title: Pain microarray analysis 1

status: Public on Mar 03 2007

city: Baltimore

first: Svetlana

middle: I

last: Novikova

address: 666 W. Baltimore st

department: Biomedical Sciences

institute: University of Maryland

summary:
Neonatal noxious insult produces a long-term effect on pain processing in adults. Rats subjected to carrageenan (CAR) injection in one hindpaw within the sensitive period develop bilateral hypoalgesia as adults. In the same rats, inflammation of the hindpaw, which was the site of the neonatal injury, induces a localized enhanced hyperalgesia limited to this paw.
This study revealed significant basal and inflammation-associated aberrations in the expression of multiple genes in the LDH of adult animals receiving CAR injection at P3 as compared to their expression levels in the LDH of animals receiving either no injections or CAR injection at P12. In particular, at baseline, twelve genes (representing GABA, serotonin, adenosine, neuropeptide Y, cholecystokinin, opioid, tachykinin and interleukin systems) were up-regulated in the bilateral LDH of the former animals. The baseline condition in these animals was also characterized by up-regulation of seven genes (encoding members of GABA, cholecystokinin, histamine, serotonin, and neurotensin systems) in the LDH ipsilateral to the neonatally-injured paw. The largest aberration in gene expression, however, was observed during inflammation of the neonatally injured hindpaws in the ipsilateral LDH, which included thirty-six genes (encoding numerous members of glutamate, serotonin, GABA, calcitonin gene-related peptide, neurotrophin, and interleukin systems). These findings suggest that changes in gene expression may be involved in the long-term nociceptive effects of neonatal noxious insult at the spinal level.
Keywords: gene expression analysis via microarray

overall_design: To gain an insight into the long-term molecular changes involved in the above-described long-term nociceptive effects of neonatal noxious insult at the spinal level, we performed DNA microarray analysis (using microarrays containing oligo-probes for 205 genes encoding receptors and transporters for glutamate, GABA, and amine neurotransmitters, precursors and receptors for neuropeptides, and neurotrophins, cytokines and their receptors) to compare gene expression profiles in the lumbar spinal dorsal horn (LDH) of adult (P60) male rats that received neonatal CAR treatment within (at postnatal day 3; P3) and outside (at postnatal 12; P12) of the sensitive period. The data were obtained both without inflammation (at baseline) and during complete Freund's adjuvant induced inflammation of the neonatally injured paw. The observed changes were verified by real-time RT-PCR.

first: Michael

last: Lidow

first: Fang


title: Time course expression data of human cell lines infected with dengue virus serotype2 NGC

status: Public on Mar 01 2007

city: Singapore

first: Martin

last: Hibberd

state: Singapore

address: 60, Biopolis Street

department: Population Genetics

country: Singapore

institute: Genome Institute of Singapore

summary:
Microarray data was used to study the gene expression changes following dengue infection at various time points to identify host genes and pathways responsible for dengue virus replication
Keywords: time course

overall_design: HepG2 cell line was infected with NGC virus and expression profiled at 3hrs, 6hrs, 12hrs,24hrs, 48 hrs and 72hrs after infection. At each time point we profiled the transcriptome using 3 biological repeats and each sample profiled using a technical repeat with dyes swapped

first: Subhash

last: Vasudevan

first: Ling

last: Ling

first: Joshy

last: George


title: Chickpea genes regulated by salicylic acid, methyl jasmonate, and aminocyclopropane carboxylic acid

city: Raleigh

first: Tristan

last: Coram

state: NC

address: 4118 Williams Hall, NC State University

country: USA

institute: USDA-ARS, NC State University

summary:
Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes and grasspea (Lathyrus sativus L.) ESTs, chickpea responses to treatments with the defence signalling compounds salicylic acid (SA), methyl jasmonate (MeJA), and aminocyclopropane carboxylic acid (ACC) were studied in four chickpea genotypes with ranging levels of resistance to ascochyta blight (Ascochyta rabiei (Pass.) L.).  The experimental system minimized environmental effects and was conducted in reference design, where samples from untreated controls acted as references against post-treatment samples.  Robust data quality was achieved through the use of three biological replicates (including a dye-swap), the inclusion of negative controls, and strict selection criteria for differentially expressed genes including a fold change cut-off determined by self-to-self hybridizations, Students t test and multiple testing correction (P<0.05).  Microarray observations were also validated by quantitative RT-PCR.  The time-course expression patterns of 715 experimental microarray features resulted in differential expression of 425 genes in at least one condition.  The A. rabiei resistant chickpea genotypes showed a more substantial range of defence-related gene induction by all treatments, indicating that they may possess stronger abilities to resist infection.  Further, the involvement of SA, MeJA, and ACC signalling was identified for the regulation of some important A. rabiei responsive genes, as well as cross-talk between these pathways.  This study also found evidence to suggest the involvement of A. rabiei-specific signalling mechanisms for the induction of several genes that were previously implicated in A. rabiei resistance.  Overall, this study characterised the regulatory mechanisms of many chickpea genes that may be important in defence against various pathogens, as well as other cellular functions.  Although the size of the microarray was limited, the results provided novel insights to the molecular control of chickpea cellular processes, which may assist the understanding of chickpea defence mechanisms and allow enhanced development of disease resistant cultivars.
Keywords: time course defence-signalling teatment analysis

platform_organism:
Cicer arietinum
Lathyrus sativus

overall_design:
Total RNA was extracted from pooled stem and leaf samples for each genotype (FLIP94-508C, ICC3996 and Lasseter) at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity and quality of the total RNA samples were assessed by OD260/OD280 ratios and gel electrophoresis respectively. Fluorescent-labeled targets were prepared and hybridized to array slides according to [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Published in Plant Biotechnology Journal]. All hybridizations were performed with six technical replicates and three biological replicates, incorporating dye-swapping (i.e. reciprocal labelling of Cy3 and Cy5) to eliminate any dye bias. Overall, 324 images were analyzed from 54 slides, resulting in 18 data points for each time-point of each genotype.

Slides were scanned at 532 nm (Cy3 green laser) and 660 nm (Cy5 red laser) at 10 µm resolution using an Affymetrix® 428™ array scanner (Santa Clara, CA), and captured with the Affymetrix® Jaguar™ software (v. 2.0, Santa Clara, CA). Image analysis was performed using Imagene™ 5 (BioDiscovery, Marina Del Rey, CA) software. Quantified spot data was then compiled and transformed using GeneSight™ 3 (BioDiscovery, Marina Del Rey, CA). Data transformations consisted of a local background correction (mean intensity of background was subtracted from mean signal intensity for each spot), omitting flagged spots, Lowess normalisation of the entire population, creating a Cy5/Cy3 mean signal ratio, taking a shifted log (base 2), and combination of duplicated spot data. To identify differentially expressed (DE) genes, expression ratio results were filtered to eliminate genes whose 95% confidence interval for mean fold change (FC) did not extend to 2.0-fold up or down, followed by Students t test with False Discovery Rate (FDR) multiple testing correction to retain only genes in which expression changes versus untreated control were significant at P < 0.05.

middle: E

first: Eddie

last: Pang


title: Genome-wide transcriptional plasticity underlies cellular adaptation to novel challenge

city: Haifa

first: Shay

last: Stern

address: Haifa

department: Chemical Engineering, Physics

country: Israel

institute: Technion- Israel Institute of Technology

summary:
Cells adjust their transcriptional state to accommodate environmental and genetic perturbations. An open question is to what extent transcriptional response to perturbations has been specifically selected along evolution. To test the possibility that transcriptional reprogramming does not need to be 'pre-designed' in order to lead to an adaptive metabolic state on physiological time scales, we confronted yeast cells with a novel challenge they had not previously encountered. We rewired the genome by recruiting an essential gene, HIS3 from the histidine biosynthesis pathway to a foreign regulatory system, the GAL network responsible for galactose utilization. Switching medium to glucose in a chemostat caused repression of the essential gene and presented the cells with a severe challenge. Using genome-wide expression arrays we show that a global transcriptional reprogramming (>1200 genes) is induced immediately following the switch into the challenging environment, allowing adaptation of the cell population. A larger environmental pressure applied directly on the recruited HIS3 gene by a competitive inhibitor, led to significantly larger expression correlations among hundreds of genes residing in different functional modules, showing that the global transcriptional response underlies the adaptation process. The correlated transcriptional pattern relaxed to the adaptive state over a long period (~10 generations). Our results show that transcriptional plasticity, involving an enhanced response of a sizeable fraction of the genome, is a natural property of the regulatory network allowing it to overcome unforeseen challenges.
Keywords: time course, chemostat

overall_design: Eight samples of cells harvested from the chemostat at time points along the different phases of the population adaptation were analyzed by expression arrays for the two experiments, with and without 3AT.

first: Tali

last: Dror

first: Naama

last: Brenner

first: Erez

last: Braun


title: Toxicogenomic responses of PCB52 in the nematode Caenorhabditis elegans

status: Public on Feb 28 2007

city: London

first: Stephen

last: Sturzenbaum

address: 150 Stamford Street

department: Analytical, Environmental and Forensic Sciences

country: United Kingdom

institute: King's College London

summary:
Although non-coplanar PCBs are ubiquitous organic chemicals known to induce numerous biological responses and thus are toxic to man and wildlife, little is known about the toxic mode of action. Using PCB52, an ortho-substituted, 2,2’,5,5’-tetrachlorobiphenyl, it was possible to pinpoint the relationship between induced gene expression and observed toxicity in the model nematode Caenorhabditis elegans.
Keywords: stress response

overall_design:
Based on the calculated EC20 for brood size (5 mg/L), whole genome DNA microarray experiments were performed using a common reference design to identify differentially expressed genes. Gene knockdown by RNAi was used to determine the consequences in reproductive fitness in the presence and absence of PCB52.

Each of the 2x4 “biological replicates” consists of two technical repeats. Each technical repeat has a cy5 value (sample) and a cy3 value (reference sample). The value for each technical repeat is calculated as a ratio and the normalized data is the mean between the ratio of the two technical repeats.

first: Ralph

last: Menzel

middle: R


title: ADH1 deletion cells upon growth on alternative carbon sources; Steady-state grown cells upon environmental perturbations

city: Rehovot

first: sagi

last: levy

address: Hertzel

department: Molecular Genetics and Physics of Complex Systems

institute: Weizmann Institute of Science

summary:
Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirement. First, the internal physiological state itself could feedback to regulated gene expression. Second, the expected physiological state could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis appears to be particularly important, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative importance of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in chemostat-grown cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmental changes presented a major selection force during yeast evolution.
Keywords: Saccharomyces_Cerevisiae, Stress response, ADH1 deletion, time courses, chemostat

overall_design: Experiment 1: ADH1 deletion cells, which grow faster on glycerol rather then glucose medium, were grown in both glucose and glycerol medium. The cells expression on glycerol is compared to their expression glucose (5 microarrays). Experiment 2: Time course of ADH1 deletion cells upon growth on glycerol and on glucose (7 microarrays). Experiment 3: Response of steady state grown cells to environmental perturbations. Cells were grown in glucose or histidine limited chemostats and reached steady state. The cells were then subjected to perturbations such as DTT, heat shock, NaCl, Clotrimazole, H2O2, and addition of limiting factors (histidine and glucose, respectively). Overall we have examined 10 timecourses with 83 microarrays.

first: Sagi

last: Levy

first: Jan

last: Ihmels

first: Miri

last: Carmi

first: Adina

last: Weinberger

first: Gilgi

last: Friedlander

last: Barkai


title: Gene expression profiles of head and neck squamous cell carcinoma (HNSCC) cell lines

city: Chiba

first: Satoya

last: Shimizu

address: 1-8-1 Inohana, Chuo-ku

country: Japan

institute: Chiba university

summary:
Chromosomal comparative genomic hybridization (CGH) and oligonucleotide microarrays were used to examine 8 HNSCC cell lines and a plot of gene expression levels relative to their position on the chromosome was produced. Three highly up-regulated genes, NT5C3, ANLN and INHBA, were identified on chromosome 7p14. These genes were subjected to quantitative real-time RT-PCR on cDNA and genomic DNA derived from 8 HNSCC cell lines. ANLN and INHBA showed a strong positive correlation between mRNA expression and genomic DNA levels and a similar relationship was shown for the known oncogene, EGFR, at 7p11.2. In clinical samples, ANLN and INHBA showed a significantly higher expression in tumors than in normal tissues. Patients with high expression levels of INHBA had a shorter disease-free survival rate. Therefore, INHBA may be a promising prognostic marker of HNSCC.
Keywords: Identification of molecular targets based on genome-wide gene expression profiling

overall_design: To identify prognostic markers of HNSCC, chromosomal CGH and oligonucleotide microarray analysis was performed on 8 HNSCC cell lines. Up-regulated genes at chromosomal gain regions detected by CGH were identified. Quantitative real-time RT-PCR on cDNA and genomic DNA from 8 HNSCC cell lines was performed for three selected genes and expression was further analyzed in clinical samples.

first: Takashi

last: Sugimoto

first: Naohiko

last: Seki


title: profiling of hypothalamus from arthritic DBA/1 mice vs. control DBA/1 mice

status: Public on May 02 2007

city: Ciudad de Buenos Aires

first: Mariano

middle: Cesar

last: Salibe

address: Av. Patricias Argentinas 435

country: Argentina

institute: Gentron LLC

summary:
The main function of the nervous system is to maintain homeostasis by sensing and reacting to signals that reach a certain threshold. For example, the brain can sense immune peripheral events through soluble compounds or the vagus nerve and can react through activation of the hypothalamus-pituitary-adrenal axis, resulting in the modulation of an ongoing immune response. Using microarrays we studied the gene expression profile of the hypothalamus in DBA/1 mice strain that developed collagen induced-arthritis vs control mice.
Keywords: gene expression profile of the hypothalamus in response to induction of arthritis

overall_design: We used two-color oligonucleotides in-house printed microarrays in a case-control direct design with two technical replicates in a dye swap design. We analyze changes in gene expression profile in the hypothalamus of arthritic vs control mice. We performed 3 samples (biological replicates)

middle: C

middle: J

last: Alvarez

first: Cintia

middle: G

last: Serra

first: Daniela

last: Celi

first: Guillermo

last: Vila-Ortiz

first: Alejandra

last: Ramirez

first: Osvaldo

middle: L

last: Podhajcer

first: Fernando

last: Pitossi


title: An expression atlas of rice mRNAs and small RNAs

status: Public on Feb 23 2007

city: St. Louis

first: Blake

middle: C.

last: Meyers

state: MO

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

summary:
Identification of all expressed transcripts in a sequenced complex genome is technically challenging, but essential for systems biology and genome analysis.  We used the transcriptional profiling technology called ‘massively parallel signature sequencing’ (MPSS) to develop a comprehensive expression atlas of rice (Oryza sativa cv Nipponbare). A total of 46,971,553 mRNA transcripts from 22 libraries, and 2,953,855 small RNAs from three libraries were sequenced. The data demonstrated widespread transcription throughout the genome, including expression for up to 25,500 annotated genes and antisense expression for nearly 9,000 annotated genes. An additional set of ~15,000 mRNA signatures mapped to unannotated genomic regions. The majority of the small RNA data represented lower abundance small-interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Among these, numerous clusters of highly-regulated small RNAs were readily observed. We developed a genome browser (http://mpss.udel.edu/rice) for public access to the transcriptional profiling data for this important crop plant.
Keywords: MPSS, mRNA, small RNA, transcriptome, rice

overall_design:
MPSS was performed to sequence small RNAs that derived from 16 untreated and 6 abiotic-treated diverse tissue libraries. 18 mRNA MPSS libraries and 3 small RNA libraries were submitted. We have total of 22 libraries (16 untreated and 6 abiotic-treated) mRNA libraries in our database, but it includes 4 mature leaf biological replicates and 2 mature root biological replicates.  In order to reduce the redundancy, we created combined sets (NL4 and NR2) and submitted those data.

The method for the MPSS sequencing of mRNAs is described in Brenner et al. (Nat Biotechnol. 2000 18:630).


title: Genomic Profiling of Malignant Melanoma using tiling resolution array CGH

status: Public on Mar 06 2007

city: Lund

first: Johan

last: Staaf

address: Medicon Village

country: Sweden

institute: SCIBLU - Swegene Centre for Integrative Biology at Lund University

type: Genome variation profiling by genome tiling array

summary:
Malignant melanoma is an aggressive, heterogeneous disease where new biomarkers for diagnosis and clinical outcome are needed. We searched for chromosomal aberrations that characterize its pathogenesis using 47 different melanoma cell lines and tiling-resolution BAC-arrays for comparative genomic hybridization. Major melanoma genes, including BRAF, NRAS, CDKN2A, TP53, CTNNB1, CDK4 and PTEN were examined for mutations. Distinct copy number alterations were detected, including loss or gain of whole chromosomes but also minute amplifications and homozygous deletions. Most common overlapping regions with losses were mapped to 9p24.3-q13, 10 and 11q14.1-qter whereas copy number gains were most frequent on chromosomes 1q, 7, 17q and 20q. Amplifications were delineated to oncogenes such as MITF (3p14), CCND1 (11q13), MDM2 (12q15), CCNE1 (19q12) and NOTCH2 (1p12). Frequent findings of homozygous deletions on 9p21 and 10q23 confirmed the importance of CDKN2A and PTEN. Pair-wise comparisons revealed distinct sets of alterations, e.g. mutually exclusive mutations in BRAF and NRAS, mutual mutations in BRAF and PTEN, concomitant chromosome 7 gain and 10 loss and concomitant chromosome 15q22.2-q26.3 gain and 20 gain. Moreover, alterations of the various melanoma genes were associated with distinct chromosomal imbalances suggestive of specific genomic programs in melanoma development.
Keywords: comparative genomic hybridization

overall_design: Genomic DNA was extracted from 47 melanoma cell lines. The DNA was labeled using Bioprime array CGH labeling kit (Invitrogen). Promega pooled male DNA was used as reference. Dye-swaps were performed for 13 cell lines. Labeled DNA was hybridized onto BAC arrays containing ~32 000 BAC clones printed in singlets. BAC arrays were produced at the SWEGENE DNA Microarray Facility at Lund University.

first: Göran

last: Jönsson

first: Christina

last: Dahl

first: Therese

last: Sandberg

first: Pär-Ola

last: Bendahl

first: Markus

last: Ringér

first: Per

last: Guldberg

first: Ake

last: Borg


title: WT vs. ACE2 knockout (grown in yeast form)

city: Dublin

first: Geraldine

last: Butler

address: Belfield

department: School of Biomolecular and Biomedical Science

country: Ireland

institute: University Colege Dublin Conway Institute

summary:
We have used DNA microarray technology to compare the expression of over 6000 genes between C. albicans WT and ACE2 knockout strains in the yeast form.
Keywords: Gene expression comparison

overall_design: We analyzed 7 arrays for the yeast-form comparison experiment. Three dye-swaps were performed.

first: Siobhan

middle: M

last: Mulhern

first: Mary

last: Logue


title: Automated array-CGH optimized for archival formalin-fixed, paraffin-embedded tumor material

city: Hamburg

first: Simon

middle: A

last: Joosse

address: Martinistrasse 52

department: Institute of Tumor Biology

country: Germany

institute: University Medical Center Hamburg-Eppendorf

summary: BACKGROUND: Array Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH. METHODS: In this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use. RESULTS: Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method. CONCLUSION: This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility.

overall_design: In this study, we optimized the BAC araay-CGH protocol for automated hybridization for FFPE breast cancer samples. We have tested hybridization temperature and duration, different hybridization buffer conditions, and post-hybridization washing.

first: Erik

middle: H

last: van Beers

first: Petra

last: Nederlof


title: Transcript analysis of aluminum tolerance responses in Medicago truncatula (A17)

status: Public on Feb 22 2007

city: St Paul

first: Kathryn

last: VandenBosch

state: MN

address: Room 250 BioSci 6022 1445 Gortner Ave

department: Plant Biology

institute: University of Minnesota

summary:
For transcript analysis of aluminum tolerance responses in Medicago truncatula (A17) we compared transcripts from 2.5 µM Al-treated and control (-Al) root tips corresponding to 12 h after Al treatment.
Keywords: One time point and one genotype

overall_design:
Experiment consists of 6 microarrays.
Experiment consists of three direct (reps1, 2, 3) and three dye-swapped (reps 4, 5, 6) technical replicates.

first: Divya

last: Chandran

first: Natasha

last: Sharopova

first: Deborah

last: Samac


title: ChIP-Chip of General Transcription factors in Halobacterium NRC-1

city: Seattle

first: Marc

last: Facciotti

state: WA

address: 1441 North 34th Street

institute: Institute for Systems Biology

type: Genome binding/occupancy profiling by array

summary:
Cells responding to dramatic environmental changes or undergoing a developmental switch typically change the expression of numerous genes.  In bacteria sigma factors regulate much of this process while in eukaryotes four RNA polymerases and a multiplicity of generalized transcription factors (GTFs) are required. Here, using a systems approach, we provide experimental evidence (including protein-coIP, ChIP-Chip, GTF perturbation and knockout, and measurement of transcriptional changes in these genetically perturbed strains) for how archaea likely accomplish similar large-scale transcriptional segregation and modulation of physiological functions.  We are able to associate GTFs to nearly half of all putative promoters and show evidence for at least 7 of the possible 42 functional GTF pairs.  This report represents a significant contribution towards closing the gap in our understanding of gene regulation by GTFs for all three domains of life and provides an example for how to utilize various experimental techniques to rapidly learn significant portions of a global gene regulatory network of organisms for which little is previously known.
Keywords: ChIP-Chip

overall_design: Halobacterium NRC-1 expressing Cmyc-tagged TFBs and TBPs were grown to an OD600 between 1 and 1.2.  The cells were cross-linked with 1.2% formaldehyde, then lysed and DNA sheared via sonication.  Next cleared cell lysate was subject to immunoprecipitation against an anti-myc antibody.  Protein-DNA complexes were purified and DNA was isolated after protease and RNAase treatment.  DNA was amplified using universal linkers, labeled directly using Kreatech 547 and 647 dyes.  Samples were grown in biological duplicate.  Each DNA sample was hybridized against 2 microarrays as dyefilps.  At least two experiments (including biological duplicates) were carried out.

middle: T

first: David

last: Reiss

first: Min

last: Pan

first: Amardeep

last: Kaur

first: Madhavi

last: Vuthoori

first: Richard

last: Bonneau

first: Paul

last: Shannon

first: Alok

last: Srivastava

first: Samuel

last: Donohoe

first: Leroy

last: Hood

first: Nitin

middle: S

last: Baliga


title: Temperature-dependent expression of internalin and internalin-like genes

city: Ithaca

first: Patrick

last: Mc Gann

state: NY

address: 412 Stocking Hall

department: Food Science

institute: Cornell University

summary:
Listeria monocytogenes, the etiological agent of listeriosis, is capable of growth and survival at temperatures ranging from 2 to 48oC, reflecting the diverse environments and host species inhabited by this organism. L. monocytogenes expresses up to 29 proteins, termed internalins, whose structure indicates they make good candidates for facilitating bacterial-host cell interactions. Considering the ubiquitous nature of this organism, we speculated that environmental temperature might serve as an important biological signal in controlling their expression. We therefore employed a subgenomic microarray to investigate the expression profiles of 24 members of the internalin gene family identified in L. monocytogenes 10403S. Competitive hybridization was performed between RNA extracted from 10403S grown to early stationary phase at 37oC, and 10403S grown to early stationary phase at 16oC, 30oC and 42oC. The data reveal that internalin genes can be divided into four broad categories such that (i) four internalin genes inlC2, inlD, lmo0331 and lmo0610 show temperature-dependent expression similar to sigB and the σB-dependent gene opuCA; (ii) three internalin genes inlA, inlB and inlC show temperature-dependent expression similar to the PrfA-dependent gene plcA; (iii) five internalin-like genes inlG, inlJ, lmo0327, lmo0514 and lmo1290 show unique temperature-dependent expression and (iv) twelve internalin genes show no difference in expression under the conditions investigated in this study. Our data also shows that the expression of many housekeeping genes can vary considerably under different temperatures, and normalization of both microarray and qRT-PCR data using housekeeping genes should be based on comprehensive experimental validation.
Keywords: Listeria monocytogenes, internalins, temperature, sub-genomic microarrays

overall_design:
To enhance the relative proportion of cells in log phase, for all experiments, each strain was initially grown in BHI at 37oC with shaking (200 rpm) to OD600=0.4, then diluted 1:100 into fresh BHI and grown to OD600=0.4.
.  A log phase culture of L. monocytogenes 1040S was diluted 1:100 in prewarmed BHI (16oC, 30oC, 37oC and 42oC) and subsequrently incubated with shaking (200 rpm) at the respective temperature until early stationary phase, defined as OD600=1.0 + 3 hrs for cells grown at 30 oC, 37 oC and 42 oC. For cells cultured at 16oC, early stationary phase was defined as OD600=1.0 + 6 hrs. Following incubation, RNA was stabilized by the addition of two volumes of RNAprotect™ (Qiagen, CA, USA). Bacterial cells were then harvested by centrifugation and stored at –80oC for no longer than 24 h before RNA isolation.
For each growth condition described above, three independent RNA isolations were performed on different days to provide distinct biological replicates. Each set of independent RNA samples was used to perform competitive hybridization microarray experiments. For each set of biological replicates, RNA from cells cultured at 37oC was hybridized to RNA from cells cultured at 16oC, 30oC and 42oC

last: McGann

first: Renata

last: Ivanek

first: Martin

last: Wiedmann

last: Boor


title: Small RNAs in Arabidopsis thaliana and its RISC complexes

status: Public on Feb 21 2007

city: Cold Spring Harbor

first: Gregory

middle: J.

last: Hannon

address: 1 Bungtown Rd

institute: Cold Spring Harbor Laboratory

type: Non-coding RNA profiling by high throughput sequencing

summary:
RNA populations in Arabidopsis thaliana and its RISC complexes
Keywords: Highly parallel pyrosequencing

overall_design: Small RNAs were prepared from Arabidopsis total extracts, AGO1 and AGO4 complexes, ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of 454 cloning primers and provided to 454 Life Sciences (Branford, CT) for sequencing. For technical details, see Qi, Y., He, X., Wang, X.J., Kohany, O., Jurka, J., and Hannon, G.J. 2006. Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443(7114): 1008-1012.

first: Yijun

last: Qi


title: Basal-like phenotype is not associated with patient survival in estrogen receptor negative breast cancers

status: Public on Feb 26 2007

city: New York

first: Sofia

middle: K

last: Gruvberger-Saal

address: 1130 St. Nicholas Ave., ICRC 406

department: Institute for Cancer Genetics

institute: Columbia University

summary:
INTRODUCTION: Basal-phenotype or basal-like breast cancers are characterized by basal epithelium cytokeratin (CK5/14/17) expression, negative estrogen receptor (ER) status and distinct gene expression signature. We studied the clinical and biological features of the basal-phenotype tumors determined by immunohistochemistry (IHC) and cDNA microarrays especially within the ER-negative subgroup.
METHODS: IHC was used to evaluate the CK5/14 status of 445 stage II breast cancers. The gene expression signature of the CK5/14 immunopositive tumors was investigated within a subset (100) of the breast tumors (including 50 ER-negative tumors) with a cDNA microarray. Survival for basal-phenotype tumors as determined by CK5/14 IHC and gene expression signature was assessed.
RESULTS: From the 375 analyzable tumor specimens, 48 (13%) were immunohistochemically positive for CK5/14. We found adverse distant disease-free survival for the CK5/14-positive tumors during the first years (3 years hazard ratio (HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was lost at the end of the follow-up period (10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19). Gene expression profiles of immunohistochemically determined CK5/14-positive tumors within the ER-negative tumor group implicated 1,713 differently expressed genes (p < 0.05). Hierarchical clustering analysis with the top 500 of these genes formed one basal-like and a non-basal-like cluster also within the ER-negative tumor entity. A highly concordant classification could be constructed with a published gene set (Sorlie's intrinsic gene set, concordance 90%). Both gene sets identified a basal-like cluster that included most of the CK5/14-positive tumors, but also immunohistochemically CK5/14-negative tumors. Within the ER-negative tumor entity there was no survival difference between the non-basal and basal-like tumors as identified by immunohistochemical or gene-expression-based classification.
CONCLUSION: Basal cytokeratin-positive tumors have a biologically distinct gene expression signature from other ER-negative tumors. Even if basal cytokeratin expression predicts early relapse among non-selected tumors, the clinical outcome of basal tumors is similar to non-basal ER-negative tumors. Immunohistochemically basal cytokeratin-positive tumors almost always belong to the basal-like gene expression profile, but this cluster also includes few basal cytokeratin-negative tumors.
Keywords: Disease state analysis

overall_design: 100 breast cancer tumor samples were analyzed on 2-color cDNA microarrays using the Stratagene Universal Human Reference RNA as the common reference sample.

first: Mervi

last: Jumppanen

first: Paivikki

last: Kauraniemi

first: Minna

last: Tanner

first: Par-Ola

last: Bendahl

first: Mikael

last: Lundin

first: Morten

last: Krogh

first: Pasi

last: Kataja

first: Ake

last: Borg

first: Marten

last: Ferno

first: Jorma

last: Isola


title: SAGE of cotton fibers and ovules.

status: Public on Feb 18 2007

city: Shanghai

first: Xiao-Ya

last: Chen

address: No. 300, Fenglin Road

department: Institute of Plant Physiology and Ecology

country: China

institute: Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

type:
Expression profiling by array
Expression profiling by SAGE

summary:
Transcriptomes fiber and ovules were compared by applying serial analysis of gene expression (SAGE).
Keywords: Tissue Comparison

overall_design: We constructed three SAGE libraries and sequenced 57321, 64188, and 69104 tags from fiber, Xu-142 ovule (ovule) and fl mutant ovules (fl) respectively of Upland Cotton, Gossypium hirsutum L. cv. Xu-142.

first: Jin-Ying

last: Gou

first: Ling-Jian

last: Wang


title: DNA methylation, RNA Pol2 binding and H3-K4me2 of human promoters in WI38 primary fibroblasts and sperm cells

status: Public on Feb 15 2007

city: Basel

first: Michaël

last: Weber

address: Maulbeerstrasse 66

department: Epigenetics

country: Switzerland

institute: Friedrich Miescher Institute

type: Genome binding/occupancy profiling by genome tiling array

type: Methylation profiling by genome tiling array

summary:
To gain insights into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase II and histone modifications at 24.000 promoters in primary human soma and germline cells.
Keywords: DNA methylation, MeDIP

overall_design: The chosen array from NIMBLEGEN (HG17_min_promoter array) represents 24134 putative human promoters, each covered by 15 oligonucleotides spanning 1.3 kb upstream and 0.2 kb downstream of the transcription start site. All experiments have been repeated two or three times, including one dye-swop.

first: Michael

middle: B

last: Stadler

last: Rebhan

first: Dirk

last: Schubeler


title: Resolving the resolution of array CGH

city: Vancouver

first: Bradley

middle: P

last: Coe

state: BC

address: 675 West 10th Ave

department: Cancer Genetics and Developmental Biology

country: Canada

institute: BC Cancer Research Centre

summary:
This entry is part of a study comparing various array CGH platforms.  In addition to the submitted data the following BT474 profiles were analyzed in this study: GSM69198 , GSM73557 and data from http://research.dfci.harvard.edu/meyersonlab/snp/snp.htm
Keywords: Comparitive Genomic Hybridization (CGH) Platform Comparison

overall_design: The samples included in this series represents an Agilent 244K CGH array dye swap pair of the breast cancer cell line BT474.  In the manuscript data was analyzed by averaging the two arrays.

first: Bauke

last: Ylstra

first: Beatriz

last: Carvalho

first: Gerrit

last: Meijer

first: Calum

last: MacAulay

first: Wan

last: Lam


title: Calcium/calmodulin-binding transcription factors in Arabidopsis

status: Public on Feb 16 2007

city: Loughborough

last: Nottingham Arabidopsis Stock Centre (NASC)

address: Sutton Bonington Campus

department: School of Biosciences, University of Nottingham

summary:
Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter.
Keywords: strain_or_line_design

overall_design: 5 samples

first: Hillel

last: Fromm


title: Expression data from MX treated Salmonella typhimurium TA100

status: Public on Feb 14 2007

city: Durham

first: William

last: Ward

address: 109 TW Alexander Dr

department: NHEERL

institute: United States EPA

summary:
We exposed Salmonella TA100 cells to 3 concentrations of MX that produced a linear concentration-response for mutagenesis with little cell killing.  We measured mutagenesis, survival, and global gene expression.  We used custom-spotted glass slides as our microarray platform and identified genes whose expressions were altered by MX treatment using three methods: (1) a Bayesian t-test, (2) an operon analysis that assumes if one gene in an operon is differentially expressed then all genes in that operon are differentially expressed, and (3) a monotonic-expression response to increasing doses of MX.  The resulting list of genes was analyzed for functional and KEGG pathway representation.
Keywords: dose response

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: Suspensions were incubated with MX at 3 concentrations with shaking at 120 rpm for 30 min at 37oC and then sampled for survival, mutagenesis, and RNA extraction.  For survival, a sample was diluted 10-6, and 100 µl of this dilution were plated in triplicate onto VBME medium supplemented with excess biotin and histidine [8].  For mutagenesis, 50 µl of the treated cells were plated in duplicate onto VBME supplemented with excess biotin and trace histidine [9].  Plates were incubated for 3 days, and the colonies were counted by an automatic colony counter.  Immediately after sampling for survival and mutagenesis, RNA was extracted and purified using the Institute of Food Research Microarray Facility protocol.

middle: O

first: Carol

middle: D

last: Swartz

first: Steffen

last: Porwollik

first: Sarah

last: Warren

first: Nancy

last: Hanley

last: McClelland

first: David

last: DeMarini


title: Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays

status: Public on Feb 13 2007

city: Amsterdam

first: Douwe

last: Molenaar

address: De Boelelaan 1085

department: Systems Bioinformatics

country: Netherlands

institute: Vrije Universiteit Amsterdam

summary:
Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-colour microarrays were used to screen for differentially expressed genes when grown on scFOS as compared to glucose (not a prebiotic). A significant up-regulation (8 to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a â-fructofuranosidase, a fructokinase, an á-glucosidase and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions, has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTS. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end-products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a â-fructofuranosidase for scFOS degradation.
Keywords: comparative transcriptomics, carbohydrate metabolism

overall_design: Two culture conditions (growth on glucose or scFOS) and 2 biological replicates per culture condition. Cultures grown on glucose were sampled at OD600=1 and cultures grown on scFOS were sampled at OD600=1 and OD600=1.5. The hybridisation scheme has a circular design in which each sample is labeled once with Cy3 and Cy5.

first: Delphine

last: Saulnier

first: Willem

last: De Vos

first: Glenn

middle: R

last: Gibson

first: Sofia

last: Kolida


title: Analysis of Rice microRNAs Expression under Drought Stress

first: Youxin

last: Jin

address: 320 Yueyang Road

institute: Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

summary:
We used this microarray to monitor rice miRNA expression profile under osmotic stress. Most of rice miRNAs have no significant change. miR-169f showed osmotic-responsive up-regulation. However, the others members of miR-169 family displays no responses to the osmotic stress.
Keywords: microRNA,rice,osmotic stress,miR-169

overall_design: The six selected time points after PEG treatment were 0h, 0.5h, 2h, 6h, 24h and 48h. For each sample, two independent small RNA libraries (biological replications) were generated and two independent reverse transcription/amplifications/hybridizations (technical replications) were performed for each library. Thus, for each sample data was collected from four independent hybridizations.

first: Botao

last: Zhao

first: Ruqiang

last: Liang

first: Liangfa

last: Ge

first: Wei

last: Li

first: Huasheng

last: Xiao

first: Hongxuan

last: Lin

first: Kangcheng

last: Ruan


status: Public on Feb 27 2007

city: Kyoto

first: Hiromu

last: Takematsu

state: Kyoto

address: Shogoin-Kawahara 53

department: Human Health Science

institute: Kyoto Univ. Sch. of Medicine

summary:
Comparison of the expression profiles of human B cell lines.
Keywords: cell type comparison

overall_design: 6 samples - all samples compared to human universal  reference RNA. No replications.

first: Yasunori

last: Kozutsumi

first: Akemi

last: Suzuki

first: Reiko

last: Fujinawa

first: Harumi

last: Yamamoto


title: Band-array detection of chromosomal rearrangements in diploid yeast cells exposed to ionizing radiation

status: Public on Feb 10 2007

first: Juan Lucas

last: Argueso

state: SP

address: 371 CARL Building, Box 3054

department: Molecular Genetics and Microbiology

institute: Duke University Medical Center

summary:
At the organismal level, genome rearrangements are usually deleterious and are often associated with disease.  Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification.  DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes.  Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes.  Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent.  The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA.  Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ).  We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired.  Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities.
Keywords: Band-array

overall_design: Each array in this series corresponds to the DNA of a yeast chromosomal band excised from a pulse-field gel (CHEF). Chromosomal aberrations identified in radiation survivors were analyzed by microarray to reveal which regions of the genome were present in the new chromosomes. The DNA enriched in the specific band appears in the arrays as continuos segments of spots with highly positive Log2 Red/Green ratios.

first: Juan

first: James

last: Westmoreland

first: Piotr

last: Mieczkowski

first: Malgorzata

last: Gawel

first: Thomas

last: Petes

first: Michael

last: Resnick


title: Comparison of S. aureus PVL-positive and PVL-negative isogenic strains in exponential and stationary phase of growth

city: Houston

first: Gabriela

last: Bowden

state: TX

address: 2121 Holcombe Blvd

department: CEMB

institute: Texas A&M

summary:
The Staphylococcus aureus Panton Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model. Here we show that PVL is sufficient to cause pneumonia and that the expression of this leukotoxin induces global changes in transcriptional levels of genes encoding secreted and cell-wall-anchored staphylococcal proteins, including the lung inflammatory factor staphylococcal protein A (Spa).
Keywords: comparative transcription profile in the presence or absence of PVL toxin

overall_design: We conducted three independent microarray experiments (biological replicates) for each strain in exponential and stationary phase of growth. We calculated the fold change as the ratio between signal averages of three PVL-positive (LUG881) cultures (starting from a single colony)  and three PVL-negative (LUG776) cultures (starting from a single colony) for each phase of growth.

first: Maria

last: Labandeira-Rey

first: Florence

last: Couzon

first: Sandrine

last: Boisset

first: Eric

last: Brown

first: Michele

last: Bes

first: Yvone

last: Benito

first: Elena

last: Barbu

first: Vanessa

last: Vazquez

first: Magnus

last: Hook

first: Jerome

last: Etienne

first: Francois

last: Vandenesch


title: Campylobacter jejuni exposure to 0.25mM GSNO

status: Public on Feb 17 2007

city: Norwich

first: Bruce

middle: MacKenzie

last: Pearson

state: Norfolk

address: Colney Lane

department: Food Safety

institute: Institute of Food Research

summary:
Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 150 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 5/10/15/45 minute exposure samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis.
Keywords: Time Course

sample_organism: Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819

overall_design:
Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 150 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure, 30 ml samples of both treated and untreated cells were  mixed immediately on ice with 3.56 ml 100% ethanol and 185 µl phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier.

Two independent RNA preparations (biological replicates) of each sample were labelled and hybridized to glass-slide microarrays. Equal quantities of RNA from control and GSNO treated cultures were labelled by using nucleotide homologues of dCTP containing either Cy3 or Cy5 florescent dye (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while another  sample was labelled with Cy5-dCTP. RNA (15 µg) was primed with 5 µg pd(N)6 random hexamers (Amersham Biosciences) and made to 12.4 µl total volume with water and supplemented on ice with 3 µl 10x RT buffer (Invitrogen), 0.6 µl 50x dNTPs mixture (25 mM each dATP, dTTP, dGTP and 10 mM dCTP), 3 µl 0.1 M DTT, 2 µl 1mM Cy3 or Cy5 (Perkin Elmer), and 200U Superscript II RNase H reverse transcriptase (Invitrogen). Incubation was  at 37 °C overnight. Labelled cDNA was purified using a Qiaquick purification kit (Qiagen) and dried before being resuspended in 19.5 µl water, 2.25 µl human Cot1 DNA (Invitrogen), 4.5 µl 20x SSC, 0.72 µl 1 M HEPES, pH 7.0, 0.68 µl 10 % SDS and 3 µl Denhardt solution. Samples were heated for 3 min in a boiling water bath, cooled at room temperature for 5 min and centrifuged at maximum speed in a microfuge for 2 min to remove particulates. This mixture was put onto the microarray slide, sealed with a coverslip in a hybridization chamber and incubated for 18 h at 63 °C. Following hybridization, microarray slides were washed briefly in pre-warmed (60 °C) 1x SSC/0.03 % SDS to remove the coverslip and then washed twice for 5 min in each of the following buffers: a) 1x SSC/0.03 % SDS (pre-warmed to 60 °C), b) 0.2x SSC and finally c) 0.05x SSC. Microarray slides were dried by centrifugation at 300 x g for 15 min before scanning.
Microarrays were scanned using an Axon 4000A scanner and images were acquired using GenePixPro 3.0 software (Axon). All microarray data were filtered to remove poor-quality data using four sequential cut-off values (Mark Reuter, IFR, personal communication). All data values were from features above 50 µm in diameter, with sum of medians above 50, regression coefficient squared values above 0.2 and with a sum of the signal : noise ratio greater than 3. A control experiment (comparison of two mRNA samples from replicated independent cultures of the wild-type strain) was used to estimate the boundaries between genes that were equally and differentially expressed in the two samples (Holmes et al., 2005). For the analyses described here, this boundary would detect changes equivalent to about 3.3-fold greater intensity in one of the fluorescence channels.

first: Claire

middle: E

last: Monk

first: Francis

last: Mulholland

first: Robert

last: Poole


title: Transcriptional Profile of Lactobacillus helveticus grown in milk vs. MRS medium

city: Madison

first: James

last: Steele

email: jlsteele@wisc.edu, vvsmeianov@wisc.edu

state: WI

address: 1605 Linden Dr.

institute: UW-Madison

summary:
L. helveticus is used to modulate cheese flavor and as a starter organism in certain cheese varieties. Our group has compiled a draft (4x) sequence for the 2.4 Mb genome of an industrial strain L. helveticus CNRZ32. The primary aim was to investigate expression of 168 completely sequenced genes during growth in milk and MRS medium using microarrays.  Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays.  Additionally, the entire draft genome sequence was used to produce tiled microarrays where the non-interrupted sequence contigs were covered by consecutive 24-mer probes.
Keywords: growth conditions response

overall_design: Milk and MRS cultures were incubated at 42oC until cells reached mid-log phase. Three independent growth experiments in milk and MRS were done; two identical samples were taken from each culture and used for the RNA isolation.  A total of six RNA samples for each growth condition were used to independently produce labeled cDNA for microarray experiments.

first: Vladimir

middle: V

last: Smeianov

first: Patrick

last: Wechter

first: Jeffery

last: Broadbent

first: Joanne

last: Hughes

first: Beatriz

last: Rodríguez

first: Tove

last: Christensen

first: Ylva

last: Ardö


title: CGH detection of chromosomal rearrangements in diploid yeast cells exposed to ionizing radiation

status: Public on Feb 08 2007

first: Juan Lucas

last: Argueso

summary:
At the organismal level, genome rearrangements are usually deleterious and are often associated with disease.  Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification.  DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes.  Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes.  Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent.  The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA.  Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ).  We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired.  Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities.
Keywords: CGH-array

overall_design: Diploid G-2 yeast cells were exposed to 80 krad of ionizing radiation and plated on rich media to obtain survivor colonies.  Genomic DNA from each of 37 survivors (Cy5/red; JW1 to JW13, and A1 to A24) was competitively hybridized to DNA from the parent diploid strain (Cy3/green).  Gains of genomic segments in the survivors were detected as continuous regions of positive Log2 Red:Green ratios, while losses were detected as negative Log2 Red:Green ratios.

first: Juan

middle: L

last: Westmoreland

first: Piotr

last: Mieczkowski

first: Malgorzata

last: Gawel

first: Thomas

last: Petes

first: Michael

last: Resnick


title: WT vs. ACE2 knockout (grown in hyphal form)

city: Dublin

first: Geraldine

last: Butler

address: Belfield

institute: University Colege Dublin Conway Institute

summary:
We have used DNA microarray technology to compare the expression of over 6000 genes between C. albicans WT and ACE2 knockout strains in the hyphal form.
Keywords: Gene expression comparison

overall_design: We analyzed 5 arrays for the hyphal-form comparison experiment. No dye-swaps were performed.

first: Siobhan

last: Mulhern

first: Mary

last: Logue


title: Circadian liver_wt and Clock mutants

city: San Diego

first: John

last: Walker

state: CA

address: 10675 John Jay Hopkins

institute: Genomics Institute of the Novartis Research Foundation

summary:
One hundred ninety wildtype male C57BL/6J mice age 7-10 weeks were purchased from Jackson Laboratory and entrained to a 12:12 light:dark cycle for 2 weeks. Mice were placed in light-tight boxes on a 12:12 LD cycle for 4 weeks, then released into constant darkness. Starting 30 hours after entry into DD (CT18), tissues from 5 (skeletal muscle) or 10 (liver or SCN) wildtype mice were collected every 4 hours for 48 hours, for a total of 12 timepoints. At timepoints 34 through 58 hours in DD, tissues from age-matched male C57BL/6J Clock/Clock homozygous mutant mice that had been treated with the same light entrainment protocol as the wildtype were collected. Tissues were collected from 5 Clock/Clock mutants at each timepoint except for 34 and 46 hours after the onset of DD, when tissues from 10 Clock/Clock mice were collected and run as independent replicates. Mice were sacrificed by cervical dislocation, and the optic nerves were severed in complete darkness; brain dissection was performed using illumination from an infrared viewer (FJW Industries, Palatine, IL). SCNs were dissected out, pooled at a density of 5 per tube in 100 ?l RNAlater (Ambion, Austin, TX), frozen on dry ice, and stored at –80?C until use.
Keywords: time course

overall_design: duplicates consisted of pools from independent mice

first: Erin

last: McDearmon

first: Satchidananda

last: Panda

first: Jessica

last: Andrews

first: Martin

last: Straume

first: Karyn

last: Esser

last: Hogenesch

first: Joseph

last: Takahashi


title: Gene Expression Profiling of Immune System Cells Stimulated with Components of Lactobacillus acidophilus

status: Public on Feb 09 2007

city: Guelph

first: Shayan

last: Sharif

state: Ontario

address: Ontario Veterinary College

department: Department of Pathobiology

institute: University of Guelph

summary:
Lactobacillus acidophilus (L. acidophilus) is one of major commensal bacteria in chicken intestine. Lactobacilli have been shown to exert health-promoting and immunostimulating activities. To examine the immunostimulating effects of probiotics, chicken cecal tonsil cells and splenocytes were stimulated in vitro with DNA, peptidoglycan, and cell envelope extracted from L. acidophilus. These bacterial constituents are known to stimulate innate defence mechanisms. Several gene clusters including chemokines and their receptors, antigen processing and presentations, apoptosis related genes were identified in the present study. These differentially expressed genes are candidates for detailed hypothesis-driven investigation of genes elucidating molecular/cellular mechanisms of effects of commensal bacteria on gut immune system in chickens.
Keywords: Gene expression profiling of stimulated and unstimulated cells

overall_design: Cells were collected at 3, 6, and 12 hrs, and total RNA was isolated. A chicken immune microarray was used to study gene expression of stimulated cells compared to unstimulated controls. Four biological replicates for each treatment group in addition controls were labelled with either Cy5 or Cy3 with dye swap. Gene signal intensity was globally normalized by LOWESS and expressed on natural log scale. A mixed model including treatment, time, array, subgrid (random effect), dye, and all interactions among treatment, time was used to identify differentially expressed genes between treatments and control, at the 5% significance level.

first: Huaijun

last: Zhou

first: Joshua

last: Gong

first: Jennifer

last: Brisbin

first: Parviz

last: Sabour

first: Hai

last: Yu

first: Aimie

middle: J

last: Sarson


title: SHREC, an Effector Complex for Heterochromatic Transcriptional Silencing

city: Bethesda

first: Shiv

last: Grewal

state: MD

address: NCI bldg 37 Rm 6068 9000 Rockville Pike

department: LBMB

institute: NCI

summary:
ChIP-chip analyses of Clr1, Clr2, Clr3, Ccq1, and Mit1 across the S. pombe genome.
Keywords: ChIP-chip

overall_design: Chromatin immunoprecipitated DNA recovered with antibody against epitope-tagged protein from wild-type asynchronous growing fission yeast cells and whole cell extracts DNA were amplified by random priming and respectively labeled with Cy5 and Cy3. Labeled DNA samples were mixed and hybridized onto a 44k pombe Agilent oligo array. Data were processed using Agilent scanner and Feature Extraction software.

first: Tomoyasu

last: Sugiyama

first: Hugh

last: Cam

first: Rie

first: Ken-ichi

last: Noma

last: Zofall

first: Ryuji

last: Kobayashi

middle: I


title: Comparison of expression profiles in response to blue, UV-A, and UV-B in the swollen hypocotyls of turnip

city: Bunkyo-ku

first: Saneyuki

last: Kawabata

state: Tokyo

address: 1-1-1 Yayoi

institute: University of Tokyo

summary:
UV-A mediated regulation of anthocyanin biosynthesis was investigated in swollen hypocotyls of the red turnip ‘Tsuda’. The shaded swollen hypocotyls which contained negligible anthocyanin were exposed to artificial light sources including low fluence UV-B, UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A, and blue plus red. Among these lights, only UV-A induced anthocyanin biosynthesis and co-irradiation of red or far-red with UV-A did not affect the extent of UV-A induced anthocyanin accumulation. The expression of phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanon 3-hydrocylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219) and anthocyanidin synthase (ANS; EC 1.14.11.-) genes were increased with time during a 24 hour exposure of UV-A. In contrast, irradiation of red, blue, UV-B, and a combination of blue with red failed to induce CHS expression. Microarray analysis showed that only a few genes, including chalcone synthase and flavanon 3-hydroxylase were induced significantly by UV-A, while a separate set of many genes was induced by low fluence UV-B. The UV-A specific induction of anthocyanin biosynthesis and the unique gene expression profile upon UV-A irradiation as compared with blue and UV-B demonstrated that the observed induction of anthocyanin biosynthesis in red turnips was mediated by a distinct UV-A specific photoreceptor, but not by phytochromes, UV-A/blue photoreceptors, or UV-B photoreceptors.
Keywords: light response

overall_design: Direct comparisons were made between blue and dark, UV-A and dark, or UV-B and dark treatments. The dark control sample was labeled with Cy5 and the other samples were labeled with Cy3. In addition to these hybridizations, three concurrent split-control hybridizations of the dark control were made to correct gene specific dye-bias. Three biological replicates were made for each comparison. For each biological replicate, RNA was extracted from more than five plants.

first: Yuhua

last: Li

first: Bo

first: Zhiru

last: Xu

first: Haifang

last: Yan


title: High-resolution genome-wide mapping of histone modifications.

status: Public on Feb 06 2007

first: Keji

last: Zhao

address: Rockville Pike

institute: NHLBI, NIH

summary:
The expression patterns of eukaryotic genomes are controlled by their chromatin structure, consisting of nucleosome subunits in which DNA of approximately 146 bp is wrapped around a core of 8 histone molecules. Post-translational histone modifications play an essential role in modifying chromatin structure. Here we apply a combination of SAGE and chromatin immunoprecipitation (ChIP) protocols to determine the distribution of hyperacetylated histones H3 and H4 in the Saccharomyces cerevisiae genome. We call this approach genome-wide mapping technique (GMAT). Using GMAT, we find that the highest acetylation levels are detected in the 5' end of a gene's coding region, but not in the promoter. Furthermore, we show that the histone acetyltransferase, GCN5p, regulates H3 acetylation in the promoter and 5' end of the coding regions. These findings indicate that GMAT should find valuable applications in mapping target sites of chromatin-modifying enzymes.
Keywords: SAGE-ChIP

overall_design: Using histones from wild-type S. cerevisiae as well as from yeast with a GCN5 deletion, we generated four GMAT libraries from the ChIP DNA with the antibodies against diacetylated (K9/K14) histone H3 and tetra-acetylated histone H4 tails.

first: Tae-Young

last: Roh

first: W

middle: C

last: Ngau

first: K

last: Cui

first: D

last: Landsman


title: Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping

summary:
The identity and developmental potential of a human cell is specified by its epigenome that is largely defined by patterns of chromatin modifications including histone acetylation. Here we report high-resolution genome-wide mapping of diacetylation of histone H3 at Lys 9 and Lys 14 in resting and activated human T cells by genome-wide mapping technique (GMAT). Our data show that high levels of the H3 acetylation are detected in gene-rich regions. The chromatin accessibility and gene expression of a genetic domain is correlated with hyperacetylation of promoters and other regulatory elements but not with generally elevated acetylation of the entire domain. Islands of acetylation are identified in the intergenic and transcribed regions. The locations of the 46,813 acetylation islands identified in this study aresignificantly correlated with conserved noncoding sequences (CNSs) and many of them are colocalized with known regulatory elements in T cells. TCR signaling induces 4045 new acetylation loci that may mediate the global chromatin remodeling and gene activation. We propose that the acetylation islands are epigenetic marks that allow prediction of functional regulatory elements.
Keywords: SAGE-ChIP

overall_design: Using GMAT, we determined the genome-wide distribution of K9/K14 diacetylated histone H3 in resting and activated human T cells.

first: S

last: Cuddapah


title: Microarray analyses that reveal the population of dendritic RNAs and their redistribution by neural activity

city: Machida-shi

first: Mineo

last: Matsumoto

address: 11 Minamiooya

institute: Mitsubishi Kagaku Institute of Life Sciences

summary:
Subcellular localization of RNA is an efficient way to localize proteins to a specific region of a cell. The dendritic localization of RNAs underlies the establishment and maintenance of the synaptic functions of neuronal cells. A requirement for dendritic RNA localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. In spite of several attempts to identify these RNAs, the population of RNA species present in dendrites as a whole has not been well described. Here we show the results of microarray analyses with RNAs isolated from heavy portion of polysome (HP) fraction where RNA granules are enriched in and synaptosome fraction, prepared from the rat brain. These analyses revealed the complex nature of the dendritic RNA population, which included RNAs that were not expected to be in the dendrites. Neural activity caused by an electroconvulsive shock triggered a redistribution of the population of dendritic transcriptome towards the area of overlap between the HP and the synaptosome, which is assumed to be neck of spine. This redistribution may accompany some changes in the translatability of those transcriptome, which suggests complex mechanisms of local translation in response to synaptic inputs.
Keywords: Comparisons of RNAs that are presumed to be derived from neuronal dendritic parts (HP and D4) with those from somas (LP and C2).

overall_design: The HP and D4 are the fractions that are assumed to be abundant of dendritic mRNAs in neurons, whereas LP and C2 are their control fractions, respectively. In both of the HP vs LP- and D4 vs C2- microarrays, two sets of the experiments were done to very the reproducibility (Replicate 1 and 2). In each of the Replicates, dye swap experiments were done to avoid a dye-specific hybridization preference. The data presented in total of four sets of 2-color microarrays out of two Replicates in each case were averaged as described in Sample_description.


title: E. coli expression data from gnotobiotic ceca 14d post-inoculation

status: Public on Feb 02 2007

city: Saint Louis

first: Christopher

middle: Swanson

last: Reigstad

address: 4444 Forest Park Blvd.

department: Center for Genome Sciences

institute: Washington University in St. Louis

summary:
Transcriptional expression of MG1655 and UTI89 harvested from monoassociated gnotobiotic mouse ceca (female C57Bl/6)
Keywords: strain comparison analysis

overall_design: Transcriptional profiling of genes shared between two different E. coli strains (after application of electronic probe mask on raw data)

first: Scott

last: Hultgren

first: Jeffrey

last: Gordon


title: Rice japonica gene expression array across eleven tissues

city: New Haven

first: Xiangfeng

last: Wang

state: CT

address: 352 OML, 165 Prospect st

department: MCDB

institute: Yale University

type: Expression profiling by genome tiling array

summary:
To validate transcription of the japonica non-exonic TARs and to understand their transcriptional relation with annotated genes, we constructed a new array (designated the re-array) to surrogate 44,385 non-TE gene models and 25,313 TARs in japonica each with five independent 36mer probes. Using the re-array, we obtained triplicate expression estimates from 11 rice tissue.
Keywords: gene expression

overall_design:
To facilitate transcriptional profiling of all transcription units (gene models and novel TARs), we constructed the re-array in which five probes were used to represent each gene model and novel TAR. To ensure that the probes are specific to its target and are free of secondary structures, we used the software OligoArray 2.1 to select probes (8). We used the coding sequence (CDS) for each gene model. For those that generate alternative transcripts, we picked the longest CDS to represent the loci. We started with 44,497 gene models and 25,346 novel TARs, which were used as the database sequence to search against. Two separate runs were initiated, one for the gene models and the other for the novel TARs, using the following parameters:
(i) Oligo length: 36,
(ii) Maximum distance accepted between the 5' end of the oligo and the 3' end of the input sequence: 2000,
(iii) Minimum oligonucleotide Tm: 80,
(iv) Maximum oligonucleotide Tm: 95,
(v) Temperature to use during secondary structure prediction (an oligo will be rejected if it can fold into a stable secondary structure at this temperature): 65,
(vi) Threshold to report putative cross-hybridizations (all targets hybridizing with a given oligo with a Tm exceeding this threshold are reported): 70,
(vii) Minimum oligonucleotide GC content: 40,
(viii) Maximum oligonucleotide GC content: 73 for gene models; 77 for TARs,
(iv) List of prohibited sequences to mask in the input sequence: Runs of five or more consecutive single nucleotides,
(x) Minimum distance between the 5' end of two adjacent oligos: 1.
For those transcription units for which the software could not generate five probes, the remaining probes were selected from the original tiling arrays that were in the 75th percentile and met most of the above criteria (hence the name ‘re-array’). For example, if the software reported three probes for a given gene model, the remaining two were picked from the tiling experiment. As a negative control, we chose from the full-genome tiling arrays 10,000 randomly selected probes and 9000 probes that exhibit intensities below the median and are within introns of PASA gene models. All probes were synthesized into a single array and hybridized in triplicates to cDNA target derived from 10 rice tissue types, namely, light-grown seedling, dark-grown seedling, seedling root, leaf, Xoo-infected leaf, flag leaf, whole flower (floret), carpel, developing seed, and suspension cultured cell.


title: Expression data from Cerebral Cortex or Hippocampus of COX-1 or COX-2 null mice

status: Public on Feb 05 2007

middle: Dennis

last: Toscano

address: 9 Memorial Drive Room 1S126

department: National Institute on Aging

institute: National Institutes of Health

summary:
To further understand the physiological role of cyclooxygenase (COX), the critical enzyme involved in the production of eicosanoids from arachidonic acid, we performed microarray analysis of gene expression in the cerebral cortex and hippocampus of mice deficient in COX-1 (COX-1-/-) or COX-2 (COX-2-/-). Expression of lipid (ATP citrate lyase, acetyl-CoA acetyltransferase, hydroxyacyl-CoA dehydrogenase) and homocysteine (methionine adenosyltransferase, S-adenosylhomocysteine hydrolase) metabolism genes was increased in the cerebral cortex of COX-2 -/- mice. Further, expression of GABAergic neurotransmission genes (GABA transporter 3, GABA-A receptor subunit ?1) was altered in the cerebral cortex and hippocampus of COX-2 -/- mice. A COX isoform specific effect was observed in the expression of Janus Kinase (JAK) 1 and 2. COX-1 -/- mice exhibited an increase in JAK1 expression while COX-2 -/- mice exhibited a decrease in JAK2 expression, an observation consistent with a previously demonstrated COX isoform specific effect on the expression of NF-kB. In summary, this study demonstrates the wide ranging effects of genetic deletion of COX isoforms in the mouse brain and suggests that inhibition of COX activity may alter the profile of gene expression in specific areas of the brain.
We used nylon cDNA microarrays to detail the global programme of gene expression in the Cerebral Cortex and Hippocampus of COX-1 and COX-2 null (knockout) mice
Keywords: brain, cerebral cortex, hippocampus, COX-1 WT, COX-1 KO, COX-2 WT, COX-2 KO and COX-2 heterozygous

overall_design: Mice were sacrificed at 12 weeks of age and the brain was dissected into cerebral cortex and hippocampus. We analyzed the overall gene expression from the cortex and hippocampus of COX-1 +/+, COX-1 -/-, COX-2 +/+, COX-2 -/-, and COX-2 +/-

first: Francesca

last: Bosetti

first: Vinaykumar

last: Prabhu

first: Kevin

middle: G

last: Becker

first: Robert

last: Langenbach


title: Global analysis of the relationship between the binding of Bas1p and meiosis-specific double-strand DNA breaks in yeast

status: Public on Feb 01 2007

city: Chapel Hill

first: Jason

middle: D.

last: Lieb

department: Biology

institute: Univeristy of North Carolina at Chapel Hill

summary:
In the yeast Saccharomyces cerevisiae, certain genomic regions have very high levels of meiotic recombination (hot spots). The hot spot activity associated with the HIS4 gene requires the Bas1p transcription factor. To determine whether this relationship between transcription factor binding and hot spot activity is general, we used DNA microarrays to map all genomic Bas1p binding sites and to map the frequency of meiosis-specific double-strand DNA breaks (as an estimate of the recombination activity) of all genes in both wild-type and bas1 strains. We identified sites of Bas1p-DNA interactions upstream of 71 genes, many of which are involved in histidine and purine biosynthesis. Our analysis of recombination activity in wild-type and bas1 strains showed that the recombination activities of some genes with Bas1p binding sites were dependent on Bas1p (as observed for HIS4), whereas the activities of other genes with Bas1p binding sites were unaffected or were repressed by Bas1p. These data demonstrate that the effect of transcription factors on meiotic recombination activity is strongly context dependent. In wild-type and bas1 strains, meiotic recombination was strongly suppressed in large (25- to 150-kb) chromosomal regions near the telomeres and centromeres and in the region flanking the rRNA genes. These results argue that both local and regional factors affect the level of meiotic recombination.
Keywords: ChIP-chip

overall_design: ChIP-chip

first: Piotr

middle: A

last: Mieczkowski

first: Margaret

last: Dominska

first: Michael

last: Buck

first: Jennifer

last: Gerton

first: Thomas

last: Petes


title: Arabidopsis thaliana small RNAs sequences identified using high-throughput 454 sequencing technology

first: James

last: Carrington

address: 975 North Warson Road

institute: Donald Danforth Plant Science Center

summary:
To determine the extent to which the major small RNA pathways functions across the Arabidopsis thaliana genome, small RNA populations from several tissues of wild-type (wt) and mutant plants were amplified by RT-PCR and sequenced using high-throughput 454 sequencing technology.
Keywords: small RNAs, high-throughput sequencing

overall_design: Amplicons were prepared by 5' and 3' adaptor ligation and RT-PCR using small RNA fractions from inflorescence tissue (containing stage 1-12 flowers) of wt Col-0 plants, mutants with defects in each DCL gene (dcl1-7, dcl2-1, dcl3-1, dcl4-2), and mutants with defects in each RDR gene for which a function has been established (rdr1-1, rdr2-1, rdr6-15).  Amplicons from whole seedlings (3 day post-germinations) were prepared from Col-0 and rdr6-15 plants. Small RNA preparations from leaf samples of Col-O that were either uninoculated or inoculated by Pseudomonas syringae pv tomato (DC3000hrcC) for 1 hr and 3 hr were also sequenced.

first: Kristin

last: Kasschau

first: Noah

last: Fahlgren

first: Elisabeth

last: Chapman

middle: M

last: Sullivan

middle: S

last: Cumbie


title: Response to plant activator in Arabidopsis

city: Kaga-gun, Okayama

first: Yoshihiro

last: Narusaka

address: 7549-1, Kibityuo-town Yoshikawa

institute: Research Institute for Biological Sciences

summary:
To develop a screening system for plant activators, which are novel substances that protect plants by enhancing their inherent disease-resistance mechanisms, we performed analysis using an Arabidopsis microarray consisting of 1200 full-length cDNA clones representing putative defense-related and regulatory genes.　A total of 1.2K potential biotic and abiotic stress-related genes were selected from the genes covered by the Arabidopsis 7K array (RIKEN, Japan) and Arabidopsis oligo microarray (Agilent Technologies, USA) for this study. Arabidopsis wild-type plants (ecotype Columbia; Col-0) were grown in soil for 28 days in a growth chamber at 22｡C under a 12-h light/ 12-h dark cycle. Arabidopsis plants were applied a foliar spray with 5 mM SA, 0.1 mM MeJA, 1 mM ethephon, 0.5 mM BTH, 10 mM BABA and 1 mM INA. Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester  and 2,6-dichloroisonicotinic acid activated plant defense responses via the salicylic acid (SA)-dependent signaling pathway, and _-aminobutyric acid  triggered a primed state in the plant that enables more efficient activation of the SA-, jasmonic acid- and ethylene-signaling pathway. These results suggest that this novel system can be used to screen for candidate plant activators.
Keywords: time course, dose response

overall_design:
A total of 1.2K potential stress-related genes that encode known or putative protein were selected for this study from the genes covered by Arabidopsis 7K array (RIKEN, Japan) and Arabidopsis oligo microarray (Agilent technologies, USA), which these genes include biotic and abiotic stress-inducible genes.
Complementary DNA was synthesized and labeled with either Cy5 for the treated samples or Cy3 the untreated plants grown in parallel. No dye swaps were carried out.The microarrays were spotted using the microarray stamping machine (model SpotArray24; PerkinElmer, Wellesley, MA, USA). Hybridization was carried out in our laboratory, and hybridized chips were scanned on a ScanArray Lite scanner (PerkinElmer, Wellesley, MA, USA)  and quantified using ScanArray Express ver.3 software. We calculated the median of the pixel intensities for each spot.

first: Mari

first: Hiroshi

last: Abe

first: Masatomo

last: Kobayashi


title: Expression data from time courses of E. coli MG1655 and UTI89 in vitro

first: Christopher

last: Reigstad

summary:
Transcriptional expression of MG1655 and UTI89 harvested from various time points during aerobic or anaerobic growth in Luria-Bertani medium
Keywords: time course analyses (aerobic and anaerobic)

overall_design: Transcriptional profiling time course analysis of genes shared between two different E. coli strains (after application of electronic probe mask on raw data)

first: Scott

last: Hultgren

first: Jeffrey

last: Gordon


title: DNA copy numbers of malignant glioma in culture

first: Philip

last: De Witt Hamer

address: PO Box 22660

department: Neurosurgery

institute: Academic Medical Center Amsterdam

summary:
Therapeutic screening of potential anticancer agents relies on representative cancer models. In vitro cell culture models have been long questioned to be representative for human malignant glioma. Therefore, in the present study genomic profiles of both short-term (2 weeks; n=8) and long-term (6 and 12 weeks; n=3) primary cell cultures and spheroid cultures were compared with their parental malignant gliomas. Cancer genomic profiles were obtained by 6400 BAC array comparative genomic hybridization. In 7 out of 8 short-term primary cell cultures, the genomic profiles clustered further from their parental tumors than the spheroid cultures. In 4 out of 8 samples, the changes were substantial and included chromosomal regions associated with prognosis and therapeutic response. The average correlation coefficients between parental tumor profiles and spheroid profiles was 0.89 (range: 0.79 to 0.97), whereas those between parental tumors and cell cultures was 0.62 (range: 0.10 to 0.96). In 2 out of 3 primary cell cultures progressive genetic changes appeared at 6 and 12 weeks after initial preservation, whereas the spheroid cultures were genetically stable throughout. It is concluded that the cancer genomic profile of primary cell cultures from malignant glioma is inconsistent with the parental tumor’s profile already after 2 weeks with subsequent progressive genetic changes. Because malignant glioma spheroids are genetically stable, biological characteristics of the parental tumor are better reflected. This indicates that the spheroid model is better for therapeutic screening.
Keywords: comparative genomic hybridization

overall_design: Two in vitro culture models (primary cell culture and organotypic spheroid culture) and their parental tumor were compared in whole-genome DNA copy number profile. Malignant glioma surgical specimens from 8 patients were divided in parental tumor (T), primary cell culture (C) and organotypic spheroid culture (S). After 2 weeks, genomic DNA was extracted from culture harvests. For 3 of 8 surgical specimens (patient 55, 58, 60) cultures were extended to 6 and 12 weeks to determine DNA copy number changes in time. Primary cell culture harvests at 2, 6 and 12 weeks were named C1, C2, C3 and organotypic spheroid cultures harvests at 2, 6 and 12 weeks were named S1, S2, S3.


title: Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in yeast

first: Jason

last: Lieb

summary:
In eukaryotes, meiotic recombination events are distributed non-randomly in the genome with certain regions having high levels of recombination (hotspots) and others having low levels (coldspots).  Species with similar DNA sequences (for example, chimpanzees and humans) can have strikingly different patterns of hotspots and coldspots.  Below, using a microarray analysis that allows us to measure the frequency of the meiosis-specific double-strand DNA breaks (DSBs) of all 6000 yeast genes, we show that mutation of a single gene (SIR2), which encodes a histone deacetylase, significantly changes DSB frequencies of 12% of yeast genes, elevating DSBs of 5% and reducing DSBs of 7%.  Many of the genes with repressed recombination are located in large (50-100 kb) regions located near, but not at, the telomeres.  Some of the genes with altered frequencies of DSBs (including the ribosomal RNA gene cluster) are known targets of Sir2p deacetylation in the wild-type strain.
Keywords: ChIP-chip

first: Piotr

last: Mieczkowski

last: Dominska


title: Interference of PTS components with PrfA of L.monocytogenes

city: Wuerzburg

first: Biju Joseph

last: Ampattu

address: Josef-Schneider Str 2

department: Department of Microbiology

institute: University of Wuerzburg

summary:
Analysis of L. monocytogenes ptsH, hprK and ccpA mutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. The hprK mutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing media whereas expression of these genes was even slightly down-regulated in the ccpA mutant when compared to the wild-type strain. The ptsH mutant could only grow in a rich culture medium and here the PrfA-dependent genes were up-regulated as in the hprK mutant. As expected, HPr-Ser-P was not produced in the hprK and ptsH mutants and synthesized at a similar level in the ccpA mutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity when L. monocytogenes was grown in minimal medium with different PTS carbohydrates.
Comparison of the transcript profiles of the hprK and ccpA mutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in the hprK mutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that not component(s) of the CCR or the common PTS pathway but rather of subsequent steps seem to be involved in the modulation of PrfA activity
Keywords: PTS components PrfA Listeria monocytogenes

overall_design: Labelled cDNA either from Hpr KP or CcpA mutant of L.monocytogenes were compared to wild type L.monocytogenes using an oligonucleotide based microarray sequence of which is available at www.operon.com

first: Sonja

last: Mertins

first: Biju

last: Joseph

first: Monika

last: Goetz

first: Regina

last: Ecke

first: Gerald

last: Seidel

first: Mareen

last: Sprehe

first: Wolfgang

last: Hillen

first: Werner

last: Goebel

first: Stefanie

last: Müller-Altrock


title: Deletion of chromosome 11q predicts response to anthracycline-based chemotherapy in early breast cancer

status: Public on Feb 07 2007

city: San Francisco

first: Joan

last: Climent

address: 2340 Sutter St

department: Cancer Research Institute

institute: UCSF Cancer Center

summary:
Despite recent consensus on eligibility of adjuvant systemic therapy in lymph-node negative breast cancer (NNBC) patients based on clinico-pathological criteria, specific biological markers are needed to predict sensitivity to the different therapeutic options. We examined the feasibility of developing a genomic predictor of chemotherapy response and recurrence risk in 185 patients with NNBC using assembled arrays containing 2,460 BAC clones for scanning the genome for DNA copy number changes. After surgery, 90 patients received anthracycline-based chemotherapy whereas ninety-five did not. Tamoxifen was administered to patients with hormone-receptor positive tumors. Association of genomic and clinico-pathological data and outcome was computed using Cox proportional hazard models and multiple testing adjustment procedures. Analysis of NNBC genomes revealed a common genomic signature. Specific DNA copy number aberrations were associated with hormonal receptor status, but not with other clinico-pathological parameters. In patients treated with chemotherapy, none of the genomic changes was significantly correlated with recurrence. In patients not receiving chemotherapy, deletion of eight BAC clones clustered to chromosome 11q was independently associated with relapse (DFS at 10 years±SE, 40±14% vs. 86±6%;p<00001). The 54 patients with deletion of 11q (29%) did not present more aggressive clinical-pathological features than those without 11q loss. The adverse influence of 11q deletion in clinical outcome was confirmed in an independent validation series of 88 NNBC patients. Our data suggest that NNBC patients with 11q deletion may benefit from anthracycline-based chemotherapy despite other clinical, pathological or genetic features. However, these initial findings should be evaluated in randomized clinical trials.

Keywords: CGH, breast cancer

overall_design:
STUDY DESIGN AND SELECTION OF PATIENTS

From September 1979 to June 2000, over 3.100 new breast cancer patients were diagnosed at the University of Valencia. Of them, 1.482 tumors were criopreserved at -80ºC. For this study, biopsies were randomly selected based on the following criteria for inclusion: (1) diagnosis of primary invasive breast carcinoma of any size; 2) treatment by modified radical mastectomy or breast-conserving surgery, including dissection of axillary lymph nodes, followed by radiotherapy if indicated; 3) the apical axillary lymph nodes were tumor-negative (pathological examination, pN0); and 4) complete clinical data were available. Over 363 samples fulfilled the criteria. Previous to DNA extraction, these frozen tumor sections were stained by hematoxylin/eosin (H&E) and reviewed for tumor infiltratrion: only those with clear >50% of tumoral cells were selected. A cohort of 185 patients fulfilled these final criteria. Clinico-pathological variables including tumor size, histological grade and subtype and ER and PR status were determined following standard methods as reported.(26) Human investigations were performed after approval by an institutional review board on scientific and ethical affairs.

 MICROARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION (ARRAY CGH)

DNA extraction, hybridization and imaging. Frozen tumors were included in OCT compound. Previous to DNA extraction, H&E stained tumor sections were examined to select samples with more than 50% of tumoral cells. Around 20-30 sections of 25Nm were used for DNA extraction. After removing the OCT with PBS washes, DNA was extracted as previously described.(26) Genome-wide analysis of DNA-copy number changes was performed using array CGH on a microchip with 2.460 BAC and P1 clones printed in triplicate (UCSF Hum Array 2.0) with a resolution of 1.4 Mb across the genome.(27) Methods and analytical procedures have been described elsewhere in detail (25, 27, 28) 6 (Figure 1). For visualization of genomic data, the TreeView program 1.60 (Stanford, CA) was used. To confirm array CGH data, CGH to chromosomes was performed in 44 biopsies included in the study.
Interphase FISH analysis. To confirm specific gains and losses of BAC clones observed in the array CGH analyses, fluorescence in situ hybridization (FISH) studies using individual BAC clones as probes on isolated nuclei from frozen tumor sections was performed using a reported technique.(29) The gene loci examined corresponded to 5 overrepresented and 4 deleted BAC clones, using appropriate centromeric probes as controls. A total of 100 cells were examined on each of the 22 tumors examined. These clones were obtained from RZPD German Resource Center (Berlin, Germany) or purchased from Vysis (Downers Grove, IL, US).

STATISTICAL DATA ANALYSIS

To process the genomic data obtained with array CGH we used a previously described analytical model.(19, 30) Arrays were normalized by subtracting the median of each array from the average log2 ratio for every clone. The array CGH data were then analyzed using Hidden Markov Model (HMM) using the Bioconductor package aCGH. Log2 ratios as ordered in the genome were segmented into regions of constant copy number. In addition, the HMM model was employed to impute missing values by using the estimated copy number ratio for the segment containing the clone(s) with missing values. Clones with missing values located between segmented regions were assigned the mean value of the segment that is closer in genomic distance. Thus, each clone was assigned a segment value referred to as its "smoothed" value. Median absolute deviation (MAD) of the difference between the observed and smoothed values was used to estimate the tumor-specific experimental variation. Clones for each array were assigned into three groups: gained (smoothed log2 ratio >3 times the MAD); lost (smoothed log2 ratio <3 times the –MAD); and not changed (the log2 ratio cannot be assigned to lost or gained groups). Smoothed data was used to study association with the following phenotypes: age, tumor size, histological grade and subtype, stage, estrogen and progesterone receptor status, and recurrence/survival. Finally, we used Fisher’s two-sided exact test 2x2 crosstabs to compare genomic events or clinical variables among both groups of treatment. To evaluate differences in disease-free survival, Kaplan-Maier survival curves for the sets of patients were examined.
Gene Ontology (GO) classification. GO is used to construct a unified and structured vocabulary for the description of genes and their products in any organism. A GO term is descriptive of the molecular function, biological process or cellular component. We then performed a statistical analysis to check if there were any GO categories that were enriched in the genes located in the region of deletion in chromosome 11q23-q24 with respect to the whole genome.

CLINICAL SERIES FOR VALIDATION OF ARRAY CGH RESULTS

To validate the possible association of chromosome 11q deletion with increased relapse rate, this was tested in a validation group of 88 tumor biopsy samples from an independent cohort of NNBC patients. These were 18 Spanish patients treated in different Institutions within the Valencia area whose genomes were analyzed with array CGH as described above. In addition, data from 70 patients were obtained from a recently published series of American breast cancer patients analyzed using similar whole-genome array CGH techniques (Chin et al, in press). All patients fulfilled the reported inclusion criteria of the study. Kaplan-Maier survival curves for the two sets of patients were evaluated. Clinicopathological characteristics of the validation series are shown in Supplemental Table S1.