

overall_design: 3 controls and 3 affected

first: Mark

middle: A

last: Oyama


title: Poorly differentiated, human liposarcoma LiSa-2 cell line response to the synthetic triterpenoid CDDO

city: Iowa City

first: Bartley

middle: Joseph

last: Brown

state: IA

institute: The University of Iowa

summary:
Analysis of poorly differentiated LiSa-2 liposarcoma (LS) cells treated with the synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) or DMSO or a combination of CDDO and DMSO for up to 11 days. Synthetic triterpenoids exert a variety of actions, including anti-proliferative and proapoptotic effects, making them potentially useful in cancer therapy. Results provide insight into the pathogenesis of LS.
Keywords: disease state analysis, CDDO effect, time course

overall_design: Temporal analysis of human liposarcoma cell line LiSa-2 subjected to treatment with a synthetic triterpenoid.


title: Eucalyptus xylem diurnal time series

city: Pretoria

first: Owen

middle: Luke

last: Solomon

state: Gauteng

address: Lab 6-20, Agricultural Building, University of Pretoria, Lynwood Rd

department: Genetics

country: South Africa

institute: University of Pretoria

summary: The daily cycle of night and day affects the behaviour and physiology of almost all living things. At the molecular level, many genes show daily changes in expression levels. To determine whether changes in transcript abundance occur in wood forming tissues of Eucalyptus trees we used a cDNA microarray to examine gene expression levels at roughly four hour intervals throughout the day.

sample_organism: Eucalyptus grandis x Eucalyptus urophylla

sample_organism: Eucalyptus camaldulensis x Eucalyptus grandis

platform_organism:
Eucalyptus grandis
Eucalyptus tereticornis

overall_design: Experiments were performed using RNA extracted from two biological replicates - GU (Eucalyptus grandis x E. urophylla) and GC (Eucalyptus grandis x camaldulensis) trees. A loop design was used, linking six time points. A dye swap was incorporated to eliminate dye bias.

first: Alexander

last: Myburg


title: Retroviral transduction of CD34+ cells with P190 and P210 BCR/ABL1 fusion genes

city: Lund

first: Petra

last: Johnels

address: Lund University Hospital

department: Clinical Genetics

country: Sweden

institute: Lund University

summary:
Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage

The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. We investigated and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under in vitro conditions favoring B-cell differentiation, neither P190 nor P210 BCR/ABL1-expressing cells formed detectable levels of CD19-positive cells. Gene expression profiling revealed that P190 BCR/ABL1 and P210 BCR/ABL1 induced almost identical gene expression profiles, and we identified a common set of 222 differentially expressed genes. Our data suggest that the early cellular and transcriptional effects of P190 BCR/ABL1 and P210 BCR/ABL1 expression are very similar when they are expressed in the same human progenitor cell population, and that STAT5 is an important regulator of BCR/ABL1-induced erythroid cell expansion.

Keywords: global gene expression profiling, BCR/ABL1, CD34+ cord blood cells, CML, Ph+ ALL

overall_design: The transcriptional effects of retroviral expression of P190 and P210 BCR/ABL1 in human cord blood (CB) CD34+ cells were compared to the expression profiles of CD34+ control cells transduced with an empty vector. Total RNA was isolated from sorted GFP+ cells isolated at day 2, 3, and 4 posttransduction in two independent experiments. RNA extraction, labeling, hybridization, washing, scanning, and feature analysis were performed as described (Järås et al., 2009, Exp Hematol).

first: Marcus

last: Järås

last: Håkansson

first: Helena

last: Ågerstam

first: Carin

last: Lassen

first: Marianne

last: Rissler

first: Patrik

last: Edén

first: Jörg

last: Cammenga

first: Tor

last: Olofsson

first: Ole

last: Weis Bjerrum

first: Johan

last: Richter

first: Xiaolong

last: Fan

first: Thoas

last: Fioretos


title: Profiling of chromosomal alterations in renal cell carcinoma using high-density single nucleotide polymorphism arrays

status: Public on May 09 2009

city: Houston

first: Xifeng

last: Wu

state: TX

address: 1155 pressler blvd.

department: Epidemiology

institute: UT M.D Anderson Cancer Center

summary: We applied Illumina’s 317K high-density SNP-arrays to profile chromosomal aberrations in clear cell renal cell carcinoma (ccRCC) from 80 patients and analyzed the association of LOH/amplification events with clinicopathological characteristics

overall_design: Log_R_Ratio and differece of Allele frequency were calculated by comparing the tumor sample to reference DNA, which is from the peripheral blood of corresponding patient

first: Meng

last: Chen

first: Yuanqing

last: Ye

first: Hushan

last: Yang

first: Pheroze

last: Tamboli

first: Surena

last: Matin

first: Nizar

last: Tannir

first: Christopher

middle: G

last: Wood

first: Jian

last: Gu


title: Affinity purification of ribosomes and associated RNAs using tagged Rpl16a and Rpl16b

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:
In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above.
A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species.

first: Regula

last: Halbeisen


title: Use of CGH microarrays to identify strain variable genomic regions in C. botulinum ATCC 3502

first: Brian

middle: H

last: Raphael

address: 1600 Clifton Road

department: Enteric Diseases Laboratory Branch

institute: Centers for Disease Control and Prevention

type: Genome variation profiling by genome tiling array

summary: Comparative genomic hybridization microarrays featuring overlapping probes spanning the entire C. botulinum type A1 strain ATCC 3502 genome were used to identify regions whose presence are variable among a diverse panel of type A C. botulinum strains.

overall_design: For each hybridization, the reference strain (C. botulinum ATCC 3502) was labeled with Cy5 and test strains were labeled with Cy3.

first: Lavin

last: Joseph

first: Loretta

last: McCroskey

first: Carolina

last: Luquez

first: Susan

last: Maslanka


title: Differentially Expressed Genes between Drought-tolerant and Drought-sensitive Barley Genotypes

city: Aleppo

first: Michael

last: Baum

state: Aleppo

address: ICARDA

department: BIGMP

country: Syria

institute: ICARDA

summary: Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at transcription levels in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme (NADP-ME) and pyruvate dehydrogenase (PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase (CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase (ALDH), ascorbate-dependant oxidoreductase (ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8 (HSP17.8) and dehydrin 3 (DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were likely constitutively expressed in drought-tolerant genotypes. Among them, 7 known annotated genes might enhance drought tolerance through signaling (such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP), anti-senescence (G2 pea dark accumulated protein GDA2) and detoxification (glutathione S-transferase (GST) pathways. In addition, 18 genes, including those encoding Δl-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C) and several chaperones, were differentially expressed in all genotypes under drought; thus, they were more likely general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley.

overall_design:
Seven flag leaves of a replication for each genotype were harvested at 0 d, 1 d, 3 d and 5 d after reach 10% of AWC in the soil to constitute a single biological replicate. These flag leaves were employed for RNA isolation by using Trizol reagent following the manufacturer’s protocol (Invitrogen, Karlsruhe, Germany). The RNA was further purified using RNeasy Kit (Qiagen, Hilden, Germany). RNA yield and quality were determined by using an Agilent 2100 Bioanalyzer (Agilent Techologies, Boblingen, Germany).
A table of the average, log2 RMA signal intensity values of three biological replicates for each Sample is linked below as a supplementary file.

first: Peiguo

last: Guo

first: Stefania

last: Grando

first: Salvatore

last: Ceccarelli

first: Guihua

last: Bai

first: Ronghua

last: Li

first: Maria

last: Korff

first: Rajeev

last: Varshney

first: Andreas

last: Graner

first: Jan

last: Valkoun


title: Systematic identification of balanced transposition polymorphisms in Saccharomyces cerevisiae

status: Public on May 07 2009

city: Chapel Hill

first: Jason

middle: D.

last: Lieb

state: NC

address: Department of Biology, #3280, Coker Hall

department: Biology and Carolina Center for Genome Sciences

institute: The University of North Carolina-Chapel Hill

summary: High-throughput techniques for detecting DNA polymorphisms generally do not identify changes in which the genomic position of a sequence, but not its copy number, varies among individuals. To explore such balanced structural polymorphisms, we used array-based Comparative Genomic Hybridization (aCGH) to conduct a genome-wide screen for single-copy genomic segments that occupy different genomic positions in the standard laboratory strain of Saccharomyces cerevisiae (S90) and a polymorphic wild isolate (Y101) through analysis of six tetrads from a cross of these two strains. Paired-end high-throughput sequencing of Y101 validated four of the predicted rearrangements. The transposed segments contained one to four annotated genes each, yet crosses between S90 and Y101 yielded mostly viable tetrads. The longest segment comprised 13.5 kb near the telomere of chromosome XV in the S288C reference strain and Southern blotting confirmed its predicted location on chromosome IX in Y101. Interestingly, inter-locus crossover events between copies of this segment occurred at a detectable rate. The presence of low-copy repetitive sequences at the junctions of this segment suggests that it may have arisen through ectopic recombination. Our methodology and findings provide a starting point for exploring the origins, phenotypic consequences, and evolutionary fate of this largely unexplored form of genomic polymorphism.

overall_design: Array CGH

first: Dina

last: Faddah

first: Eric

middle: W

last: Ganko

first: Caroline

last: McCoach

first: Joseph

middle: K

last: Pickrell

first: Sean

last: Hanlon

first: Frederick

last: Mann

first: Joanna

last: Mieczkowska

first: Corbin

middle: D

last: Jones

first: Todd

last: Vision


title: Effect of LacI (lac repressor) on transcription in Salmonella

city: Bangalore

first: Dipshikha

last: Chakravortty

state: Karnataka

address: C.V.Raman AVENUE

department: Microbiology and Cell Biology

country: India

institute: Indian Institute of Science

summary: Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonella’s close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid.  As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

overall_design:
Overall design	The total RNA was isolated from Salmonella using ‘RNeasy Mini Kit’ (QIAGEN).
Organism used: Salmonella enterica serovar Typhimurium  LT2
* Slides: Agilent’s arrays (8x15k) AMADID: NO: 17809
* Starting material: Salmonella RNA in nuclease-free water
* RNA Samples used:
Wild type (WT), WT + pTrc99A+LacI (PTRC), WT + pTrc99A-LacI(Delta lac)
* Labeling kit: MessageAmp™ II-Bacteria RNA Amplification Kit from Ambion Cat # AM1790
* Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA
* Total RNA and cRNA Purification Kit: Qiagen’s RNeasy minikit Cat#74104
* Hybridization Kit: Agilent’s In situ Hybridzation kit 5184-3568
* RNA quality was checked using Bioanalyzer.
The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with mutant and rescue experiments and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.

first: DrDipshikha

first: DrSandeepa

last: M E


title: Gene expression analysis during metamorphosis and the onset of calcification in the scleractinian coral M. faveolata

status: Public on May 06 2009

city: San Francisco

first: Michael

last: DeSalvo

state: CA

department: Department of Anesthesia

institute: UC San Francisco

summary: Similar to many marine invertebrates, scleractinian corals experience a dramatic morphological transformation, as well as a habitat switch, upon settlement and metamorphosis. At this time, planula larvae transform from non-calcifying, demersal, motile organisms into sessile, calcifying, benthic juvenile polyps. We performed a gene expression microarray analysis between planulae, aposymbiotic primary polyps, and symbiotic adult tissue to elucidate the molecular mechanisms underlying coral metamorphosis and early stages of calcification in the Robust/Short clade scleractinian coral Montastraea faveolata. Among the annotated genes, the most abundant upregulated transcripts in the planula stage are involved in protein synthesis, chromatin assembly and mitochondrial metabolism; the polyp stage, morphogenesis, protein catabolism and organic matrix synthesis; and the adult stage, sexual reproduction, stress response and symbiosis. Additionally, our results indicate that metamorphosis in M. faveolata planulae is likely regulated by: 1) a mechanism that resembles that described for hydrozoan cnidarians involving the neuropeptide LWamide; and 2) conserved cell adhesion and apoptosis mechanisms. Our results also suggest that calicoblast differentiation pathways may be regulated by transforming growth factors from the BMP family and Notch signalling pathway. We also present evidence showing that the planula and adult transcriptomes are more similar to each other than to the polyp trancriptome. Lastly, our results point to a large number of uncharacterized adult coral-specific genes likely involved in coral-specific functions such as symbiosis and calcification.

overall_design:
Collection of samples -- Montastraea faveolata gamete bundles and adult fragments were collected on 3 September 2007 during the annual spawning event in Puerto Morelos, Quintana Roo, Mexico. Gamete bundles were collected and fertilized as described in Miller and Szmant 2005. After 1 hour, excess sperm was rinsed away, and the eggs were examined for signs of cleavage.  Embryos were maintained in 1 μm filtered seawater, with water changes several times per day, until they reached the planula stage. At this point, a subset of planula larvae was collected and preserved in RNAlater (Ambion) for posterior RNA extraction and microarray hybridizations. Remaining planulae were transferred to polycarbonate culture bins and allowed to metamorphose and settle. After a week, settled polyps were collected and preserved in RNAlater. Three adult fragments were obtained using a hammer and chisel from one colony of M. faveolata at 2.7 m depth near Puerto Morelos (20o52’28.77”N and 86o51’04.53”W) on 31 July 2007. Fragments were transported back to the station and acclimated to a flow-through (~0.6 L/min) 50 L aquarium for 27 days. Mean temperature of the aquarium was 28.4 ± 0.9oC (as measured by a HOBO Light/Temperature Data Logger – Onset Corp.). The fragments were frozen in liquid nitrogen prior to and during transport to Merced, CA. All samples were exported to the USA through a CITES permit (MX-HR-007-MEX).

Microarray data analysis -- The experiment followed a loop design in which all life stages (i.e. planula, polyp, adult) were directly compared against each other.  Hybridizations were performed in triplicate (n=3 for each developmental stage) and included dye swapping between technical replicates. Pre-processing of microarray intensity data was performed using TIGR TM4 software. Briefly, spot-finding was performed in SpotFinder 3.11 with background-subtracted median intensities being extracted. Using MIDAS 2.19, the data were LOWESS normalized, and the dye swap pairs were collapsed (thus leaving an output of 9 data files collapsed from 18 total arrays). Secondly, the in-slide duplicates were collapsed (also in MIDAS 2.19). Resulting intensities in both channels (for nine total microarrays) were compiled into a spreadsheet (AR_matrix1.xls). Genes were included in subsequent statistical analysis only if there were data in two out of three replicates for each developmental stage. Of 1310 possible genes, 1243 passed filtering criteria. The ratio between the fluorescence intensity of the two channels was used as input for BAGEL (Bayesian Analysis of Gene Expression Levels). Normalized, filtered data is provided as  another matrix (AR_matrix2.xls). Note this file contains the ratio fo both channels NOT log2 transformed.

first: Alejandro

last: Reyes-Bermudez

first: Christian

middle: R

last: Voolstra

first: Shinichi

last: Sunagawa

first: Alina

last: Szmant

first: Roberto

last: Iglesias-Prieto

first: Monica

last: Medina


title: S. pyogenes wild type vs. CvfA- mutant

status: Public on May 08 2009

city: Carbondale

first: KyuHong

last: Cho

state: IL

address: 1125 Lincoln Dr. Room 131

department: Microbiology

institute: Southern Illinois University Carbondale

summary: The overall gene expression of a CvfA- mutant of Streptococcus pyogenes grown in C medium was compared to that of the wild type (HSC5spc).  Stationary phase cells (8 hrs post-inoculation) were used because the gene expression of the CvfA- mutant was most different from that of the wild type at stationary phase, while the difference of gene expression between the CvfA- mutant and the wild type was negligible at exponential phase.

overall_design:
Two biological replicates (two different RNA sets extracted from the CvfA- mutant and the wild type at different day were used): 399773,399887,399888 vs. 399889,399890,399891
Technical replicates (Microarrays were hybridized with the same RNA with the same dye): 399773 and 399887; 399889 and 399890
Dye swap (Microarrays were hybridized with the same RNA but Cy3 and Cy5 dyes were swapped): 399773, 399887 vs. 399888; 399889, 399890 vs. 399891

last: Caparon


title: microRNA expression and opioid signaling

status: Public on May 04 2009

city: Minneapolis

first: Xiaoxiao

last: Zhang

address: 312 Church St. SE

institute: University of Minnesota

summary:
To study the relationship between microRNAs and μ-opioid receptor (MOR) signaling, we examined microRNA expression after chronic morphine or fentanyl treatment in rat primary hippocampal neurons and in mouse hippocampus. Mouse cerebellum region was also tested as a negative control to eliminate microRNA expression changes unrelated to MOR signaling, as the cerebellum is essentially devoid of MOR.  We identified a number of microRNAs that altered their expression upon treatment with both morphine and fentanyl in the rat and mouse systems.  There were, however, some microRNAs that changed in response to morphine, or fentanyl, but not both.

Keywords: Expression profiling

platform_organism:
Caenorhabditis elegans
Drosophila melanogaster
Danio rerio
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: There are up to three biological replicates (indicated by 1, 2, and 3) of primary hippocampal neurons from new born rats and the cerebellum and hippocampus regions from adult mice treated for three days (control, morphine, and fentanyl).  The biological replicates were from experiments performed on different dates.  Each biological replicate contained cells or tissues collected from multiple animals so that enough RNA could be extracted for RNA analysis.  RNA was labelled with a green dye, mixed with a reference DNA sample labelled with a red dye.  The reference DNA contained a number of synthetic DNA oligos with mature microRNA sequences that served to verify microarray hybridization.  RNA signals were in ch1, DNA signals ch2.

first: Yan

last: Zeng

first: Hui

last: Zheng

first: Ping-Yee

last: Law


title: Wolbachia mCGH of Five Supergroup A Strains

status: Public on May 11 2009

city: Baltimore

first: Julie

middle: C

last: Dunning Hotopp

state: MD

address: 20 Penn St.; HSF-II; S203B

department: Institute for Genomic Sciences

institute: Univeristy of Maryland Baltimore

summary: Using microarray-based comparative genome hybridizations (mCGH), the genomic content of Wolbachia pipientis wMel from Drosophila melanogaster was compared to the Wolbachia from D. innubila (wInn), D. santomea (wSan), and three strains from D. simulans (wAu, wRi, wSim).

overall_design: Cy3- and Cy5-labeled probes were synthesized from a pool of three distinct GenomiPhi amplifications to reduce bias. One flip-dye experiment (two hybridizations) was performed with each of three independent pools, yielding a total of six hybridizations/strain. With 4-8 printed replicates per slide this yielded 24-48 replicated spots per gene to compare across the study. Ratios were normalized using iterative log mode centering. The geometric mean ratio was calculated for all good spots in each flip-dye experiment.

first: Nadeeza

last: Ishmael

first: Panagiotis

last: Ioannidis

first: Sarah

last: Biber

first: Joyce

last: Sakamoto

first: Stefanos

last: Siozios

first: Vishvanath

last: Nene

first: John

last: Werren

first: Kostas

last: Bourtzis

first: Seth

last: Bordenstein

first: Herve

last: Tettelin


title: Brucella melitensis global gene expression

city: College Station

first: Leslie

middle: Garry

last: Adams

address: 402 Raymond Stotzer Blvd.

department: Veterinary Pathobiology

institute: Texas A&M University

summary:
In this study we report that B. melitensis at the late logarithmic phase of growth are more invasive for HeLa cells than at mid logarithmic or stationary growth phases. Microarray analysis of B. melitensis gene expression identified 414 up- and 40 down-regulated genes in late-log growth phase compared to the stationary growth phase. The vast majority of the up-regulated genes in late-log cultures were those associated with DNA replication, transcription and translation, intermediate metabolism, energy production and conversion, membrane transport and cell envelope, biogenesis and outer membrane, while the down-regulated genes were distributed among several functional categories. This first Brucella global gene expression study provides novel information on growth phase-specific gene regulation important not only for understanding Brucella physiology but also the initial molecular interactions between Brucella and its host.
Keywords: Comparison bacterial growth phase normalized to genomic DNA

overall_design: There are two kind of samples consisting of RNA isolated from Brucella melitensis grown logarithmically or at stationary phase.  There are four biological replicates of each sample.  Every Brucella melitensis open reading frame was printed in triplicate on each microarray, thereby providing three technical replicates for each biological replicates.  Each replicate was normalized against labeled Brucella melitensis genomic DNA.

first: Carlos

last: Rossetti

first: Cristi

last: Galindo

first: Sara

last: Lawhon

first: Thomas

last: Ficht

first: Harold

last: Garner

first: L


title: Reproducibility of Quantitative RT-PCR Array in miRNA Expression Profiling and Comparison with Microarray Analysis

city: San Antonio

first: Jonathan

middle: Adam

last: Gelfond

address: Mail Code 7933, 7703 Floyd Curl

department: Epidemiology & Biostatistics

institute: UT Health Science Ctr San Antonio

type:
Other
Non-coding RNA profiling by array

summary: High reproducibility with TaqMan microRNA array (qPCR-array) was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array.

overall_design:
Replicate preparations (n =4) using different aliquots of a single C2C12 RNA sample (500 ng) were used to determine the reproducibility of the reverse transcription process as well as the results of the same reverse transcription products performed on different days. TaqMan microRNA Array A was used for this purpose.
To assess the reliability of pre-amplification, miRNA expression profiles obtained with and without pre-amplification using MiRNA TaqMan Array B were performed.
Independent miRNA expression profiling studies using uParaflo microfluidic biochips were performed by an independent company to determine the relationship between results obtained with qPCR-array and microarrays. Aliquots of the same C2C12 RNA were used in both the qPCR-array (500 ng) and microarrays (8 µg).

first: Yongxin

last: Chen


title: Whole Genome Expression Profile of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro

status: Public on May 01 2009

city: Silver Spring

first: Rasha

last: Hammamieh

address: 503 Robert Grant Ave

department: Molecular Pathology

institute: Walter Reed Army Institute of Research

summary: hole Genome Expression Profile of Human Peripheral Blood Mononuclear cells Exposed to Bacillus anthracis in vitro. Peripheral blood mononuclear cells exposed to a 1 MOI (multiplicity of infection pathogenic) of the B. anhracis spores.

first: Marti

last: Jett


title: Telomere behavior in a hybrid yeast

city: Seattle

first: Maitreya

middle: J.

last: Dunham

state: WA

address: Foege Building, S403B, Box 355065

department: Genome Sciences

institute: University of Washington

summary: Telomeres and the protein/RNA complexes involved in maintaining them are rapidly evolving systems across eukaryotes. Using two Saccharomyces species, S. cerevisiae and S. bayanus, we provide evidence that the telomere systems of these two closely related yeasts have evolved significantly apart and that the gene in one species can not maintain the set-point of telomere length of the other species in the hybrid.

sample_organism: Saccharomyces bayanus x Saccharomyces cerevisiae

platform_organism:
Saccharomyces bayanus
Saccharomyces cerevisiae

overall_design: Each array has co-hybridized RNA vs DNA for the indicated species (BB9=S. bayanus, CC5=S. cerevisiae) or hybrid (BC11).  The cultures for RNA preparation were grown to midlog phase at the indicated temperature.

first: Yixian

last: Zheng

first: Ona

last: Martin

first: Christopher

last: DeSevo

first: Douglas

last: Koshland

first: Benjamin

last: Guo

middle: J


title: Comparative analysis of transcripts associated with all-stage resistance to stripe rust in wheat

city: Raleigh

first: Tristan

last: Coram

address: 4118 Williams Hall, NC State University

institute: USDA-ARS, NC State University

summary:
Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a destructive disease of wheat worldwide. Genetic resistance is the preferred method for controlling stripe rust, of which two major types are race-specific and race non-specific resistance. Race-specific resistance includes the qualitatively inherited all-stage resistance, controlled by single major resistance (R) genes. Conversely, adult-plant resistance is race non-specific, inherited quantitatively, and is durable. Previously, we characterized the gene expression signatures involved in Yr5-controlled all-stage resistance and Yr39-controlled adult-plant resistance using the Affymetrix Wheat GeneChip. For this study, we designed and constructed custom oligonucleotide microarrays containing probes for the 116 and 207 transcripts that we had found important for the Yr5 and Yr39 resistance responses, respectively. We used this custom microarray to profile the resistance responses of eight wheat genotypes with all-stage resistance (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15, and Yr17). The aim of this analysis was to identify common and unique gene expression signatures involved in race-specific resistance accross genotypes, which were used to infer information regarding the general pathways involved in all-stage resistance.

Keywords: Stress response

overall_design: Eight wheat genotypes with defined single gene all-stage resistance to particular stripe rust (Pst) isolates were selected for this study, as well as the Pst susceptible genotype ‘Avocet Susceptible’ (AVS). Near Isogenic Lines (NILs) for each of eight Yr resistance genes (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15 and Yr17) were developed at the Plant Breeding Institute, Sydney, Australia, by backcrossing the Yr gene donors with the recurrent susceptible spring wheat genotype (Triticum aestivum L.) AVS six times (AVS*6/Yr*) and selecting for the appropriate resistance at each generation. Both virulent and avirulent isolates of Pst were selected for each NIL, except for Yr15, for which no virulent isolate is currently known. Care was taken to select the fewest isolates needed to satisfy the spectrum of virulence/avirulence required, resulting in the use of six isolates identified as PST-17, PST-21, PST-43, PST-45, PST-78 and PST-AUS. Each isolate was selected and maintained on susceptible genotypes. For each of three biological replications, individual genotypes were planted in separate 25 X 42.5-cm flats using a potting mix (6 peat moss: 4 vermiculite with lime: 3 sand: 3 commercial potting mix: 2 perlite: 1.7 g/L lime: 3.3 g/L Osmocote: 2.2 g/L ammonium nitrate). Each flat consisted of three rows of six seedlings, with rows randomly assigned one of two harvest times (24 and 48 h post-inoculation). Seedlings from the 3rd row were used to monitor the expected disease responses to inoculation. Seedlings were grown to the second leaf stage (Feekes 1.2, emergence with second leaf unfolded, ~10 days after planting) in a greenhouse with a diurnal temperature cycle of 10ºC (2:00 am) to 25ºC (2:00 pm) and a 16 h light/8 h dark cycle. Inoculation was performed by misting the plants with sterile water and applying a 1:20 urediniospore:talc mixture to leaves with a sterile brush. Talc was used to aid in the spread and adhesion of spores over leaf surfaces. Control flats were treated the same way except for the absence of spores in the talc. All treatments for each biological replication were performed at 9 am Pacific Standard Time. To promote spore germination, all flats were transferred to a dew chamber (100% RH) operating at 10ºC in the dark for 24 h, before being placed in a growth chamber with a diurnal temperature cycle of 4ºC (2:00 am) to 20ºC (2:00 pm) and a 16 h light/8 h dark cycle. Rows of plants were harvested from all flats at the assigned times for RNA extraction.

first: Xueling

last: Huang

first: Lisa

last: Orfe

first: Doug

last: Call

first: Xianming


title: Delayed cell cycling in DS Ts1Cje neural precursor cells results in gene expression dysregulation

status: Public on Apr 30 2009

city: Paris

first: Luce

last: Dauphinot

address: 47 boulevard de l'hôpital

department: ICM

country: France

institute: INSERM U1127-CNRS UMR7225-UPMC

summary:
Down syndrome neurophenotypes are characterized by mental retardation and a decreased brain volume. In order to identify whether deficits in proliferation, differentiation or survival could be responsible for this phenotype, neural precursor cells (NPCs) were isolated from the developing E14 neocortex of Down syndrome partial trisomy Ts1Cje mice and euploid (WT) littermates. Proliferation, cell differentiation and cell death assays revealed that Ts1Cje NPCs proliferated at a slower rate, due to a longer cell cycle and that a greater number of cells were positive for glial fibrillary acidic protein. An increase in Ts1Cje NPC cell death was also noted. Gene expression profiling was conducted on RNA extracted from Ts1Cje and WT NPCs. Approximately 54% of triploid gene expression ratios were significantly greater than the expected diploid gene ratio of 1.0. A number of diploid genes associated with differentiation, glial function and proliferation were dysregulated. The evidence points to a delay in cellular cycling that could exert stress on the NPC population, which might result in cellular death and a mobilization of glial cell survival responses. Importantly, these phenotypic changes, which mimic those seen in Down syndrome individuals, do not require over-expression of amyloid precursor protein (App) or soluble superoxide dismutase 1 (Sod1). In conclusion, early developmental proliferation deficits in Down syndrome result in secondary morphological changes that can impact on cognitive development and function.
Keywords: Down syndrome, Neocortical precursor cells, transcriptome, proliferation

overall_design: Neural precursor cells (NPCs) were isolated from mouse E14 neocortex of Down syndrome Ts1Cje mice and euploid littermates. We compared gene expression profiles from trisomic and wild-type cells using pangenomic microarrays.

first: Randal

last: Moldrich

first: Julien

last: Laffaire

first: Tania

last: Vitalis

first: Yann

last: Hérault

first: Philip

last: Beart

first: Jean

last: Rossier

first: Stylios

last: Antonarakis

first: Corinne

last: Gehrig

first: Robert

last: Lyle

first: Marie-Claude

last: Potier


title: Physiological stability of Bifidobacterium longum NCC2705 under continuous culture conditions

city: Zurich

first: Valeria

last: Mozzetti

address: Schmelzbergstrasse 7

department: D-AGRL

institute: ETH

summary:
A continuous culture of Bifidobacterium longum NCC2705 was carried out in a 2.5-l reactor (Bioengineering AG, Wald, Switzerland), equipped with a Biospectra control system (Biospectra AG, Schlieren, Switzerland) and containing 2 l of MRS, added of 0.05% cysteine, inoculated with 2 % (v/v) preculture. The temperature was maintained at 37°C and the pH at 6.0 by addition of 5 M NaOH. The culture was stirred constantly at 250 rpm using two rushton type propellers. Anaerobic conditions were maintained by flushing the headspace of the reactor with CO2. After 8 h in batch mode the culture was run in continuous mode at a dilution rate of 0.1 h-1. Fresh medium was added using a peristaltic pump (Alitea, Bioengineering AG, Wald, Switzerland), and fermented broth harvested with a second peristaltic pump (Alitea, Bioengineering AG, Wald, Switzerland) set at a slightly higher flow rate. A stabilization period of 90 h (corresponding to nine reactor volume changes) was operated prior culture monitoring (t=0). Aliquots of 2 ml taken at t=31, 134 and 211 h were centrifuged (4,000 g, 1 min, room temperature) for transcriptomic analysis. Supernatants were discarded and cell pellets snap frozen in liquid nitrogen and stored at -80ºC until RNA-extraction.

Keywords: Time course of Bifidobacterium longum in continuous culture

overall_design: Bifidobacterium longum NCC2705 at time 31 versus time 134 h and versus time 211 h in continuous culture. Two technical replicares with dyes swaps

first: Deborah

last: Moine

first: Bernard

last: Berger


title: flagellar regulon studies

city: Madison

first: Kai

last: Zhao

state: WI

address: 2100 University Avenue

department: Oncology

institute: Mcardle Laboratory for Cancer Research

summary:
This study examined the genes under the control of FlhDC and sigmaF in E. coli.
Keywords: wild-type, deletion and overexepression strains

overall_design: Under defined steady-state growth condition, we used two different genetic approaches to alter the modulator concentration in cells; (1) moderately expressing FlhDC or sigmaF from anhydrotetracycline (aTc) inducible and Tet repressor-controlled PLtet promoter in a plasmid-borne flhDC or fliA gene; (2) disrupting the expression of FlhDC or sigmaF in flhDC or fliA deletion mutant strains. Samples were taken from culture with wild-type or deletion strains at mid log phase (OD=0.2) or from overexpression strains at mid log phase (OD=0.2) before or 5 minutes after moderate induction. Samples were then RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChip E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).

first: Mingzhu

last: Liu

first: Richard

last: Burgess


title: Trans. Profiling of Immortalized Melanocyte Cell Lines Identifies GPNMB as a Melanoblast-Expressed, MITF-Dependent Gene

city: Bethesda

first: Christopher

last: Pan

address: Rm 5228, Bldg 50, South Dr.

department: NHGRI

institute: NIH

summary:
Gene expression profiling of tissues derived from different genetic backgrounds can provide insight to potential phenotype-related changes in gene expression.  We have compared wildtype C57BL/6 (melan-Ink4a) melanocyte cell gene expression to that of, “brown“ Tyrp1b/ b (melan-Ink4a-b) and “chocolate” Rab38 cht/cht (melan-cht) melanocyte derived lines. We observed alterations in gene expression consistent with a physiological response of melan-Ink4a-b cells to endoplasmic reticulum protein-folding impairment. Additionally, a set of loci was identified that are expressed in a similar manner to known melanocyte genes. Importantly, the collection of 254 genes is enriched for expression in the multiple melanocyte lines relative to control cell populations. This set contains Tyr, Tyrp1, Dct, Si, Melna, Slc24a5, Slc45a2, Slc24a4, Mitf, and Sox10, in addition to genes not previously attributed to pigmentation and genes with unknown function. Further evaluation of one gene in this cohort, Gpnmb, demonstrated an expression pattern similar to Dct, Si and Mitf. This expression is vastly reduced in MitfMi mutants embryos, indicating that it is dependent on Mitf for in vivo expression. Consistent with this notion, a highly-conserved MITF binding site resides directly upstream of human GPNMB, and deletion of this sequence dramatically reduces in vitro enhancer activity.
Keywords: NIH embryo fibroblast cell line, melanocyte cell line, Ink4a, Tyrp1b, Rab38

overall_design: A 32k expression arrays were generated by printing Oligo nucleotides from Operon Mouse oligo set (MV3) onto epoxy slides. Total RNA from immortalized melanocyte cell lines derived from C57BL/6J Ink4a-/- (melan-Ink4a-1), C57BL/6J Ink4a-/-; Tyrp1b/b (melan-Ink4a-b) and 2 separate C57BL/6J Ink4a-/-; RaB38cht/cht lines (melan-cht4),  (melan-cht5) and NIH3T3 were labeled with Cy5/3 and co-hybridized with a total RNA universal reference (Stratagene, Inc) labeled with Cy3/5. Each comparison was done in Triplicate including one dye swap.

first: Stacie

last: Loftus

first: Anthony

last: Antonellis

first: Ivana

last: Matera

first: Gabriel

last: Renaud

first: Laura

middle: L

last: Baxter

first: Duncan

last: Reid

first: Tyra

last: Wolfsberg

first: Yidong

last: Chen

first: ChenWei

last: Wang

first: Megana

last: Prasad

first: Seneca

last: Bessling

first: Andrew

middle: S

last: McCallion

first: Eric

last: Green

first: Dorothy

last: Bennett

first: William

last: Pavan


title: Barley root gene expression during colonisation by Piriformospora indica

status: Public on Apr 29 2009

city: Giessen

first: Patrick

last: Schaefer

address: Heinrich-Buff-Ring 26-32

country: Germany

institute: Institute of Phytopathology and Applied Zoology

summary: The fungal mutualist Piriformospora indica is colonising barley roots thereby mediating various beneficial effects to its host. The interaction is characterised by an initial biotrophic interaction stage which is followed by a cell death-dependent colonisation phase. We used microarrays to identify the global programme of gene expression during the colonisation process of barley roots by P. indica and  to obtain informations into plant defense and metabolic reprogramming.

overall_design: In three independent experiments plants were inoculated with Piriformospora indica. Samples from inoculated roots were taken at 1, 3, and 7 days after inoculation. Samples from uninfected control plants were taken at the same time points.


title: Transcriptome Analysis of Cold Acclimation in Barley Albina and Xantha Mutants1

city: Riverside

first: Livia

last: Tommasini

address: 2150 Batchelor Hall

department: Botany and Plant Science

institute: University of California, Riverside

summary:
we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes
Keywords: stress response

overall_design: five barley genotypes with two treatments and three replicates were analysed

first: Jan

middle: T

last: Svensson

first: Cristina

last: Crosatti

first: Chiara

last: Campoli

first: Roberto

last: Bassi

first: Antonio

middle: M

last: Stanca

first: Timothy

last: Close

first: Luigi

last: Cattivelli


title: Transcriptome analysis of barley anthers: effect of mannitol treatment on microspore embryogenesis

summary:
we analyzed the anther transcriptome before and after 4 days of mannitol treatment using the 22k Barley1 GeneChip
Keywords: stress response

overall_design: one genotype (DH line 46), two treatments (4 days of mannitol treatment and control) and three repeats each treatment

first: Maria

last: Munoz-Amatriain

first: Ana-Maria

last: Castillo

first: Luis

last: Cistue

first: Maria-Pilar

last: Valles


title: Expression data from renal primary epithelial cells

city: Mannheim

first: Li

last: Li

address: Theodor-Kutzer-Ufer

institute: University Heidelberg

summary:
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in the ADPKD genes PKD1 and PKD2 account for nearly all the cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1, the product of PKD1, regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can acts as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. In this study we utilized different kidney cell lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 activity and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of STAT-1/p21 pathway. Genome-wide expression analysis in these cells revealed that expression of the cyclin-dependent kinase inhibitor, p57 is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.   Our results indicate the probable involvement of p57 on epithelial cell proliferation in polycystic kidney disease. In addition, it confirms that PKD2-induced proliferation is a multifactorial process.
We used microarrays to study the modulation of gene expression by mutated PKD2 in the primary tubular epithelial cells of a transgenic rat mode

Keywords: transgene

overall_design: Gene expression in the primary tubular epithelial of a transgenic rat mode with mutated PKD2 was compared with that of SD control rats

first: Kyriacos

middle: N

last: Felekkis

first: Panayiota

last: Koupepidou

first: Evdokia

last: Kastanou

first: Chang

middle: X

last: Bai

first: Ralph

last: Witzgall

first: Leonidas

last: Tsiokas

first: Norbert

last: Gretz

first: Constantinos

last: Deltas


title: Low temperature stress in cv. Dicktoo

first: Livia

last: Tommasini

summary:
In this study we used the Affymetrix Barley 1 GeneChip to investigate transcriptome responses of barley cv. Dicktoo to low temperature, including triplicated measurements of cold, freeze/thaw cycles and de-acclimation over 33 days.
Keywords: stress response

overall_design: Plants were grown at 20ºC for seven days and subject to a symmetrical cycle of acclimation, cold, freeze-thaw, and deacclimation. Chilling began by decreasing the temperature overnight from 20ºC to 4ºC at a rate of 1.3ºC•h-1 and maintaining temperatures of 4 ºC in the day and 2ºC at night for 5 days. Freeze-thaw cycling lasted 12 days with day temperatures of 4ºC and night temperatures gradually decreasing from -2ºC the first night to -4ºC for three nights and -10ºC for four nights, then recovering to -4ºC for three nights and -2ºC for one night. This treatment was designed to allow daily freeze-thaw cycling and protein synthesis. Chilling conditions (4ºC day, 2ºC night) were resumed for five days, followed by deacclimation with increasing temperature to 20ºC overnight and maintaining for three days. Sampling was done at four different times, each at the 11th hour of light to avoid circadian effects: 1) before chilling treatment, 2) five days after initiation of chilling treatment, 3) eight days into freeze-thaw treatment and 4) three days into de-acclimation.

first: Jan

last: Svensson

first: Edmundo

last: Rodriguez

first: Tim


title: ABA experiment

summary:
In this study we used the Affymetrix Barley 1 GeneChip to investigate transcriptome responses of barley cv. Morex to ABA treatment, at two time points, each including triplicated measurements
Keywords: ABA response

overall_design: Plants were grown at 20ºC for seven days.

last: Close


title: Genome Variation in the Symbiotic Nitrogen-Fixing Bacterium Sinorhizobium meliloti

city: Hamilton

first: HONG

last: GUO

state: Ontario

address: 1280Main St. West

department: BIOLOGY

country: Canada

institute: MCMASTER UNIVERSITY

summary:
Differences in genome size and gene content are among the most important signatures of microbial adaptation and genome evolution. Here, we investigated the patterns of genome variation among strains of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Using the sequenced strain Rm1021 as a reference, the genome size and gene content variations were analyzed among ten diverse natural strains, through pulse field gel electrophoresis (PFGE) and whole-genome microarray hybridizations. Our PFGE analysis showed a genome size range of 6.45-7.01Mbp, with the greatest variation arising from the pSymA replicon, followed by that of pSymB. No observable size difference was evident among the chromosomes. Consistent with this pattern of size differences, 41.2% of ORFs on pSymA were variably absent/present, followed by 12.7% on pSymB, and 3.7% on the chromosome. However, the percentages of ORFs that were variably duplicated were more evenly distributed among the three replicons, 11.0%, 16.5% and 15.3% respectively for ORFs on pSymA, pSymB and the chromosome. Among the 10 strains, the percentages of absent ORFs ranged from 1.51% to 6.35% and those of duplicated ORFs ranged from 0.27% to 8.56%. Our analyses showed that host plants, geographic origins, multilocus enzyme electrophoretic types, and replicon sizes had little influence on the distribution patterns of absent or duplicated ORFs. The proportions of ORFs that were either variably absent/present or variably duplicated differed greatly among the functional categories, for each of the three replicons as well as for the whole genome. Interestingly, we observed positive correlations among the three replicons in their numbers of absent ORFs as well as the numbers of duplicated ORFs, consistent with coordinated gene gains/losses in this important bacterium in nature.
microarray:Sm6kOligo

overall_design: A total of 12 strains were included for the microarray analyses in this study. The 12 strains included two reference strains and ten other natural strains. Reference strain Rm1021 was the strain with a completely sequenced genome, from which the whole genome microarray was based on (Krol et al. 2004). The second reference strain RmF909 was derived from strain Rm1021 but with a deletion of 575 ORFs located on the megaplasmid pSymB (genomic location 106724-735431, Charles et al. 1991). The two reference strains were used for positive and negative controls in our microarray hybridization experiments and for establishing threshold values for determining whether an ORF is present or absent among natural strains.

first: Hong

last: Guo

first: Sheng

last: Sun

first: Bertrand

last: Eardly

first: Turlough

last: Finan

first: Jianping

last: Xu


title: NFkB dependent gene expression in a Mdr2-KO hepatocellular carcinoma (HCC) mouse model using a IkB-Superrepressor

city: Stanford

first: Daniel

last: Haag

state: Santa Clara

address: 265 Campus Drive

department: ISCBRM

institute: Stanford School of Medicine

summary:
This study aims on the identification of the NF-kB dependent gene regulatory network during inflammation-associated liver carcinogenesis using the well-established Mdr2 knockout mouse model. We could identify 367 differentially expressed genes comparing expression profiles of tumor samples from Mdr2-KO mice to tumor samples derived from mice with an additional hepatocyte specific expression of an IkB-superrepressor. This IkB-superrepresser is undegradable upon ubiquitinylation initialized by Ikk dependent phosphorylation and therefore impedes NFkB activity.
Keywords: NFkB, IkB-superrepressor, inflammation-associated liver carcinogenesis, Mdr2-KO mouse model, hepatocellular carcinoma, gene expression, microarray

overall_design: In the experiments shown here, we compare gene expression profiles of HCCs from 4 idividual mice harboring a biallelic Mdr2 knockout to HCC specimen of 6 individual mice with Mdr2 knockout and a hepatocyte specific expression of the IkB-superrepressor. Each individual sample was hybridized aginst a universal reference mouse pool (Stratagene) on a two-color microarray. Furthermore was each sample/reference hybridization conducted as a dye swap experiment yielding two technical replicates with inverted dye channel orientation per sample. In addition a biological control using wildtype liver tissue from mice of the same genetic background was hybridized the same way aginst a universal reference. The latter was used to build a sample/control ratio for displays of indirect comparison.

first: Julia

last: Németh


title: Methylation detection Oligonucleotide Microarray Analysis: high resolution method for CpG island methylation detection 2

status: Public on Apr 28 2009

city: Cold Spring Harbor

first: Robert

last: Lucito

address: 1 Bungtown Road

department: Genome Center

institute: Cold Spring Harbor Laboratory

type: Methylation profiling by genome tiling array

summary: Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25,000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.

overall_design: We have developed a method to profile genome wide methylation. 7 ovarian normal samples and 11 tumor samples from other individuals were analyzed for CpG methylation. After inter array normalization, the tumor samples were taken together and the methylation compared to that of the normal samples to identify regions of the CpG islands that are significantly altered between the two datasets. Some of these regions were validated for their methylation as a proof of principle for the method.

first: Chunlao

last: Tang

first: James

last: Byrnes

last: Hicks

first: Michael

last: Riggs

first: Sitharthan

last: Kamalakaran


title: Genome wide screen of estrogen receptor α regulated microRNAs reveals a negative autoregulatory feedback loop

city: London

first: geraint

last: barton

address: South Kensington Campus

department: Bioinformatics Support Service

country: United Kingdom

institute: Imperial College

summary: Estrogen receptor alpha (ERα) is a nuclear receptor linked to progression of the majority of human breast cancers. Following activation ERα regulates the transcription of target genes via DNA binding. Through a genome wide approach we have identified a subset of microRNAs (miRNAs or miRs) modulated by ERα. Among them, miRNAs encoded from 2 paralogous clusters, miR-17-92 and miR-106a-363, were up-regulated. In addition, increased expression of miR-424, miR-542-3p and miR-450, located within the same genomic region and down-regulation of miR-181 family members were observed during the estrogenic response. We observed that ERα alone is sufficient for sustained transcription of miR-17-92 and we show that levels of this cluster increase earlier than miRNAs encoded by it, implicating post-transcriptional regulation. Since miR-17-92 a pri-miRNA, is immediately cleaved by DROSHA to pre-miR-18a, this regulation occurs during the formation of the mature molecule from the precursor. Moreover, pre-miR-18a was significantly up-regulated in ERα-positive breast cancers compared to ERα-negative breast cancers. We also demonstrate that the miRNAs belonging to these paralogous clusters target and down-regulate ERα, while a subset of cluster-derived miRNAs inhibit protein translation of its major oncogenic transcriptional p160 co-activator by targeting AIB1 revealing a negative autoregulatory feedback loop. This pathway adds to the highly regulated cellular response to estrogen that fine tunes ERα transcriptional activity and cell proliferation.

overall_design: 4 time points (0hr,3hr,6hr,12hr) and 4 treatments (TO,JP,TO+TET,JP13+TET) x 3 biological replicates of each condition = 24 arrays

first: Leandro

last: Castellano

first: Georgios

last: Giamas

first: Jimmy

last: Jacob

first: Walter

last: Lucchesi

first: Paul

last: Thiruchelvam

first: Geraint

last: Barton

first: Long

last: Jiao

first: Robin

last: Wait

first: Jonathan

last: Waxman

first: Justin

last: Stebbing


title: Methylation detection Oligonucleotide Microarray Analysis: high resolution method for CpG island methylation detection 1

last: Lucito

overall_design: We have developed a method to profile genome wide methylation. 12 breast normal samples and matching tumors from these individuals and an additional 28 tumor samples from other individuals were analyzed for CpG methylation. After inter array normalization, the tumor samples were taken together and the methylation compared to that of the normal samples to identify regions of the CpG islands that are significantly altered between the two datasets. Some of these regions were validated for their methylation as a proof of principle for the method.

first: Tang

last: Chunlao

first: Byrnes

last: James

first: Hicks

first: Riggs

last: Michael

first: Kamalakaran

last: Sitharthan


title: DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro.

city: San Jose

first: Jomar

middle: Patrício

last: Monteiro

address: 2260 Clove Drive

department: Division of Infectious Diseases and Geographic Medicine

institute: Stanford University

type: Expression profiling by genome tiling array

summary: Paracoccidioides brasiliensis is a thermally dimorphic fungus, which causes the most prevalent systemic mycosis in Latin America.  Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form.  Information regarding gene expression by either form has been rarely addressed with respect to multiple time points of growth in culture.  Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the organism’s genome, and its utilization in identifying genes and gene expression patterns during growth in vitro.  Cloned, amplified inserts from randomly sheared gDNA and known control genes were printed onto glass slides to generate a microarray of over 12,000 elements.  To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days.  Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from mycelia, especially at earlier time-points, and that mycelial gene expression changed less than it did on yeasts over time.  Genes, upregulated in yeasts, were found to be involved in methionine/cysteine metabolism, respiratory processes, metabolic processes (sugars, amino acids, proteins and lipids), transporters (small peptides, sugar, ions and toxin), regulatory proteins and transcription factors.  Mycelia genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transporters showed differential expression.  Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence.  We also analyzed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes coding for ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast.  These findings expand knowledge about the different morphologic forms of P. brasiliensis during growth in culture.

overall_design:
Briefly we aimed at generating and comparing gene expression patterns of P. brasiliensis mycelia and yeasts during growth in liquid modified McVeigh/Morton media for 5, 8 and 14 days.
We extracted total RNA from two cultures per time point for each morphologic form.
Hybridizations were done twice for each total RNA sample.
We used sheared genomic DNA as reference sample (Cy3) on every slide.
Normalization was done as follows:
Raw data underwent global normalization using the R statistical package to perform quantile normalization of the global reference channel by computing the centroid of all global reference signatures, and performing a loess fit of each individual global reference signature to the centroid.  The loess curve for a given sample was then used to scale both channels for each gene in that sample.
After that data was loaded in GeneSpring GX 7.3.1 and underwent the following normalization steps:
-per spot to the gDNA channel
-per slide using 102 positive controls (listed below)
-per gene to each gene’s respective median

Positive controls:
JM_2d11
JM_5a12
JM_22h5
JM_120a12
JM_61c4
JM_15g2
JM_13g9
JM_27c1
JM_55a6
JM_42a8
JM_14e1
JM_66c4
JM_13a9
JM_109e5
JM_20c1
JM_100a11
JM_62d8
JM_105c11
JM_22e8
JM_6a7
JM_66a5
JM_1e7
JM_42f5
JM_99h9
JM_9c7
JM_123g1
JM_33e5
JM_112a2
JM_25g6
JM_114h6
JM_98b1
JM_38h9
JM_30e11
JM_10e10
JM_22g11
JM_120c9
JM_120g4
JM_87g2
JM_70e12
JM_119e12
JM_79b12
JM_99a8
JM_67b5
JM_41b11
JM_76a2
JM_131e11
JM_65c2
JM_104e12
JM_26h3
JM_18c11
JM_56a7
JM_111c8
JM_21c9
JM_5e1
JM_3g12
JM_64e7
JM_115e5
JM_74c5
JM_117a5
JM_17b12
JM_10a5
JM_118e2
JM_15e5
JM_73e8
JM_89c6
JM_1c5
JM_5c8
JM_2e1
JM_83f10
JM_81e10
JM_83f11
JM_116a3
JM_5b11
JM_4c4
JM_86g6
JM_66e10
JM_44a6
JM_22a1
JM_22b4
JM_16a2
JM_22b5
JM_79g2
JM_26a11
JM_94b5
JM_25c2
JM_17e3
JM_66g1
JM_54a9
JM_84a11
JM_74b11
JM_115c6
JM_14a6
JM_122c9
JM_133e9
JM_11b9
JM_51b4
JM_24c7
JM_52b2
JM_66f3
JM_10c8
JM_57c1
JM_8b2

middle: P

first: Karl

middle: V

last: Clemons

first: Laurence

middle: F

last: Mirels

first: John

middle: A

last: Coller

first: Thomas

last: Wu

first: Jata

last: Shankar

first: Catalina

last: Lopes

first: David

last: Stevens


title: Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke

city: Genoa

first: Alberto

last: Izzotti

address: A. Pastore, 1

department: Dept. of Health Sciences

country: Italy

institute: University of Genoa

summary:
While microRNAs have extensively been investigated in cancer research, little is known  regarding their response to noxious agents in apparently healthy tissues. We analyzed the  expression of 484 miRNAs in the lung of rats exposed to environmental cigarette smoke (ECS)  for 28 days. ECS downregulated 126 miRNAs (26.0%) at least two-fold and 24 miRNAs more  than three-fold. We previously demonstrated that 107 of 4858 genes (2.9%) and 50 of 518  proteins (9.7%) were upregulated by ECS in the same tissue, consistently with the role of  microRNAs as negative regulators of gene expression. The most remarkably downregulated  microRNAs belonged to the families of let-7, miR-10, miR-26, miR-30, miR-34, miR-99, miR-  122, miR-123, miR-124, miR-125, miR-140, miR-145, miR-146, miR-191, miR-192, miR-219,  miR-222, and miR-223, which regulate stress response, apoptosis, proliferation, angiogenesis,  and expression of genes. In contrast, miR-294, an inhibitor of transcriptional repressor genes,  was upregulated by ECS. There was a strong parallelism in dysregulation of rodent microRNAs  and their human homologues, which are often transcribed from genes localized in fragile sites  deleted in lung cancer. Five ECS-downregulated microRNAs are known to be affected by single  nucleotide polymorphisms. Thus, changes in microRNA expression are an early event following  exposure to cigarette smoke.

Keywords: microRNAs • gene expression • environmental cigarette smoke

platform_organism:
Arabidopsis thaliana
Homo sapiens
Mus musculus

overall_design: Sprague–Dawley rats (Harlan Italy, Correzzana, Milan, Italy), weighing 315-320 g, were  either exposed to ECS for 4 weeks or kept in filtered air for the same period of time (sham).

first: George

last: Calin

first: Patrizio

last: Arrigo

first: Vernon

middle: E

last: Steele

first: Carlo

last: Croce

first: Silvio

last: De Flora


title: TGF-beta responsive gene in primary osteoblast.

city: Tsukuba

first: Tomoki

last: Chiba

state: Ibaraki

address: 1-1-1 Tennodai

country: Japan

institute: University of Tsukuba

summary:
Gene expression profiling of a total of 3,774 genes in primary osteoblastic cells treated with TGF-beta1

Keywords: cytokine response

overall_design: Primary osteoblasts cultured under serum-starved condition were treated or untreated with TGF-beta1 for 24 hr.


title: Chip-chip from pro-B cells with H3K4me3, H3K4me2 and H3K9ac

status: Public on Apr 27 2009

city: Vienna

first: Meinrad

last: Busslinger

address: Dr. Bohrgasse 7

country: Austria

institute: Instutute of Molecular Pathology

summary:
The transcription factor Pax5 represses B-lineage-inappropriate genes and activates B-cell-specific genes in B-lymphocytes. Here we have identified 170 novel Pax5-activated genes. Conditional mutagenesis demonstrated that the Pax5-regulated genes require continuous Pax5 activity for normal expression in pro-B and mature B cells. Expression of half of the Pax5-activated genes is either absent or significantly reduced upon Pax5 loss in plasma cells. Direct Pax5 target genes were identified based on their protein synthesis-independent activation by a Pax5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of Pax5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Pax5 directly activates the chromatin at promoters or putative enhancers of Pax5 target genes. The novel Pax5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation and germinal center B cell formation, thus revealing a complex regulatory network, which is activated by Pax5 to control B cell development and function.

Keywords: Chip-chip, cell type comparison

overall_design: comparison of Pax5-/-Rag2-/- vs Rag2-/- pro-B cells

first: Shane

last: McManus


title: Digital DNA methylation analysis of stem cell reprogramming by targeted bisulfite sequencing

city: La Jolla

first: Kun

last: Zhang

address: 9500 Gilman Drive, MC0412

department: Bioengineering

institute: University of California at San Diego

type: Methylation profiling by high throughput sequencing

summary: We report a method for specific capture of an arbitrary subset of genomic targets for single molecule bisulfite sequencing, and for digital quantitation of DNA methylation at a single nucleotide resolution. We used targeted bisulfite sequencing to characterize the changes of DNA methylation during the de-differentiation of human fibroblasts into hybrid stem cells, and into induced pluripotent stem cells. We compared the methylation level of approximately 66,000 CpG sites within 2020 CpG islands on chromosome 12, chromosome 20, and 34 selected regions. A total of 288 differentially methylated regions were identified between fibroblasts and pluripotent cells. Methylation cluster analysis revealed distinct methylation patterns between fibroblasts and pluripotent cells. Furthermore iPS cells are globally more methylated than human embryonic stem cells, which could be due to the reprogramming process. This targeted bisulfite sequencing method is particularly useful for efficient and large-scale analysis of DNA methylation in organisms with large genomes.

overall_design: Comparison of DNA methylation on 2020 CpG islands and 34 other selected regions among eleven human ES, iPS and fibroblast lines.


title: SMAUG is required for zygotic transcription during the maternal-zygotic transition MZT

status: Public on Apr 23 2009

city: Mississauga

first: Zak

last: Razak

address: 3359 Mississauga Rd

institute: Canadian Drosophila Microarray Centre

summary: Microarray analysis of zygotic gene expression in 2-to-3 hour wild-type (wt) and smg mutant embryos.  Expression is relative to mature, stage 14 oocytes, which contain the full maternal pool of mRNA.   Strictly maternal genes that are not transcribed at the MZT contribute approximately 80% of transcripts in early embryos, and are not shown.  Class I zygotic genes showed high levels of expression in 2-to-3 hour embryos.  142 of the 166 Class I genes were not expressed in smg mutants.  The remaining zygotically expressed genes were also present in oocytes.  These genes were divided into two classes, based on analysis of 4-to-6 hour old unfertilized eggs (4-6h unf), which are transcriptionally inactive.  Class-II genes produce maternal transcripts that are stable in unfertilized eggs and show significantly increased expression in 2-to-3 hour post-fertilization embryos. 358 of 395 Class-II genes require SMAUG for zygotic expression.  Class-III genes produce maternal transcripts that are degraded in unfertilized eggs and show significantly increased expression in 2-to-3 hour post-fertilization embryos.  65 of 408 Class-III genes require SMAUG for expression in 2-to-3 hour embryos.

overall_design:
mRNA was extracted from staged fertilized or unfertilized eggs, as well as stage 14 oocytes as described previously (Tadros et al., 2007a). To assay mRNA quality, known stable (rpA1) and unstable (Hsp83) transcripts were probed on Northern blots. Total RNA was then reverse transcribed with random primers (Tadros et al., 2007a) and labeled asÊdescribed in the Indirect Labeling of Total RNA for Microarray Hybridization protocol at http://www.flyarrays.com.  The fluorescently labeled cDNA probes were hybridized to 12Kv1 microarrays obtained from the Canadian Drosophila Microarray Centre (http://www.flyarrays.com; GEO platform accession number GPL1467:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL1467).  Hybridization and scanning were performed using a PerkinElmer/GSI ScanArray 4000 scanner and the ScanArray software as previously described (Neal et al., 2003). The 16 bit TIFF image files were quantified using QuantArray Version 3 (PerkinElmer), using the adaptive quantification algorithm and analyzed using GeneTraffic Duo3.2 (Iobion Informatics/Stratagene). The 12Kv1 arrays were normalized using the rank invariant LOWESS extrapolation method (Schadt et al., 2001).

first: Chun

middle: H

last: He

first: Linan

last: Chen

first: J

last: Westwood

first: Howard

middle: D

last: Lipshitz


title: TU-tagging: cell type specific RNA isolation from intact complex tissues

status: Public on Apr 24 2009

city: Eugene

first: Michael

last: Miller

state: OR

address: 1229 University of Oregon

department: Institute of Molecular Biology

institute: University of Oregon

summary: We show that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil (4TU) delivery can be used to label and purify cell type specific RNA from intact complex tissues. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in any organism where UPRT can be spatially expressed, including mammals and other vertebrates.

overall_design: To confirm that thio-labeled RNA was from UPRT-expressing cells and demonstrate the utility of TU-tagging for cell type-specific RNA isolation, we purified TU-tagged and untagged RNA and compared them using microarrays. We chose to isolate RNA from larval glia, which are present in low number, are highly dispersed, and have a complex cell morphology, making them one of the most difficult cell types to isolate by dissection or dissociation methods. We used reversed polarity (repo)-GAL4 to drive expression of UAS-UPRT specifically in glial cells of the larval brain. We purified TU-tagged and untagged RNA from 72-96h larval brains, and hybridized them to custom Agilent microarrays.  We performed two biological replicate experiments.

middle: R

first: Chris

middle: Q

last: Doe


title: Identification of differentially expressed genes between clinical and bovine-biased Escherichia coli O157:H7 genotypes

city: East Lansing

first: Thomas

last: Whittam

state: MI

address: 165 Food Safety & Tox

department: Microbiology

institute: Michigan State University

summary: Escherichia coli O157:H7 strains have been classified into different genotypes based on the presence of specific shiga toxin-encoding bacteriophage insertion sites.  Genotypes that are predominant in clinical isolates are named clinical genotypes and those that are isolated mostly from bovine sources are bovine-biased genotypes. To determine whether inherent differences in gene expression could possibly explain the variation in infectivity of these genotypes, we compared the expression patterns of O157:H7 strains isolated from cattle, which belonged to either clinical genotype 1 or bovine-biased genotype 5. Important virulence factors of O157, including locus of enterocyte effacement, enterohemolysin, and pO157 plasmid encoded genes, showed increased expression in clinical genotype. Genes essential for acid resistance such as gadA, gadB, and gadC and other stress fitness-associated genes were up-regulated in the bovine-biased genotype 5. Overall, these results suggest that clinical genotype 1 strains more commonly cause human illness because of an enhanced ability to express O157 virulence factors known to be important for disease pathogenesis.  By contrast, strains of the bovine-biased genotype 5 appear to be more resistant to adverse environmental conditions, which enable them to survive well in bovines without causing disease.

overall_design: The results are based on O157:H7 clinical and bovine-biased genotype cultures grown in DMEM medium to exponential phase. Four strains were selected from each genotype and strains were considered as biological replicates. A double loop microarray design was used for comparing the samples. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to strain (clinical or bovine-biased) using the following linear model: array+dye+sample (biological replicate)+strain+error. We incorporated the dye-swaps among the biological replicates.

first: Sivapriya

last: Kailasan Vanaja

first: Shannon

last: Manning

first: Cody

last: Springman

last: Besser


title: Suppressing the barley uroporphyrinogen gene by Ds activation tagging element generates developmental photosensitivity

city: Canberra

first: Andrew

last: Spriggs

state: ACT

address: GPO Box 1600

department: Bioinformatics Group

country: Australia

institute: CSIRO Plant Industry

summary: Chlorophyll production involves the synthesis of photoreactive intermediates that are toxic when in excess due to the production of reactive oxygen species (ROS). A novel activation tagged barley mutant is described that results from antisense suppression of a uroporphyrinogen III synthase (Uros) gene, the product of which catalyses the sixth step in the synthesis of chlorophyll and heme. In homozygous mutant plants uroporphyrin(ogen) I accumulates by spontaneous cyclization of hydroxyl methylbilane, the substrate of Uros. Accumulation of this tetrapyrrole intermediate results in photosensitive cell death due to the production of reactive oxygen species. The efficiency of Uros gene suppression is developmentally regulated, being most effective in mature seedling leaves compared with newly emergent leaves. A comparison of RNA from mutant and wild type seedlings grown under 30% and 3% light, shows reduced transcript accumulation of a number of nuclear-encoded photosynthesis genes occurs in the mutant even under 3% light conditions. This is consistent with a retrograde plastid-nuclear signalling mechanism arising from Uros gene suppression. No evidence for a direct signalling role by uroporphyrin I was observed suggesting that nuclear gene transcriptional suppression was mediated by ROS accumulation.

platform_organism:
Hordeum vulgare
Triticum aestivum

overall_design: Two replicates samples were used for barley mutant 70a3 grown under 3% and 2 replicate sample of the mutant grown under 30% light, barley cv Golden Promise was used as the control.

last: Ayliffe

first: Dominicoantonio

last: Agostino

first: Bryan

middle: C

last: Clarke

first: Robert

last: Furbank

first: Susanne

last: von Caemmerer

first: Anthony

middle: J

last: Pryor


title: nasopharyngeal carcinoma versus nasopharyngeal phlogistic tissue reference

status: Public on Apr 21 2009

city: Shanghai

first: xuping

last: fu

country: China

institute: Institute of Genetics, Fudan University

summary:
Nasopharyngeal carcinoma (NPC) remains a majoy health problem worldwide, specially in Southeast China. In order to find the new candidate genes and molecular markers that are associated with nasopharyngeal carcinoma (NPC), this study focused on the screening NPC relative genes by gene expression profile.
Keywords: disease state analysis

overall_design: 23 NPC biopsies and 15 nasopharynx chronic phlogistic biopsies were used to screen NPC relative genes by BioStarH-141s (2004) profile gene chips which contained 14112 points of full length human genes. The tumor samples were labeled with Cy5-dUTP.The nasopharyngeal phlogistic tissues  were labeled with Cy3-dUTP. Biostatistics and bioinformatics were also used to analyse the differently expressed genes.

first: guo

last: yi


title: Lactococcus lactis stringent response

city: Toulouse

first: Muriel

last: Cocaign-Bousquet

address: 135 avenue de Rangueil

department: UMR5504 INSA/CNRS - UMR792 INSA/INRA

institute: Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés

summary:
The stringent response was defined in Lactococcus lactis through transcript profiling after the addition of a chemical inductor, the norvaline. Gene expression was measured in the exponential growth phase (reference sample) and at 1.6 h after norvaline addition. Four hundred and sixty one differentially expressed genes were identified and constituted the stringent response regulon.
Keywords: stress response, time course

overall_design: Stringent response was imposed through norvaline addition during the growth of Lactococcus lactis IL1403 under controlled conditions (30 °C, pH 6.6, nitrogen atmosphere). Cell samples were harvested in exponential phase and 1.6 h after norvaline addition. Total RNA was extracted from these samples and radiolabelled cDNA were prepared and hybridized on nylon arrays. 2053 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. The 2 time-points were analyzed simultaneously and 3 independent repetitions were performed.

first: Emma

last: Redon

first: Pascal

last: Loubiere


title: Expression profiling of skeletal muscle following acute β2-adrenergic stimulation

city: St Lucia

first: Michael

middle: Andrew

last: Pearen

state: Queensland

address: The University of Queensland

institute: Institute for Molecular Bioscience

summary: Systemic administration of β-adrenoceptor (β-AR) agonists has been found to induce skeletal muscle hypertrophy and significant metabolic changes. In the context of energy homeostasis, the importance of β-AR signaling has been highlighted by the inability of β1–3-AR-deficient mice to regulate energy expenditure and susceptibility to diet induced obesity. However, the molecular pathways and gene expression changes that initiate and maintain these phenotypic modulations are poorly understood. Therefore, the aim of this study was to identify differential changes in gene expression in murine skeletal muscle associated with systemic acute administration of the β2-AR agonist formoterol. Skeletal muscle gene expression (from murine tibialis anterior) was profiled at both 1 and 4 hours following systemic administration of the β2-AR agonist formoterol, using 46K Illumina(R) Sentrix BeadArrays. Illumina expression profiling revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress.

overall_design: Total RNA obtained from tibialis anterior of mice at 1 or 4 hours following a single intraperitoneal injection of formoterol or saline.

first: James

middle: G

last: Ryall

first: Gordon

middle: S

last: Lynch

first: George

last: Muscat


title: Promoter CpG island methylation data from A2780 (cisplatin-sensitive) and Round5 A2780 (cisplatin-resistant) cell lines.

status: Public on Apr 17 2009

city: Bloomington

first: Meng

last: Li

state: IN

address: 1001 E 3rd Street

department: Medical Sciences

institute: Indiana University

summary: Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, subsequently resulting in a poor long-term prognosis.  To model the onset of drug resistance, and investigate the DNA methylation alterations associated with cisplatin resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin.  After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation microarray analyses.

overall_design: We treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin.  After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation analyses by differential methylation hybridization (DMH) using a customed 44K promoter CGI microarray.

first: Kenneth

last: Nephew


status: Public on Apr 16 2009

city: San Francisco

first: Esther

middle: T

last: Chan

state: California

address: 1500 Owens Street, Suite 500

institute: Celgene Corporation

summary: Expression of known and predicted genes in tissues of Xenopus tropicalis (frog) pooled from multiple healthy individuals.

overall_design:
Two-colour experiments with two different tissues hybridized to each array. Each tissue is arrayed in replicate with dye swaps.
Tissues: Brain, Cartilage, Esophagus, Eye, Fat body, Femur, Gallbladder, Heart, Kidney, Large intestine, Liver, Lung, Muscle, Ovary, Oviduct, Skin, Small intestine, Spleen, Stomach, Testis

first: Gerald

last: Quon

last: Chua

first: Tomas

last: Babak

first: Miles

last: Trochesset

first: Ralph

last: Zirngibl

first: Jane

last: Aubin

last: Ratcliffe

first: Andrew

last: Wilde

last: Brudno

first: Quaid

last: Morris

first: Timothy

last: Hughes