title: O1-O139 serogroup conversion / phage selected (2nd half)

first: Melanie

last: Blokesch

summary:
These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain MO10, an O139 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjees El Tor phage V (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected for opaque morphotype and agglutination by O139-specific antiserum. Four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone.    Strain Names: AIIIpO139#1 / AIIIpO139#3 / AIIIpO139#4 / AIIIpO139#5 are four clones analyzed after the second experiment; AIVpO139#2 / AIVpO139#4 / AIVpO139#5 / AIVpO139#8 are four clones analyzed after the fourth independent experiment. Two MA replicates for each clone were done.
A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's.
Keywords: all_pairs, array CGH

overall_design: Computed


title: Effects of capture-release events in wild dolphins: PBL analysis

status: Public on May 03 2007

city: Charleston

first: MarineGenomics

last: Project

state: SC

address: 331 Fort Johnson Rd

country: USA

summary:
Goal: To determine the effects of capture-release events in wild dolphins (Tursiops truncatus).
Methods:  An analysis of the Peripheral Blood Leukocyte (PBL) transcriptome was conducted on a group of 20 animals. The samples were collected in 2 different locations along the US east coast (Charleston, SC; Indian River Lagoon, FL) and 2 blood samples were collected for each dolphin 1) immediately after the capture event (*pre*) and 2) just before the animal was released (*post*). In between *pre* and *post* blood collections (30-40 minutes) additional samples were collected from the animals for physiological, chemical and biochemical analysis. RNA extracted from *pre* and *post* blood samples was used for micorarray hybridizations and transcriptome analysis using a species-specific PBL cDNA microarray (Mancia *et al*., 2007).
Keywords: blood cells (PBL)

overall_design: time course, field collection/wild caught organisms,  For multiple hybridizations: serial, biological replicates

first: Annalaura

last: Mancia


title: H3 ChIP-chip

status: Public on Apr 17 2007

city: Los Angeles

first: Steve

middle: E

last: Jacobsen

state: CA

address: 621 Charles E. Young Dr. South

department: Molecular Cell and Developmental Biology

institute: University of California, Los Angeles

type: Genome binding/occupancy profiling by genome tiling array

summary:
Trimethylation of histone H3 lysine27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. Here we identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways such as DNA methylation or RNAi. Unlike in animals where H3K27m3 occupies large genomic regions, we found that H3K27m3 domains in Arabidopsis were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.
Keywords: ChIP-chip

overall_design: Nucleosomal DNA was isolated with ChIP using anti-H3 antibody

first: Steven


title: Exploration of the Drug Action of Capreomycin on Mycobacterium tuberculosis

status: Public on Apr 25 2007

city: IRVINE

first: Li

last: Fu

address: 8 Corporate Park, Suite 300

institute: Pacific Tuberculosis and Cancer Research Organization

summary:
Multi-drug resistance and latent infection are two major issues in current tuberculosis (TB) control and management. Capreomycin is an important drug used for TB with multi-drug resistance. A recent study also indicates that this drug possesses unique bactericidal activity against non-replicating TB bacilli among known anti-TB drugs. Thus, there is an urgent need for investigating the full-spectrum action of capreomycin. Here we conduct the first microarray-based study on capreomycin using the high-resolution Affymetrix oligonucleotide GeneChip system. The results indicate that capreomycin primarily acts on the information pathways but it also significantly affects cell wall, cell processes, intermediate metabolism and respiration in Mycobacterium tuberculosis. This study not only transcriptionally validates the specific molecular target, 16S rRNA, but also discovers potential new targets of capreomycin, including genes operating at the DNA level, such as Rv0054 (ssb) and Rv3715c (recR), as well as genes involved in cell division like Rv3260c (whiB2). In addition, the nuo gene cluster and the ATP synthase gene cluster are repressed.
Keywords: Drug-induced Differential gene expression analysis

overall_design: The RNA collected from the cells before drug treatment was the baseline sample to which the drug-treated sample was compared. The drug-induced gene response was deduced from the differential gene expression between the test and baseline samples. The difference call computed by the Affymetrix system between the two samples indicates increase (I), decrease (D), marginal increase (MI), marginal decrease (MD), and no change (NC). Only decreases (D) and increases (I) are considered in this report. The increase call signifies up-regulation of gene activity from the baseline to test condition, whereas the decrease call suggests down-regulation. The difference call is statistically based on four system parameters reflecting differences across multiple (40) probes implementing each gene between two conditions.

middle: M

first: Thomas

last: Shinnick


title: Input DNA

summary:
Trimethylation of histone H3 lysine27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. Here we identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways such as DNA methylation or RNAi. Unlike in animals where H3K27m3 occupies large genomic regions, we found that H3K27m3 domains in Arabidopsis were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.
Keywords: genomic

overall_design: Input DNA hybridization signals


title: O1->O37 serogroup conversion; phage selected

city: Stanford

address: 279 Campus Drive

department: Medical School

institute: Stanford University

summary:
These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to 37. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain ATCC25872, an O37 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjee's El Tor phage III (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected by non-agglutination with O1-specific antiserum. One to four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone.   Strain Names: AIpO37#1 / AIpO37#4 / AIpO37#6 / AIpO37#8 are clones analyzed from the first experiment; AIIpO37#9 / AIIpO37#13 / AIIpO37#16 are clones analyzed from the second independent experiment; AIIIpO37#9A is a clone analyzed from the third independent experiment; AIVpO37#1A / AIVpO37#3A / AIVpO37#4A / AIVpO37#8A are clones analyzed from the fourth independent experiment. Two MA replicates per clone were done. CGHs of A1552 versus ATCC25872 are provided as control.
A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's.
Keywords: genotyping_design


title: ArrayCGH experiments involving patients with T-ALL

status: Public on Apr 15 2007

city: Leuven

first: Rekin's

last: Janky

address: Herestraat 49 Box 816

department: Nucleomics Core

country: Belgium

institute: VIB

type: Genome variation profiling by genome tiling array

summary:
We consider a data set consisting out of 27 arrayCGH dye-swap experiments (54 arrays) of individuals with T-ALL versus normal individuals.
Aim was to detect novel unbalanced genomic rearrangements in T-ALL.
Analysis of the patients was done using an array with genomic BAC and PAC probes with an overall resolution of 1Mb over the entire genome and with additional probes around known and candidate oncogenes.
Keywords: comparative genomic hybridization, genotype

overall_design: 27 dye-swap experiments of patient samples versus normal, reference samples.

first: Idoya

last: Lahortiga

first: Kim

last: De Keersmaecker

first: Pieter

last: Van Vlierberghe

first: Carlos

last: Graux

first: Barbara

last: Cauwelier

first: Frederic

last: Lambert

first: Nicole

last: Mentens

first: H.

last: Berna Beverloo

first: Rob

last: Pieters

first: Frank

last: Speleman

first: Maria

last: D. Odero

first: Marijke

last: Bauters

first: Guy

last: Froyen

first: Peter

last: Marynen

last: Vandenberghe

first: Iwona

last: Wlodarska

first: Jules

last: P.P. Meijerink

first: Jan

last: Cools


title: Human Uterine Leiomyomata microRNA gene expression

city: Newark

first: Tongsheng

last: Wang

state: NJ

address: 225 Warren Street

department: Center For Applied Genomics

institute: Public Health Research Institute

summary:
MicroRNAs (miRNAs) are a class of small, non-coding RNAs. MiRNAs regulate developmental activity and may be aberrantly expressed in human diseases, including cancer. Expression of miRNAs is tissue or organ specific. Human uterine leiomyomas (ULMs) are the most common neoplasms of women, characterized by the ovarian sex hormone dependence, worse morbidity in black women and genetic heterogeneity.  The high incidence of the disease may be associated with developmental defects in uteri. In this study, we examined miRNA expression in 55 ULMs with matched myometrium using spotted microarray platform.
Keywords: Disease state analysis

overall_design:
We selected 206 well-characterized human microRNAs to evaluate miRNA expression in 41 normal myometrium and 55 usual type leiomyomas.  The tumor samples were derived from 41 hysterectomies for fibroid uteri. To minimize the variations between cases, tissue samples of tumors and matched normal myometrium from each case was labeled with Cy3 (myometrium) and Cy5 (tumor) respectively and hybridized on the same chip. To reduce the possible spot to spot variation miRNAs were printed in triplicates on the arrays.  To identify miRNA whose expression was significantly different between normal and tumor samples, we analyzed all detectable miRNA using both t-test and Significance Analysis of Microarray (SAM) software.
We compared miRNA expression by age, tumor size, ethnicity, tumor phase and number of tumors.

Tumor size:
tumor size < 3cm: GSM114934, GSM114944, GSM114945, GSM114950, GSM114956, GSM114957, GSM114958, GSM114959, GSM114961, GSM114962, GSM114964, GSM114973, GSM114974, GSM114976, GSM114978
tumor size 4-9 cm: GSM114936, GSM114942, GSM114948, GSM114949, GSM114951, GSM114965, GSM114966, GSM114967, GSM114968, GSM114970, GSM114971, GSM114980
tumor size > 10 cm: GSM114935, GSM114937, GSM114938, GSM114939, GSM114940, GSM114941, GSM114943, GSM114946, GSM114947, GSM114952, GSM114953, GSM114954, GSM114955, GSM114960, GSM114963, GSM114969, GSM114972, GSM114975, GSM114977, GSM114979, GSM114981, GSM114982, GSM114983, GSM114984

Ethnicity:
ethnicity-black: GSM114936, GSM114937, GSM114938, GSM114940, GSM114941, GSM114946, GSM114948, GSM114950, GSM114951, GSM114953, GSM114958, GSM114959, GSM114962, GSM114963, GSM114964, GSM114965, GSM114966, GSM114968, GSM114982, GSM114983
ethnicity-white: GSM114934, GSM114935, GSM114939, GSM114943, GSM114944, GSM114945, GSM114949, GSM114952, GSM114954, GSM114955, GSM114960, GSM114961, GSM114970, GSM114971, GSM114973, GSM114974, GSM114975, GSM114976, GSM114978, GSM114979, GSM114981, GSM114984
ethnicity-other: GSM114942, GSM114947, GSM114956, GSM114957, GSM114967, GSM114969, GSM114972, GSM114977, GSM114980

Age:
<40: GSM114938, GSM114946, GSM114967, GSM114973, GSM114975, GSM114976, GSM114977, GSM114981, GSM114982
40-50: GSM114934, GSM114935, GSM114936, GSM114937, GSM114939, GSM114940, GSM114941, GSM114945, GSM114948, GSM114949, GSM114950, GSM114951, GSM114956, GSM114957, GSM114958, GSM114959, GSM114960, GSM114963, GSM114964, GSM114965, GSM114966, GSM114969, GSM114971, GSM114972, GSM114974, GSM114980
>50: GSM114942, GSM114943, GSM114944, GSM114947, GSM114952, GSM114953, GSM114954, GSM114955, GSM114961, GSM114962, GSM114968, GSM114970, GSM114978, GSM114979, GSM114983, GSM114984

Tumor phase:
tumor phase-proliferative endometrium: GSM114934, GSM114936, GSM114937, GSM114941, GSM114943, GSM114945, GSM114951, GSM114954, GSM114955, GSM114956, GSM114958, GSM114959, GSM114960, GSM114961, GSM114965, GSM114966, GSM114967, GSM114968, GSM114969, GSM114970, GSM114971, GSM114972, GSM114973, GSM114975, GSM114976, GSM114977, GSM114978, GSM114984
tumor phase-secretory endometrium: GSM114938, GSM114939, GSM114940, GSM114946, GSM114949, GSM114950, GSM114957, GSM114963, GSM114964, GSM114980, GSM114981, GSM114982
tumor phase-inactive endometrium: GSM114935, GSM114942, GSM114944, GSM114947, GSM114948, GSM114952, GSM114953, GSM114962, GSM114974, GSM114979, GSM114983--

Number of tumors:
number of tumors <5: GSM114935, GSM114936, GSM114944, GSM114949, GSM114952, GSM114957, GSM114960, GSM114973, GSM114974, GSM114975, GSM114977, GSM114979
number of tumors 5-20: GSM114939, GSM114940, GSM114941, GSM114942, GSM114943, GSM114945, GSM114946, GSM114947, GSM114948, GSM114950, GSM114951, GSM114954, GSM114956, GSM114959, GSM114961, GSM114963, GSM114964, GSM114965, GSM114967, GSM114969, GSM114970, GSM114972, GSM114976, GSM114978, GSM114981, GSM114983, GSM114984
number of tumors >20: GSM114934, GSM114937, GSM114938, GSM114953, GSM114955, GSM114958, GSM114962, GSM114966, GSM114968, GSM114971, GSM114980, GSM114982

first: Xinmin

last: Zhang

first: Laura

last: Obijuru

first: Virginie

last: Aris

first: Peng

last: Lee

first: Khush

last: Mittal

first: Patricia

last: Soteropoulos

first: Jian-jun

last: Wei


title: O1->O139 Serogroup conversion / KanR selected (15%)

first: Melanie

last: Blokesch

summary:
These experiment were performed to show a serogroup conversion in Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 WT = A1552 was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (2 ug each) from strain VCO139-Kan was added after 24h and the cells grwon further for 24h. The VCO139-Kan strain is a MO10 derivative (both O139 serogroup) which harbors a Kanamycin cassette in the O139 region (as part of the operon between wbfA and wbfB w/o own promotor) for better selection. Transformants were selected on LB+Kan plates.   Two groups of transformants were gained:  Group I had a full exchange of the O1 region by the O139 region (clones serogroup-converted: SGC#1-3); the crossovers for the homologous recombination event had occurred within or upstream of the gmhD gene and in most instances within or downstream of the homolog gene of VC0271. This implies an exchange of an at least 33 kb spanning O1 genomic region by more than 42 kb of the O139 region.  Group II had only half of the O139 region transfered and therefore half of the O1 region kept (clones HSGC#4-6). We analyzed their genotype and found that all of them had undergone a homologous recombination event with one crossover in or upstream of the gmhD gene and the second one inside the VC0254 and IS1358 gene.  The transformation experiment was done three independent times (I - III). Three clones from group I and group II were selected from each experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone.
A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's.
Strain Name: -III for experimental replicates (transformation); clones #1 -#3 full region exchanged; clones #4-#6 half region exchanged. Two MA replicates for each clone
Keywords: genotyping_design, array CGH


title: ChIP-chip analyses of H3K9 methylation in rrp6 deletion and rrp6 alp13 double mutant S.pombe cells.

status: Public on Apr 23 2007

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 H3K9 methylation in rrp6 deletion (exosome mutant) and in rrp6 alp13 double mutant S.pombe cells.
Keywords: ChIP-chip

overall_design: Chromatin immunoprecipitated DNA recovered with antibody against methylated H3K9 from 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 custom Agilent 44K in situ 60mer DNA microarray that tiles the entire Schizosaccharomyces pombe genome every 300 bp alternately on both strands. Data were processed using Agilent scanner and Feature Extraction software.

first: Estelle

last: Nicolas

first: Takatomi

last: Yamada

first: Hugh

middle: P

last: Cam

middle: C

last: FitzGerald

first: Ryuji

last: Kobayashi

middle: I


title: Reovirus MyRV2-CpC18 responsive genes in Cryphonectria parasitica

status: Public on Apr 20 2007

city: Rockville

first: Donald

middle: Lee

last: Nuss

address: 9600 Gudelsky Drive

department: Center for Biosystems Research

institute: University of Maryland Biotechnology Institute

summary:
Infection of the chestnut blight fungus, Cryphonectria parasitica, by hypovirus CHV1-EP713 or by reoviruses MYRV1-Cp9B21 or MYRV2-CpC18 results in reduced fungal virulence (hypovirulence). However, additional phenotypic changes caused by the two groups of mycoviruses are quite different. CHV1-EP713 infection results in depressed pigmentation and conidiation while reovirus infection has little effect on these processes. We now report that loss of female fertility and resulting absence of virus transmission through sexual spores observed after hypovirus infection was not observed for reovirus infected C. parasitica. Reovirus-infected strains were male and female fertile and able to transmit virus to ascospore progeny at a high rate when serving as a female parent. Consistent with this result, real-time RT-PCR revealed that expression of two genes involved in sexual reproduction, the pheromone precursor gene, mf2-1 and yeast Ste12-like transcriptional factor gene, Cpst12, were less reduced in reovirus-infected strains than in hypovirus-infected strains. Analysis with a custom microarray cDNA chip containing EST clones representing ca 2,200 unique C. parasitica genes identified 140 and 128 host genes that were responsive to MYRV1-Cp9B21 and MYRV2-CpC18 infection, respectively. Comparison of these virus-responsive genes revealed an overlap of 85 genes even though the overall degree of nucleotide sequence identity of the two reoviruses is less than 50%. Significantly, 84 of the 85 genes were altered in the same direction. Further comparison revealed that 51 % and 48% of the genes that were responsive to reoviruses MYRV1-Cp9B21 and MYRV2-CpC18 infection were also responsive to CHV1-EP713 infection. Finally, similar to results reported for hypovirus infection, a high percentage (59% and 66%) of the mycoreovirus responsive genes were also differentially expressed following disruption of the cellular G-protein signal transduction pathway. These data support the hypothesis that hypovirus and reovirus infections perturb common and specific C. parasitica regulatory pathways to cause hypovirulence and distinct sets of phenotypic changes.
Keywords: Mycoreovirus, hypovirus, gene expression, microarray.

overall_design: The transcriptional profiles between reovirus MyRV2-CpC18 infected and isogenic wild-type strain EP155 were compared in this experiment. Two sets of RNA samples with dye-swab (total of four hybridizations) were analyzed.

first: Fuyou

last: Deng

first: Todd

middle: D

last: Allen

first: Bradley

last: Hillman

middle: L


title: ChIP-chip analyses of H3K9 acetylation in WT, alp13 deletion and pst1-1 mutant S.pombe cells.

summary:
ChIP-chip analyses of H3K9 acetylation in WT, alp13 deletion (Clr6-complex II mutant) and pst1-1 (Clr6-complex I mutant) S.pombe cells.
Keywords: ChIP-chip

overall_design: Chromatin immunoprecipitated DNA recovered with antibody against acetylated H3K9 from 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 custom Agilent 44K in situ 60mer DNA microarray that tiles the entire Schizosaccharomyces pombe genome every 300 bp alternately on both strands. Data were processed using Agilent scanner and Feature Extraction software.

first: Peter


title: ChIP-chip analyses of H3K9 methylation in alp13 deletion and clr6-1 mutant S.pombe cells.

summary:
ChIP-chip analyses of H3K9 methylation in alp13 deletion (Clr6-complex II) and clr6-1 mutant (complexes -I and -II) S.pombe cells.
Keywords: ChIP-chip


title: Epigenetic activation of a subset of mRNAs identified using polyribosome microarray analysis

status: Public on Apr 21 2007

city: Stockholm

first: Ola

last: Larsson

department: Department of oncology-pathology

country: Sweden

institute: Karolinska Institutet

summary:
Background: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5 prime cap–binding protein, plays a major role in translational control. To understand how eIF4E affects cellular proliferation and cell survival, we identified mRNA targets that are translationally responsive to eIF4E.

Methodology/ principal findings: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Induction of eIF4E expression resulted in increased translation of a defined set of mRNAs; many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth–related factors. eIF4E overexpression also led to augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum–mediated apoptosis.

Conclusions/ significance: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a therapeutic option for the development of novel anti-cancer drugs.
Keywords: time course

overall_design:
Comparison of total and polysomal RNA upon eIF4E iinduction in NIH3T3/parental and NIH3T3/eIF4E cells

Each of the following pairs were generated from one hybridization:

GSM153931 GSM153932
GSM153933 GSM153934
GSM153935 GSM153936
GSM153937 GSM153938
GSM153939 GSM153940
GSM153941 GSM153942
GSM153943 GSM153944
GSM153945 GSM153946
GSM153947 GSM153948
GSM153949 GSM153950
GSM153951 GSM153952
GSM153953 GSM153954
GSM153955 GSM153956
GSM153957 GSM153958
GSM153959 GSM153960
GSM153961 GSM153962


title: Whole WRKY gene family transcription analysis in rice during biotic and abiotic stress.

status: Public on Apr 10 2007

city: Milan

first: Stefano

last: Berri

address: via Celoria 26

department: biomolecular sciences and biotechnology

country: Italy

institute: University of Milan

summary:
The WRKY gene family has a very ancient origin but has faced extensive duplication only in the plant kingdom so much that Arabidopsis (Arabidopsis thaliana) has 74 copies of WRKY genes encoding transcription factors while 109 can be found in Rice (Oryza sativa L.). Several studies in the last decade has pointed their involvement in an heterogeneous number of biological processes, from development to hormone signalling, dormancy and senescence, but a wide number of WRKY genes are transcriptionally regulated during biotic or abiotic stresses. To investigate involvement of WRKY genes upon host and non-host infection (different strain of Magnaporthe grisea) and osmotic stress in Rice, we performed a gene family transcription analysis using custom microarray. Results indicate that a relevant part of WRKY genes are involved during at least one of these stresses, that there is little difference in transcriptional regulation between host and non-host infection or between different tissues upon the same osmotic stress. Moreover, are evident groups of genes that, often with opposite behaviour, are co-regulated in all or most of the studied conditions. We thus formulated the hypothesis that WRKY genes might be part of co-regulatory networks with other WRKY genes.
Keywords: stress response

overall_design: We analyzed 40 arrays and tested 6 conditions: BR29 (Non-host Pathogen), BR32 (Non-host Pathogen), FR13 (Host pathogen), Osmotic leaves 5 hours, Osmotic roots 1 hour and Osmotic roots 5 hours. 2 biological replicated were analyzed and between 2 to 4 technical replicates applied for each biological sample.

first: Pietro

last: Piffanelli

first: Ana

last: Brasilero

first: Odile

last: Faivre-Rampant

first: Mario

last: Pè


title: The effect of ectopic expression of Dorsal and Dif on Drosophila Gene Expression

status: Public on Apr 16 2007

city: College Park

first: Subhamoy

last: Pal

address: 5126 Plant Sciences Building

institute: University of Maryland

summary:
This study explores the effect of ectopic expression of Dorsal and Dif on Drosophila larval gene expression. The abstract of the manuscript we are submitting follows:

Microarray analyses reveal possible new targets for the Rel protein Dif in Drosophila melanogaster

The fruit fly Drosophila melanogaster express three closely related NF-κB-like transcription factors: Dorsal, Dif, and Relish. They share significant sequence identity in their DNA binding Rel homology domain and bind similar DNA sequence motifs. These factors play an important role in the transcription of Drosophila humoral immune proteins such as antimicrobial peptides during infection. To study their roles in vivo, we used microarrays to determine the effect of null mutations in individual Rel transcription factors on larval gene expression. Of the 188 genes that were significantly upregulated in wildtype larvae upon bacterial challenge, overlapping but distinct groups of genes were affected in the Rel mutants. We also ectopically expressed Dorsal or Dif and used cDNA microarrays to determine the genes that were up-regulated in the presence of these transcription factors. Combining this data, we identified novel genes that may be specific targets of Dif.
Keywords: Genetic Modification

overall_design: Staged larvae containing a transgenic HS-Dorsal or HS-Dif were subjected to 37C for one hour, and their gene expression was compared to wildtype Drosophila treated in the same way.  The experiment was repeated in triplicate.

first: Junlin

last: Wu

first: Louisa


title: chpT depletion data

status: Public on Apr 05 2007

city: Cambridge

first: Michael

last: Laub

state: MA

address: 31 Ames St.

institute: MIT

summary:
chpT depletion ML808 versus wild type CB15N
Keywords: genetic modification

overall_design: three independent colonies were grown to mid-log phase in PYE + xylose and RNA harvested for analysis on microarrays

first: Emanuele

middle: G

last: Biondi

middle: T


title: Expression profiling in alp13 deletion, clr6-1 mutant and pst1-1 mutant, versus WT S. pombe cells.

first: Shiv

last: Grewal

summary:
Expression analyses in alp13 deletion (Clr6-complex II mutant), clr6-1 partial mutant (Clr6 complexes I and II mutant) and pst1-1 partial mutant (Clr6-complex I mutant) S.pombe cells.
Keywords: Expression

overall_design: Wild type or mutant cDNA reverse transcribed from total RNA was labeled with either Cy3 or Cy5. Four experiments were performed for each genotype with two experiments in which the dyes were swapped. Agilent 44K in situ 60mer DNA microarray that tiles the entire Schizosaccharomyces pombe genome every 300 bp alternately on both strands was used to probe expression of transcripts up-regulated in alp13 null, clr6-1 and pst1-1 cells versus wild-type cells.

first: Estelle

last: Nicolas

first: Takatomi

last: Yamada

first: Hugh

last: Cam

first: Ryuji


title: ChIP-CHIP data for oleate-specific transcriptional regulatory network analysis

status: Public on Apr 19 2007

city: Seattle

first: Jennifer

middle: Joy

last: Smith

state: WA

address: 1441 North 34th Street

institute: Institute for Systems Biology

summary:
The goal of the study was to determine the direct targets of four oleate-responsive transcription factors (Oaf1p, Pip2p, Oaf3p, Adr1p) in the presence and absence of the fatty acid oleate.
This data is part of a larger study outlined below:
A novel network topology-based clustering approach was used to characterize a dynamic transcriptional regulatory network responsive to the fatty acid oleate.  Condition-specific large-scale chromatin localization data were generated for four fatty-acid related regulators, and used to construct physical interaction networks.  Data integration and simple statistical analysis of dynamic network architecture led to the identification of a regulatory role for a previously uncharacterized protein, and unique mechanism for coordination and regulation of multiple biological responses through the formation of related parallel combinatorial control motifs.  Temporal coordination and specificity are achieved through the ability of a regulator (Oaf1p) to have multiple context-specific activities under the same condition.  The analysis suggests that active paths for a transcriptional response can be highly interconnected in parallel and involve multiple functions for individual regulators through combinatorial control.
Keywords: ChIP-chip, growth condition

overall_design: For each experiment (i.e. IP of one factor under one growth condition), there were 3 biological replicates, each hybridized to one array.  As each array contains four replicates of each intergenic region, the total number of replicates for each condition is 12.

middle: J

first: Stephen

middle: A

last: Ramsey

first: Marcello

last: Marelli

first: Bruz

last: Marzolf

first: Daehee

last: Hwang

first: Ramsey

last: Saleem

first: Richard

last: Rachubinski

first: John

last: Aitchison


title: Gene expression profiling of chickpea responses to cold stress

status: Public on Apr 03 2007

city: Bundoora

first: Nitin

last: Mantri

state: Victoria

address: Bldg 223, Level 1, Plenty Road

department: Biotechnology & Environmental Biology

country: Australia

institute: RMIT University

summary:
‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to cold stress. Two groups of a tolerant and susceptible accession were challenged with cold stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaves and flowers/buds/early pods tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf and flower tissues separate) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1.
The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress.

Globally, the level of 221 transcripts was affected in response to cold stress in all the genotypes and tissue types studied. The DE transcripts in response to cold stress fell into various functional categories, indicating a broad response. Sixteen out of the 221 DE transcripts were consistently expressed in cold tolerant/susceptible genotypes. All these transcripts were repressed and none was found to be consistently induced in response to cold-stress. Most of the putative genes were identified in leaves of tolerant genotypes, and included a beta-galactosidase (DY475141) transcript that was possibly indicative of disaccharide (e. g. sucrose) retention with the effect of protecting cell membranes during cold stress.  Several protein synthesis/modification and energy/metabolism transcripts were also repressed (e.g. DY475282, DY396371 and DY475555), which was likely due to the impairment of photosynthesis and respiration at low temperature. Other consistently repressed transcripts in tolerant genotypes included putative signalling (DY396262, DY475384 and DY396307) and defence-related proteins (CV793589 and DY396343), which may be involved in the repression of cell death mechanisms that are absent in tolerant genotypes.  In susceptible genotypes, a putative superoxide dismutase precursor protein (DY475397) and sorting nexin protein (DY475523) were the only known transcripts to be consistently repressed.
Keywords: Chickpea, Cold stress, Tolerant, Susceptible, cDNA microarray

platform_organism:
Cicer arietinum
Lathyrus sativus
Lens culinaris

overall_design:
Total RNA was extracted from separately pooled leaf and flowers/buds/early pods tissues for each genotype {Sonali (Tolerant1), Amethyst (Susceptible1), ILC 01276 (Tolerant2) and DOOEN (Susceptible2)} 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 as described [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Plant Biotechnology Journal. 4(6), 647–666]. 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, 288 images were analyzed from 48 genotype x tissue type x treatment/control x biological replication condition.

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-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.

first: Edwin

last: Pang

first: Rebecca

last: Ford

first: Tristan

last: Coram


title: Reovirus MyRV1-Cp9B21 responsive genes in Cryphonectria parasitica

first: Donald

last: Nuss

summary:
Infection of the chestnut blight fungus, Cryphonectria parasitica, by hypovirus CHV1-EP713 or by reoviruses MYRV1-Cp9B21 or MYRV2-CpC18 results in reduced fungal virulence (hypovirulence). However, additional phenotypic changes caused by the two groups of mycoviruses are quite different. CHV1-EP713 infection results in depressed pigmentation and conidiation while reovirus infection has little effect on these processes. We now report that loss of female fertility and resulting absence of virus transmission through sexual spores observed after hypovirus infection was not observed for reovirus infected C. parasitica.  Reovirus-infected strains were male and female fertile and able to transmit virus to ascospore progeny at a high rate when serving as a female parent. Consistent with this result, real-time RT-PCR revealed that expression of two genes involved in sexual reproduction, the pheromone precursor gene, mf2-1 and yeast Ste12-like transcriptional factor gene, Cpst12, were less reduced in reovirus-infected strains than in hypovirus-infected strains. Analysis with a custom microarray cDNA chip containing EST clones representing ca 2,200 unique C. parasitica genes identified 140 and 128 host genes that were responsive to MYRV1-Cp9B21 and MYRV2-CpC18 infection, respectively. Comparison of these virus-responsive genes revealed an overlap of 85 genes even though the overall degree of nucleotide sequence identity of the two reoviruses is less than 50%. Significantly, 84 of the 85 genes were altered in the same direction. Further comparison revealed that 51 % and 48% of the genes that were responsive to reoviruses MYRV1-Cp9B21 and MYRV2-CpC18 infection were also responsive to CHV1-EP713 infection. Finally, similar to results reported for hypovirus infection, a high percentage (59% and 66%) of the mycoreovirus responsive genes were also differentially expressed following disruption of the cellular G-protein signal transduction pathway. These data support the hypothesis that hypovirus and reovirus infections perturb common and specific C. parasitica regulatory pathways to cause hypovirulence and distinct sets of phenotypic changes.
Keywords: Mycoreovirus, hypovirus, gene expression, microarray.

overall_design: The transcriptional profiles between reovirus MyRV1-Cp9B21 infected and isogenic wild-type strain EP155 were compared in this experiment. Two sets of RNA samples with dye-swab (total of four hybridizations) were analyzed.

first: Fuyou

last: Deng

first: Todd

last: Allen

first: Bradley

last: Hillman


title: Identification of breast cancer tumor associated antigens

city: Torino

first: Raffaele

last: Calogero

state: To

address: Via Nizza 52

department: Molecular Biotechnology Center

institute: University of Torino

summary:
Four prototypic situations, corresponding to progressive stages of mammary carcinogenesis, were analyzed. Total RNA from whole-mount for  10, 15, 19 and 22 weeks old BALB-neuT animals was extracted.
Differential expression between the various prototypic conditions indicates the presence of changing in level of mRNAs due to the increment in the tumor mass (Astolfi et al. Cancer Immunol Immunother 2005, 54:599-610). Since the same amount of RNA is hybridized on all arrays the change in composition of mRNA species associated to the increment of the tumor cellular population allows the tagging as “putative tumor associated antigens” (Cavallo et al. BMC Bioinformatics 2005 6(Suppl 4):S7) for all transcripts progressively up-modulated as the age of the animals increases.
Keywords: Disease state analysis

overall_design: Four prototypic situations, corresponding to progressive stages of mammary carcinogenesis, were analyzed. Total RNA from whole-mount for  10 (5 animals), 15 (2 animals), 19 (4 animals) and 22 (4 animals) weeks old BALB-neuT animals was prepared and hybridized on AB1700 mouse arrays.

first: Federica

last: Cavallo

first: Elena

last: Quaglino

first: Silvia

last: Saviozzi

first: Marco

last: Lo Iacono

first: Nicola

last: Cirenei

first: Francesca

last: Cordero

first: Guido

last: Forni


title: H3K27me3 ChIP-chip

first: Steve

last: Jacobsen

summary:
Trimethylation of histone H3 lysine27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. Here we identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways such as DNA methylation or RNAi. Unlike in animals where H3K27m3 occupies large genomic regions, we found that H3K27m3 domains in Arabidopsis were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.
Keywords: ChIP-chip

overall_design: Genomic DNA associated with H3K27me3 was isolated by ChIP with anti-H3K27me3 antibody

first: Steven


title: X chromosome cDNA microarray screen of lymphoblasts from males with XLMR

city: Baltimore

first: tao

last: wang

address: 733 N. Broadway, BRB513

institute: Johns Hopkins University

summary:
The goal of this study is to identify genes that show altered transcript levels in lymphoblasts from male patients with XLMR as candidate genes that are responsible for XLMR.
Keywords: disease state

overall_design: Using a custom X chromosome cDNA microarray, we studied X chromosome gene expression in lymphoblasts from 43 patients with XLMR and identified those genes with altered transcript levels as compared to that in 6 normal controls.


title: An Ustilago maydis gene involved in H2O2 detoxification is required for virulence

city: Marburg

first: Lazaro

last: Molina

address: Karl-von-Frish

department: Organismic interactions

country: Germany

institute: Max Planck Institute

summary:
The fungus Ustilago maydis is a biotrophic pathogen of corn. In its genome we have identified an ortholog of YAP1 from Saccharomyces cerevisae which regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H2O2 than wild type cells and their virulence was significantly reduced. U. maydis yap1 could partially complement the H2O2 sensitivity of a yap1 deletion mutant of S. cerevisiae and a Yap1-GFP fusion protein showed nuclear localization after H2O2 treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two cysteine residues prevented accumulation in the nucleus and the respective mutant strains showed the same virulence phenotype as Dyap1 mutants. DAB staining revealed an accumulation of H2O2 around yap1 mutant hyphae which was absent in wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection Yap1 showed nuclear localization after penetration up to 2-3 days after infection. Through array analysis a large set of yap1 regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its yap1 controlled H2O2 detoxification system for coping with early plant defense responses.
Keywords: Oxidative stress, yap1 dependent genes, Ustilago maydis

overall_design: Two independent overnight cultures of U. mayidis FB1 and FB1Dyap1 grown in CM-glucose (OD600 0.8) were diluted in 100 ml of the same medium (OD600 0.2) and growth at 28° C until an OD600 0.6. The cultures were divided and one half was supplemented with 5mM H2O2. After one hour of exposition to H2O2, cells were harvested by centrifugation and frozen in liquid nitrogen. RNA extraction, purification, cDNA generation, purification and labeling were performed according to standard protocols (Affymetrix). DNA array analysis was performed performed on two biological replicates each, using custom-designed Affymetrix chips (MPIUstilagoA).  Data were analysed using a GeneArray Scanner (Agilent/Affymetrix) and the GeneChip Expression Analysis software (GCOS)

first: Regine

last: Kahmann


title: Microarray was used to study the gene expression of P. stutzeri A1501 under different growth conditions

city: Beijing

first: yongliang

last: yan

address: zhongguancun south 12th

country: China

institute: CAAS

summary:
Nitrogen fixation is a highly energy-demanding process and highly regulated at multiple levels. The two major signals that regulate nitrogen fixation in most diazotrophs are oxygen and ammonia. In order to study the complex regulated mechanism and to highlight the complete nitrogen fixing system in genome level, here we present the transcriptional profiles of the nitrogen fixation genes of P.stutzeri A1501 in different growth conditions with a genome-wide DNA microarray. In this study, the three samples of "P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension","P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension-2" and "P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension-3" were three repeat experiments, while, the other three samples of "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-1", "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-2" and "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-3" were three repeat experiments. The gene expressions under these two growth phases were compared to investigate which genes' expression were effected by different ammonia concentrations.
Keywords: nitrogen fixation, nitrogen repression

overall_design: P.stutzeri whole genome microarray, which was based on our sequenced strain A1501. The microarray used in this study featured 3988 of the 4145 ORFs identified in P.stutzeri strain A1501, because 157 ORFs were not on the microarray of the inability to amplify these products. ORFs were amplified with specific primer pairs (Invitrogen). To ensure that the elements of our array would detect specifically their corresponding genes and no others, the ORF coordinates fed into the primer program were circumscribed such that they would exclude regions of any ORF that contained significant similarity to any other ORF. Agarose gel electrophoresis was used to perform quality control on all PCR products. Oligonucleotides were removed from the PCR mix by PCR 96 cleanup plate (Millipore). DNA was resuspended in 12 μl of spotting solution containing 50% dimethyl sulfoxide. PCR products were spotted onto gamma amino propylsilan coated GAPII slides (Corning) with a OmniGrid ™ microarrayer (GeneMachines).The negative control genes were from the type III secretion system genes of shigella flexneri 2a and the commercial Arabidopsis genes (SpotReport™ cDNA Array Validation System, Stratagene). The positive control genes were from the 16S rRNA of P.stutzeri A1501. We used Cy3-labeled genomic DNA and Cy5 dye-labeled cDNA, for hybridization. The cDNA was synthesized by reverse transcription of 5 μg of total RNA. The labeled cDNA and genomic DNA samples were mixed and hybridized at 65℃ for 16 hours. Genomic DNA was used as a universal internal control for the quality of the microarray and also allowed for the comparison of results across multiple experiments. Each experiment was repeated three times from the beginning of inoculation.

first: Yongliang

last: Yan

first: Yuetan

last: Dou

first: Min

last: Lin


title: Profiling of interferon beta immediate transcriptional effects in humans

status: Public on Apr 18 2007

city: San Francisco

first: Sergio

last: Baranzini

address: 513 Parnassus Ave

department: Neurology

institute: University of California San Francisco

summary:
In this study we used high frequency sampling and DNA microarrays to analyze the longitudinal transcriptional profile of blood cells taken from two individuals only hours after interferon beta administration and up to a week later as compared to samples taken from healthy controls taken at coordinated time points.
Keywords: Time series

overall_design: Repeated blood draws were conducted into Paxgene tubes (Qiagen, Valencia, CA) in two individuals after administration of a therapeutic dose of IFNB1a (Avonex, Biogen) in accordance to institutional guidelines. In one individual (P1), samples were obtained at baseline and at 3.5h, 6.25h, 9.5h, 11.5h, 16.5h, 25h, 49h, and 156h. In the other (P2), blood was collected at 7.25h, 10.25h, 13h, 15.5h, and 33h. Samples from four healthy volunteers were also collected. Because samples from the two IFNB-treated individuals were collected at slightly different times, we matched the collection times of two controls to follow each time series.

first: Guy

middle: H

last: Fernald

first: Andrew

middle: R

last: Pachner

first: Stacy

last: Caillier

first: Kavitha

last: Narayan

first: Jorge

last: Oksenberg


title: Genome-wide detection of recurring sites of uniparental disomy in folicular and transformed follicular lymphoma

status: Public on Apr 02 2007

city: London

first: Jude

last: Fitzgibbon

address: Charterhouse Square

department: Centre Medical Oncology

country: United Kingdom

institute: Barts and the London Medical School

type:
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary:
Follicular lymphoma (FL) is the second most common B-cell malignancy representing one quarter of Non-Hodgkin's Lymphomas. Although the disease has a relatively long median survival, the illness follows a fluctuating course of progression punctuated by remissions of variable duration. For as many as half of patients, transformation to a more aggressive lymphoma (t-FL) may occur; this event is often associated with a particularly poor response to treatment. SNP analysis has revealed the presence of large regions of homozygosity in the absence of copy number change. These events can result in the selection of of daughter cells made homozygous for a pre-existing mutation.

We have used SNPs array technology to investigate regions of loss of hetrozygosity in the absence of copy number change in order to establish the contribution of these abnormalities to the evolution of FL and t-FL.
Keywords: DNA copy number, Loss of heterozygosity

overall_design: DNA from 26 pairs of follicular and transformed follicular lymphoma samples and 3 cell lines were analysed using Affymetrix 10K SNP arrays. The genotype data of transformed follicular  lymphoma samples were compared with the data from the corresponding follicular lymphoma sample.

first: Sameena

last: Iqbal

last: Davies

first: Derville

last: O'Shea

first: Emanuela

last: Carlotti

first: Tracy

last: Chaplin

first: Janet

last: Mathews

first: Manoj

last: Raghavan

last: Norton

first: T

last: Lister

first: Bryan

last: Young


title: Wobbler modifier

status: Public on Apr 01 2007

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum München GmbH

summary:
The vacuolar-vesicular protein sorting (vps) factors are involved in vesicular trafficking in eukaryotic cells. The wobbler mouse is an animal model of amyotrophic lateral sclerosis. Motoneuron survival and spermiogenesis are severely compromised in the wobbler mouse. The effect of the wobbler modifier in Chr. 10 on gene expression levels in testis.
Keywords: cDNA array, Wobbler, testis

overall_design:
DNA chip technology has been exploited for RNA expression profiling analyses. Close to genome-wide microarrays were used for identification of changes in gene expression levels in testis. Pools of 3 individuals were used for hybridisation experiments.
In total 12 chip hybridisations were performed including 50% dye swap experiments. (wt vs wr/wr; wt vs wr/mod; wr/wr vs wr/mod).

first: Johannes

last: Beckers

first: Marion

last: Horsch


title: Expression analysis of proliferative phase Arabidopsis thaliana endosperm

city: Dunedin

first: Robert

last: Day

state: Otago

address: Cumberland Street

department: Biochemistry

country: New Zealand

institute: University of Otago

summary:
Laser capture microdissection (LCM) provides a useful method for isolating specific cells or tissues from biological samples. Here, we adapted microdissection protocols to allow high-resolution transcript analysis of different tissues from developing Arabidopsis seed. However, to obtain enough RNA for microarray analyses it was necessary to amplify the RNA. Microarray analyses, using endosperm derived RNA amplified by two-round IVT, reproducibly identified endosperm enriched marker genes.
Keywords: LCM Endosperm Arabidopsis

overall_design: We carried out four hybridisations from three replicate endosperm samples microdissected from sections of 4 DAP siliques. We hybridised the endosperm samples against a universal control obtained from the tissues remaining post-microdissection scraped from one of the sample slides. This approach was used to minimise alterations in the measured differential expression due to possible differences in RNA quality between LCM prepared samples and conventional samples. Endosperm rep1 was labelled with Cy3, rep3 with Cy5 and rep2 was hybridised twice as dye swaps ie once labelled with Cy3 and once with Cy5.

first: Les

last: Mcnoe

first: Richard

last: Macknight


title: Wobbler: Spinal cord and testis

summary:
The vacuolar-vesicular protein sorting (vps) factors are involved in vesicular trafficking in eukaryotic cells. The wobbler mouse is an animal model of amyotrophic lateral sclerosis. Motoneuron survival and spermiogenesis are severely compromised in the wobbler mouse.
Keywords: dual colour hybridisation on cDNA microarrays, Wobbler, Vps54, spinal cord, testis

overall_design:
DNA chip technology has been exploited for RNA expression profiling analyses. Close to genome-wide microarrays were used for identification of changes in gene expression levels in spinal cord and testis of 10 wr/wr mice.

Spinal cord: Pooled RNA of 4 individual mutant mice were hybridised in repetitions against a pool of 10 reference animals. In total 10 chip hybridisations were performed including 50% dye swap experiments.

Testis: Individual mutant mice were hybridised in repetitions against a pool of 10 reference animals. In total 40 chip hybridisations were performed including 50% dye swap experiments.

first: Thomas

last: Schmittjohn


title: Xenopus cDNA microarray identification of genes with endodermal organ expression

status: Public on Mar 31 2007

city: POHANG

first: JIN-KWAN

last: HAN

state: KYUNGBUK

address: SAN 31, HYOJA-DONG, NAM GU

department: LIFE SCIENCE

country: South Korea

institute: POSTECH

summary:
The endoderm is classically defined as the innermost layer of three Metazoan germ layers. During organogenesis, the endoderm gives rise to the digestive and respiratory tracts as well as associated organs such as the liver, pancreas, and lung. At present, however, how the endoderm forms the variety of cell types of digestive and respiratory tracts as well as the budding organs is not well understood. In order to investigate the molecular basis and mechanism of organogenesis and to identify the endodermal organ-related marker genes, we carried out microarray analysis using Xenopus cDNA chips. To achieve this goal, we isolated the Xenopus gut endoderm from three different stages of Xenopus organogenesis, and separated each stage of gut endoderm into anterior and posterior region. Competitive hybridization of cDNA between the anterior and posterior endoderm regions, to screen genes that specifically expressed in the major anterior organs, revealed 891 candidates. We then selected 104 clones for in situ hybridization analysis. Here, we report the identification and expression patterns of the 104 Xenopus endodermal genes, which would serve as useful markers for studying endodermal organ development.
Keywords: Xenopus, endoderm, microarray, organogenesis

overall_design: In order to isolate the endodermal organ-related genes, we carried out a microarray analysis using high-density Xenopus cDNA chip. To this aim, we isolated the Xenopus endoderm from three different stages (St. 30, 39, and 46) and manually dissected the each stage of endoderm to anterior and posterior region. The anterior endoderm will later become major organs such as the lung, oesophagus, duodenum, stomach, pancreas, liver, and gallbladder, and the posterior endoderm will become the intestine. Duplicate sets of three individual microarray experiments were performed using anterior endoderm cDNAs of each stage, labeled by Cy5, and the posterior endoderm cDNAs of each stage, labeled by Cy3.  The normalization strategy involves condensing the duplicate array data for each of the three developmental stages, thus giving rise to three GEO Samples.

first: EDMOND

last: PARK

first: TADAYOSHI

last: HAYATA

first: KEN

middle: W

last: CHO


title: The sigmaB regulon of B. cereus ATCC14579

city: Wageningen

first: Menno

middle: van der

last: Voort

address: Bomenweg 2

department: Agrotechnology and Food Sciences Group

country: Netherlands

institute: Wageningen UR

summary:
The alternative sigma factor sigmaB has an important role in the acquisition of stress-resistance in many gram-positive bacteria. In the foodborne pathogen Bacillus cereus sigmaB is activated strongly upon a heat shock and other stress conditions. Here we describe the identification of the set of sigmaB-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. A total of 24 genes could be identified as being sigmaB-dependent as witnessed by (i) significantly lower expression-levels of these genes in mutants deleted for sigB and rsbY (encoding the alternative sigma factor sigmaB and a PP2C phosphatase that acts as a crucial positive regulator of sigmaB-activity, respectively) than in the parental strain B. cereus ATCC 14579, and (ii) increased expression of these genes upon a heat shock. Newly identified members of the sigmaB-regulon of B. cereus include an ECF-sigma factor (sigmaZ), a histidine kinase and two genes that have predicted functions in spore germination. Our data indicate that the sigmaB-regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses of sigmaB in which sigmaB in the B. cereus group was suggested to be close to the ancestral form of sigmaB in gram-positive bacteria.
Keywords: stress response, comparative genomics

overall_design: Three comparisons were performed: WT-DsigB, WT-DrsbY and WT30degrees-WT42degrees

first: Willem

last: van Schaik

last: van der Voort

last: de Vos

first: Tjakko

last: Abee


title: Characterization of the HP1021 regulon of Helicobacter pylori

city: Wuerzburg

first: Dagmar

last: Beier

address: Am Hubland

institute: Universität Wuerzburg

summary:
The genome of the gastric pathogen Helicobacter pylori harbors a remarkably low number of regulatory genes, including three and five open reading frames encoding two-component histidine kinases and response regulators, respectively, which are putatively involved in transcriptional regulation. Inactivation of the response regulator gene hp1021 resulted in a severe growth defect, as indicated by a small-colony phenotype. Recently we found that phosphorylation of the receiver domain HP1021 is not needed for its response regulator function and may not occur at all. No target genes have been identified so far. In this study we define the HP1021-dependent regulon consisting of 79 genes (51 activated, 28 repressed) by global transcriptional profiling of an HP1021-deficient H. pylori mutant.
Keywords: Identification of an HP1021-Regulon

overall_design:
Transcriptome analyses were performed using a whole-genome microarray containing 1649 PCR products generated with specific primer pairs derived from the genome sequences of H. pylori 26695 (Tomb et al., 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547) and J99 (Alm et al., 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180) which were spotted in duplicate. Microarrays were produced as described by Gressmann et al. (Gressmann et al., 2005. Gain and loss of multiple genes during the evolution of Helicobacter pylori. PLoS Genet 1(4):e43). To determine genes which are differentially expressed in the HP1021-deficient mutant 26695/1021::km, cDNA was prepared from RNA extracted from H. pylori 26695 WT and 26695/1021::km. A total of eight RNA samples from two independent RNA preparations from strain 26695 WT and 26695/HP1021::km, respectively, was used for cDNA labelling und hybridisation. Dye reversal colour swaps were performed as follows: one cDNA sample was generated using Cy3-dCTP and the other using Cy5-dCTP resulting in four labelled cDNAs per colour swap. Cy5-dCTP and Cy3-dCTP labelled cDNAs were combined and hybridized to the H. pylori microarray. The slides were scanned using ScanArray HT and analysed by using the ScanArray express software (Perkin Elmer, version 3.0). Spots were flagged and eliminated from analysis when the signal to background ratio was less then three or in obvious instances of high background or stray fluorescent signals. Median intensities of spots were background corrected and differences in dye bias were normalized by using the LOWESS algorithm (Yang et al., 2002. Normalization for cDNA microarray data: a robuste composite method addressing single and multiple slide systematic variation. Nucleic Acid Res. 30:e15). The signal ratios as measure of differential expression between the red and green channels were obtained from processed signal intensities. Ratios were further analysed with Microsoft Excel (Microsoft) and SAM software for statistic significance (Tusher et al., 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98:5116-5121).
To determine the significance of differential expression RNA was isolated from the H. pylori 26695 WT grown in BHI broth, and 20 µg of this RNA were labelled either with Cy3-dCTP or with Cy5-dCTP. The two cDNA probes generated were hybridized onto the same slide, and the data were analysed as mentioned above. Signal ratios < 0.5 and > 2.0 were analyzed further.

first: Michael

last: Pflock

first: Hans

last: Mollenkopf

middle: F

last: Meyer


title: Liver transcriptome (BXD)

status: Public on Apr 09 2007

city: Hidaka

first: Itoshi

last: NIKAIDO

state: Saitama

address: 1397-1 Yamane

country: Japan

institute: Saitama Medical School

summary:
Expression profile of Liver transcriptome of BXDs, C57BL6/J and DBA2/J. We use expression QTL analysis to discover disease loci and regulatory pathways regulating serum lipid levels.
Keywords: strains

overall_design: 18 BXDs, DBA2/J C57BL6/J were sacrificed at 10-20 weeks of age.  Liver tissues were collected from three unfasted males for each of the mouse  used for these analyses. We then measured differences in gene expression profile between each RI/parents and B6 17.5 day embryos.

first: Yasushi

last: Okazaki


title: Differentially Expressed Genes in the Skin of Tumor Susceptible K14-Agouti Transgenic Mice

city: Oak Ridge

first: Yesim

last: Aydin Son

state: TN

address: ORNL, Bethel Valley Road, Building 1061,MS-6445

department: Functional Genomics Group

institute: UT-ORNL Graduate School of Genome Science and Technology

summary:
The mouse agouti gene is normally expressed in the skin and regulates coat color pigmentation. Dominant regulatory mutations in the agouti gene, such as viable-yellow (Avy), cause ubiquitous over-expression of the wild-type gene product in every tissue in the body. As a result of this altered agouti expression, viable-yellow mice have solid yellow hair, they are obese and diabetic, and they have increased susceptibility to spontaneous and chemically induced tumors in a wide variety of tissues (e.g., skin, lung, liver, mammary gland and urinary bladder).  Additionally, liver-specific expression of the agouti gene was shown to promote hepatocellular carcinogenesis, even in the absence of obesity and diabetes.

Our lab recently extended these findings to show that the agouti gene also promotes skin cancer in the absence of obesity and diabetes. In K14-Agouti transgenic mice, the wild-type agouti gene is over-expressed in the skin under the regulatory control of the keratin 14 (K14) promoter. Over-expression of agouti in the skin results in yellow coat color without the obesity and diabetes. In two-stage skin carcinogenesis experiments the agouti protein was shown to act as a tumor promoter since it promotes the development of skin tumors in the K14-Agouti transgenic mice, but only after the skin was initiated with a single dose of 7, 12-dimethylbenz[a]anthrance (DMBA). These experiments set the stage for determining the mechanism of action of agouti protein in tumor promotion, which remains completely uncharacterized.

As a first step in determining the role of agouti in skin cancer, we used cDNA microarray and quantitative real time polymerase chain reaction (QRT-PCR) analysis to identify and validate a set of differentially expressed genes in the skin of K14-Agouti transgenic mice at the promotion stage of carcinogenesis.
Keywords:  promotion stage of carcinogenesis, biological replicates,

overall_design: Total RNA from three different eight-week-old male K14-Agouti transgenic mice was hybridized individually against a pooled total RNA sample from three sex-matched, age-matched non-agouti control mice. All three hybridizations were repeated with dye swapping to minimize dye bias in the microarray analysis. Since each one of the NIA 15K mouse ESTs is spotted in duplicate on the microarray slides, 12 data points were generated for the expression level of each gene


title: Comparative genomic analysis of T.whipplei strains reveals that diversity is mainly related to the WiSP proteins

status: Public on Apr 06 2007

city: Marseille

first: My-Van

last: La

address: 27 BD Jean Moulin

country: France

institute: Unité des Rickettsies (CNRS-UMR 6020)

summary:
In this work we analyzed the genomic diversity of several Tropheryma whipplei strains by microarray based-comparative genomic hybridization. Fifteen clinical isolates originating from biopsy samples recovered from different countries were compared with the T. whipplei Twist strain. For each isolate, the genes were defined as either present or absent/divergent using the GACK analysis software. Genomic changes were then further characterized by PCR and sequencing. Obtained results revealed a limited genetic variation between these T. whipplei isolates with at most 2.24 % of the probes exhibiting differential hybridization against the Twist strain. The main variation was found in genes encoding for the WiSP family proteins supporting the view of these membrane proteins as key actors of immune evasion. This work also evidenced a 19.2 kb-pair deletion within T. whipplei DIG15 strain. This deletion takes place in the same region as the large genomic rearrangement previously described between Twist and TW08/27 which can thus be considered as a major hot-spot for intra-specific T. whipplei differentiation. Analysis of this deleted region confirmed the role of WND-domains in generating T. whipplei diversity
Keywords: Comparative genomic hybridization

overall_design:
*15 test strains (Endo5, Endo7, Slow1B, Slow2, ART1, Neuro1, Neuro2, Dig7, Dig9, Dig10, DigADP11, Dig Neuro14, Dig15, DigMusc17, DigNeuro18)
*1 reference strain (T. whipplei strain Twist)
*3 biological replicates per condition
*4 technical replicates per microarray
*Dual-labeling using a reference design: reference sample labeled with Cy5-dCTP and test sample labeled with Cy3-dCTP

first: Nicolas

last: Crapoulet

first: Pascal

last: Barbry

first: Didier

last: Raoult

first: Patricia

last: Renesto


title: Complex interactions of stress-associated regulons in an mprAB deletion mutant of Mycobacterium tuberculosis

status: Public on Mar 28 2007

city: Newark

first: Saleena

last: Ghanny

address: 185 South Orange Ave

institute: Rutgers University

summary:
Using a deletion mutant lacking portions of the response regulator, MprA, and the histidine kinase, MprB, we demonstrated by DNA microarrays that MprAB activates sigE and sigB under SDS stress and during logarithmic growth.
Keywords: effects of 0.05% SDS on Mtb gene expression

overall_design: The expression of SigE-regulated genes {Manganelli, 2004 #212} was evaluated in SDS-treated and control cultures of H37Rv and Rv-D981, using array analyses, for each pair of samples in each experiment, dye flips were performed

first: Xiuhua

last: Pang

first: Phong

last: Vu

first: Thomas

last: Byrd

last: Soteropoulos

first: Galina

middle: V

last: Mukamolova

first: Shiping

last: Wu

first: Buka

last: Samten

first: Susan

last: Howard


title: Partial hepatectomy 1, 2, 4, 6h

city: Philadelphia

first: Egle

last: Juskeviciute

state: PA

address: 1020 Locust st. JAH 527

department: Pathology

institute: Thomas Jefferson University

summary:
Adult male Sprague-Dawley rats (275-350 g) were anesthetized and subjected to two-thirds PHx. Liver sections removed by partial hepatectomy (PHx) were collected and used both as controls (time=0) and as individual reference material for each animal to reduce the error introduced by animal-to-animal variability. At 1, 2, 4, and 6 hours following PHx, rats were killed and liver samples were harvested.
Keywords: Time series PHx (1, 2, 4, 6h)

sample_organism:
Rattus norvegicus
synthetic construct

overall_design: Reference Design with 4 biological repeats for each time-point.


title: Effect of heat shock at 500 atm and pressure shock to 500 atm on Methanocaldococcus jannaschii

city: Berkeley

first: Boonchai

last: Boonyaratanakornkit

address: 201 Gilman Hall #1462

department: Chemical Engineering

institute: UC Berkeley

summary:
Growth and transcriptional profiles of the barophilic methanarchaeon Methanocaldococcus jannaschii were studied at temperatures up to 98C and pressures up to 500 atm. Application of 500 atm of hyperbaric pressure shifted the optimal growth temperature upwards, and heat shock from 88C to 98C at 500 atm resulted in termination of growth. Pressure shock of M. jannaschii from 7.8 to 500 atm over 15-min, the first pressure upshift reported for a barophile, did not accelerate growth. Transcriptional profiles indicated a similar pressure response under growth and heat shock at 500 atm and pressure shock to 500 atm suggesting that the commonly affected genes are important for high-pressure adaptation. Factorial microarray design allowed de-convolution of the interacting effect of elevated pressure and heat shock on expression profiles, thus suggesting genes that may contribute to the organism’s survival in the turbulent in situ conditions of deep-sea hydrothermal vents.
Keywords: stress response, time course, high pressure, heat shock, pressure shock

overall_design: The cDNA arrays that were used contained three technical replicate spots for each ORF in the genome of M. jannaschii. Twenty-four sub-arrays were hybridized with the following pairs of samples: four biological-replicate pairs of samples extracted from 7.8 atm and 500 atm, both without heat shock, four biological-replicate pairs of samples extracted from 7.8 atm without heat shock and 7.8 atm with heat shock (0.5 h and 1.5h), four biological-replicate pairs of samples extracted from 500 atm without heat shock and 500 atm with heat shock (0.5 h and 1.5h), and four biological-replicate pairs of samples extracted from 7 atm and 500 atm, both heat shocked (0.5 h). Each of the four pairs of biological replicates included two pairs of dye-swapped samples. Six sub-arrays were hybridized with the following pairs of samples: three biological-replicate pairs of samples extracted from cells pressure shocked to 500 atm (0.5 h and 1.5 h) and from cells grown at 7 atm without heat shock.

middle: B

first: Liyan

last: Miao

first: Douglas

middle: S

last: Clark

first: Anjana

last: Simpson

first: Najib

last: El-Sayed

first: Vivian

last: Peng


title: Expression data from premalignant breast cancers

city: St Louis

first: Sangjun

last: Lee

state: MO

address: 4940 Parkview Place

institute: Washington University at St. Louis

summary:
Enlargement of normal terminal duct lobular units (TDLUs) by hyperplastic columnar epithelial cells is one of the most common abnormalities of growth in the adult female human breast.  These hyperplastic enlarged lobular units (HELUs) are important clinically as the earliest histologically identifiable potential precursor of breast cancer.  The causes of the hyperplasia are unknown but may include estrogen-simulated growth mediated by estrogen receptor alpha, which is highly elevated in HELUs and may be fundamental to their development.
This study used DNA microarray technology and RNA from microdissected pure epithelial cells to learn more about changes in gene expression and molecular pathways associated with the development of HELUs from TDLUs
Keywords: Identification of early cancer precursors

overall_design: Samples for the microarray studies were derived from 8 formalin-fixed paraffin-embedded tissue (FFPET) biopsies containing paired normal TDLUs and well-developed HELUs from non-cancerous adult female human breasts obtained within the past 3 years.  Populations of nearly pure (>95%) epithelial cells were isolated from the TDLUs and HELUs in each biopsy (approximately 25,000 cells/sample) by laser capture microdissection (LCM)

first: Dan

last: Medina

first: Anna

last: Tsimelzon

first: Syed

middle: K

last: Mohsin

first: Sufeng

last: Mao

first: Yun

first: D

middle: C

last: Allred


title: miRNA expression during UCB-CD34 cells erythropoiesis

city: Singapore

first: Meng Ling

last: Choong

address: 31 Biopolis Way, #03-01 Nanos

department: Drug Discovery

country: Singapore

institute: Experimental Therapeutics Centre

summary:
We investigated the miRNA expression in ex vivo human erythroid cultures from umbilical cord blood (UCB)-derived CD34 cells.  Hypothetically, the decline of certain miRNAs may promote erythropoiesis by unblocking expression of key functional proteins while the up-regulation of other miRNAs may block commitment to non-erythroid lineages. Therefore, discovering the patterns and sequence of miRNA expression during hematopoietic differentiation will provide insights about the functional roles of these tiny noncoding genes.
Keywords: miRNA expression profiling

overall_design: miRNA probeset was purchased from Ambion and printed onto glass slides. miRNA was extracted from the cells at regular intervals throughout the induction period using commercial miRNA isolation kit (Ambion), labeled with cy-dye (Amersham Pharmacia) and hybridized onto the slides as described by the manufacturer's protocols (Ambion). Samples from all other days were compared to sample from Day 0 (untreated).

first: MengLing

first: Henry

last: Yang

first: Ian

last: McNiece


title: Gene expression profiling of chickpea responses to high-salinity stress

city: Bundoora

first: Nitin

last: Mantri

address: Bldg 223, Level 1, Plenty Road

institute: RMIT University

summary:
‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to high-salinity stress. Two groups of a tolerant and susceptible accession were challenged with high-salinity stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaf/shoot and root tissues were collected at 24 and 48 h post-treatment (hpt) and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (shoot and root tissues separate for each time point) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1.
The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress.

Globally, the level of 409 transcripts was affected in response to high-salinity stress in all the genotypes and tissue types studied. Of the transcripts consistently expressed in tolerant genotypes in response to high-salinity stress, the annotation of transcripts at 24 hpt suggest a reduction in energy production in shoots and roots by repression of putative genes including P700 chlorophyll a-apoprotein (DY475501) and NADH-plastoquinone oxidoreductase subunit I (DY475287), cytosolic fructose 1,6-bisphosphatase (DY475548) and splicing factor-like protein (DY396290). The ATHP3 (DY396300) and protein kinase (DY475077) that are potentially involved in signalling cascades responsible for sensing and relaying osmotic stress signals, were consistently repressed in tolerant genotypes in response to high-salinity. Additionally a glycine rich protein (DY396342) that is associated with lignification of cell walls in response to wounding and pathogen attack was repressed in tolerant genotypes.

In tolerant genotypes at 48 hpt, two energy and metabolic-related transcripts were consistently repressed in roots, including a carbonic anhydrase (DY475403) and putative thiazole biosynthetic enzyme (DY475242). In shoots, only a pathogenesis-related protein (DY396301) was consistently repressed at 48 hpt, but a similar transcript (DY396281) was induced in roots of tolerant genotypes at the same time.

Only four transcripts were DE in susceptible genotypes at 24 hpt, all occurring in root tissue.  Two of these were unknown/unclear, but the others included a proline oxidase transcript (DY475225) and a nuclear transport factor 2 (DY396436). At 48 hpi, a putative splicing factor-like protein (DY396290) and polyubiquitin (DY396328) were repressed in roots of susceptible genotypes. Interstingly, a putative xylosidase (DY475408) was induced in susceptible roots at 48 hpi.

Finally, several unknown/unclear transcripts were DE in both tolerant and susceptible genotypes.  Of interest were two unknowns (DY475293 and DY475521) that were consistently expressed in tolerant genotypes and may be important for high-salinity tolerance.
Keywords: Chickpea, High-salinity stress, tolerant, susceptible, cDNA microarray

overall_design:
Total RNA was extracted from separately pooled shoot and root tissues for each genotype {CPI 060546 (Tolerant1), CPI 60527 (Susceptible1), ICC 06474 (Tolerant2) and ICC 08161 (Susceptible2)} 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 as described [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Plant Biotechnology Journal. 4(6), 647–666]. 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, 576 images were analyzed from 96 genotype x tissue type x treatment/control x biological replication condition.

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-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.

first: Edwin

last: Pang

first: Rebecca

last: Ford

first: Tristan

middle: E

last: Coram


title: Gene expression profiling of chickpea responses to drought stress

summary:
‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to drought stress. Two groups of a tolerant and susceptible accession were challenged with drought stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaf and flower/bud tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf and flower tissues separate) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1.
The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress.
Globally, the level of 114 transcripts was affected in response to drought stress in all the genotypes and tissue types studied. The DE transcripts in response to drought stress coded for various functional and regulatory proteins, most of which were repressed. Protein and other solute transport was repressed in susceptible-1 flowers but induced in tolerant-2 flowers, as evidenced by the induction of a protein-transport facilitation protein (DY475074) in flowers of tolerant-2 and repression of a aquaporin-like membrane channel protein (DY396334) and DNA-J like protein involved in intracellular protein transport (DY475488) in flowers of susceptible-1. Two putative auxin-repressed proteins (DY396289, DY396359) were highly repressed in flowers and leaves of tolerant-2, whilst being induced in flowers and leaves of susceptible-1. Defence-related genes including pathogenesis-related proteins (DY396305 and DY396343), nematode-resistance protein (CV793603), Cf-9 gene cluster (DY396352) and disease resistance response proteins (DY396265 and DY396276) were repressed in flowers of tolerant and susceptible genotypes. A Pea (pi230) disease resistance response protein (DY396390) and a multi-resistance protein ABC transporter (CV793605) were induced in flowers of both tolerant genotypes. Some genes involved in energy metabolism (DY396279 and DY475316) were repressed in leaves and flowers of tolerant and susceptible genotypes. Additionally, some transcripts related to senescence, including auxin responsive protein IAA9 (DY396315), senescence-associated protein DIN 1 (DY396338), and dehydration stress-induced protein (DY396321) were repressed in leaves and flowers of the tolerant genotypes. Of the 114 DE transcripts expressed in drought tolerant and susceptible genotypes, only two were consistently expressed. These included a cytosolic fructose 1,6-bisphosphatase (DY475548) and a gene with unknown function, which were repressed in flowers of both susceptible genotypes.
Keywords: Chickpea, Drought stress, Tolerant, Susceptible, cDNA microarray

overall_design:
Total RNA was extracted from separately pooled leaf and flower tissues for each genotype {BG 1103 (Tolerant1), Kaniva (Susceptible1), BG 362 (Tolerant2) and Genesis 508 (Susceptible2)} 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 as described [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Plant Biotechnology Journal. 4(6), 647–666]. 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, 288 images were analyzed from 48 genotype x tissue type x treatment/control x biological replication condition.

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-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: L


title: Salmonid host response to Hematopoietic Necrosis virus: cellular receptors, viral control and novel pathways of defence

city: Nanaimo

first: Shaorong

last: Li

state: British Columbia

address: 3190 Hammond Bay Road

department: Salmon and Freshwater Ecosystems Division

country: Canada

institute: Fisheries and Oceans Canada

summary:
The transcriptional response of Atlantic salmon (Salmo salar) to infectious hematopoietic necrosis (IHN) virus was elucidated using 16,008 gene GRASP cDNA microarrays.   S. salar were exposed to the IHN virus in a waterborne challenge, and kidney samples from five fish sampled on each of days 0, 1, 5 and 9 were analysed in the microarray study.  Validation of nine of the significant genes was conducted on fish sampled on alternate days from 1 to 13 and compared to the unchallenged control sampled on day 0.  The key pathways up-regulated in response to the virus included endosomal transport, type I and II interferon responses, the alternative complement pathway, apoptosis, phagocytosis and phagocytic cell oxidation, natural killer cell activity, antiviral replication via iron sequestering, virally-induced disruption of the cell cycle, retroviral DNA integration, T-cell activation and cellular immunity.  Fish that did not contain viral titer on days 5 and 9 responded in a similar manner to those with titer, but further induced a number of genes involved in anti-viral replication (Glutathione peroxidase 1 and ferritin H), anti-proliferation (PTEN and B-cell receptor associated protein 32), lysosomal response (CD63 antigen), pro-inflammatory response (lipopolysaccharide binding protein, stress activated JNK1, thiol peroxidase), and T-cell activation (T-cell activation Rho-GTPase activating protein) that may increase resistance to the virus.   Three genes that were potentially co-opted by the virus to enhance infectivity were also identified, including uPAR (angiogenesis), CypA (viral replication and infectivity), and BAF1 (viral protein biosynthesis).  Perhaps the most notable finding was the up-regulation of uPAR which can function to increase the density of fibronectin, the receptor of the IHN virus, on the cell surface, hence facilitate viral entry into the cell.
Keywords: Immunlogical response to IHN virus challenge

platform_organism:
Oncorhynchus mykiss
Salmo salar

overall_design: A preliminary microarray experiment was run on the waterborne-challenged S. salar whereby the five individuals sampled on a given day were combined and run on a single slide, and we compared the expression profiles from each day (days 0 (unchallenged control) through 11).  From this experiment, we noted that the most profound changes were occurring around days 1, 5, and 9, so we performed subsequent experiments at these time points, with five fish per day and 5 control fish analysed in total.

first: Kristina

last: Miller

first: Garth

last: Traxler

first: Karia

last: Kaukinen

first: Jon

last: Richard

first: Norma

last: Ginther


title: Profiling of genes regulated by TGF-beta in lung carcinoma (A549) and immortalized lung epithelial (HPL1D)cells.

status: Public on Apr 04 2007

city: Bangalore

first: Paturu

last: Kondaiah

address: C.N.R. Rao Circle

department: Molecular Reproduction, Development and Genetics

country: India

institute: Indian Institute of Science

summary:
TGF-beta is one of the key cytokines implicated in various disease processes including cancer.  TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis.  It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations.  Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells.

Results: Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells.  In addition, 267 genes are commonly regulated in both the cell lines.  Semi quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data.

Conclusion: These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells.
Keywords: cell type comparison in response to TGF-beta

overall_design: To compare the genes regulated by TGF-beta in non transformed and transformed cells, we chose an immortalized lung epithelial cell line, HPL1D and a lung adenocarcinoma cell line, A549.  Cells were plated and propagated in designated media and TGF-beta treatments were carried out in serum free conditions at various time intervals, 1hr, 4hr and 12hr.  The RNA was extracted and expression profiling was performed using microarray experiments on 19K cDNA arrays after labeling the cDNAs with Cy3 and Cy5 dyes for normal and tumor cell RNAs respectively. The image and data analysis was performed using Quantarray (P.E. Life Sciences, USA) and Avadis (Strand Life Sci. India) softwares.  The results are computed as log2 transformed fold changes between treated and untreated respective cell lines.  The data of regulated genes of each cell line was compared with the other cell line and exclusively and commonly regulated genes have been determined.

first: Prathibha

last: Ranganathan

first: Animesh

last: Agrawal

first: Raghu

last: Bhushan

first: Ravikiran

last: Kalathur

first: Takashi

last: Takahashi