Comparison of lymphoblastoid cell gene expression with immunofluorescent chromosome banding
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ABSTRACT: Metaphase chromosome staining was used to provide a high level overview of the pattern of histone modifications (H3K27ac, H3K27me3 and H3K4me3) at a single cell level in human lymphoblastois cells. These epigenomic banding patterns were related to various genomic features including gene and CpG island density as well as gene expression. A 3 array study using RNA extracted from 3 separate cultures of human lymphoblastoid cells as biological replicates
Project description:Investigation of whole genome gene expression level differences of Fusarium fujikuroi between wild-type and a Ffvel1 (velvet) deletion mutant in liquid medium with minimal nitrogen between 24 hr, 72 hr and 120 hr of growth using an array based on a F. verticillioides gene call set. Fusarium fujikuroi produces a number of secondary metabolites including gibberellins, bikaverin, fumonisin and fusarin C that are influenced by nitrogen availability and the velvet global regulatory complex. A twelve chip study using total RNA recovered from six cultures of wild-type Fusarium fujikuroi and six cultures of Ffvel1 F. fujikuroi deletion mutant. Each chip measures the expression level of over 13,000 putative genes with twelve 60-mer probes per sequence.
Project description:Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10M-BM-5g/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended. 12 samples
Project description:The goal of this analysis is to define the anaerobic regulon for the global transcription factor CRP (catabolite repressor protein) in the periodontal pathogen Aggregatibacter actinomycetemcomitans. The objectives were met by comparing the mRNA profile of a wild type strain, JP2, to that of an isogenic mutant deleted of the CRP gene. Both strains were grown anaerobically. A three biological replicates study using total RNA recovered from three separate cultures of wild-type Aggregatibacter actinomycetemcomitans strain JP2 and three separate cultures of strain AAM167, an isogenic deletion mutant in which the gene for cAMP-Receptor-Protein (CRP) has been replaced by a spectinomycin resistance gene cassette. The cells were grown anaerobically in pairs (wild type paired with mutant). Each chip measures the expression level of 2,116 genes from A. actinomycetemcomitans strain HK1651. There were 11 different 60-mer probes /gene for 94% of the genes, and the entire probe set (22,537 probes) was replicated (technical replicates) three times on each array. Each probe set is contained on a quarter of a chip and there were also 4,426 random probes per array. Wild type and mutant RNAs from cultures #1 and #2 were hybridized to the same chip and the RNAs from cultures #3 were hybridized in different quadrants of a second chip.
Project description:The conductive pili of Geobacter sulfurreducens are essential for optimal extracellular electron transfer to Fe(III) and long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene for PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, slow rates of Fe(III) reduction were detected after extended (> 30 days) incubation in the presence of Fe(III) oxide. After seven consecutive transfers the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, proteomic, and gene deletion studies indicated that this adaptation was associated with greater production of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every three days, the wild-type strain out-competed the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron-shuttling producing Fe(III) reducers in most soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current; consistent with the concept that long-range electron transport through G. sulfurreducens biofilms cannot be achieved without PilA-pili. An eight-chip study using total RNA recovered from four separate cultures of Geobacter sulfurreducens JS-1 (experimental condition) or Geobacter sulfurreducens KN400 (control condition) grown with acetate (10mM)-Fe(III) oxide (100 mmol l-1) exponential growth. Each chip measures the expression level of 3,328 genes from Geobacter sulfurreducens KN400 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Investigation of the whole genome gene expression level changes relative to exponential phase growth in Nitrosomonas europaea ATCC19718 after 12 hours ammonia starvation, 144 hours ammonia starvation, and 20 minutes following ammonia addition to starved cells. The ammonia monooxygenase of chemolithotrophic ammonia oxidizing bacteria (AOB) catalyzes the first step in ammonia oxidation by converting ammonia to hydroxylamine. The monooxygenase of Nitrosomonas europaea is encoded by two nearly identical operon copies (amoCAB1,2). Several AOB, including N. europaea, also posess a divergent monocistronic copy of amoC (amoC3) of unknown function. Previous work suggested a possible functional role for amoC3 in N. europaea during recovery from extended ammonia starvation as part of the σE- stress response regulon during the recovery of N. europaea from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of amoC3 is part of a general post-starvation cellular response system in N. europaea. We also found that amoC3 is required for efficient exit from prolonged ammonia starvation, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions. Deletion of the σ32 global stress response regulator demonstrated that the heat shock regulon also plays a significant role in mediating the recovery of N. europaea from starvation. These findings provide the first described phenotype associated with the divergent AmoC3 subunit which appears to function as a stress responsive subunit capable of maintaining ammonia oxidation activity under stress conditions. A twelve chip study using total RNA recovered from four timepoints for each of three biological replicates of wild-type cultures of Nitrosomonas europaea ATCC 19718. Total RNA was obtained from each biological culture replicate during exponential growth, following 12 hours ammonia starvation, 144 hours ammonia starvations, and 20 minutes following ammonia addition to starved cells.
Project description:The goal of this analysis is to define the aerobic and anaerobic regulons for the transcription factor Mlc (Makes Large Colonies) in the periodontal pathogen Aggregatibacter actinomycetemcomitans. The objectives were met by comparing the mRNA profile of a wild type strain, JP2, to that of an isogenic mutant deleted of the Mlc gene. Both strains were grown aerobically and anaerobically. The results show that Mlc is involved in the regulation of dozens of genes in both growth conditions. Half of the study (anaerobic samples) was a four biological replicates study using total RNA recovered from four separate anaerobic cultures of wild-type Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans strain JP2 and four separate anaerobic cultures of strain AAM155, an isogenic deletion mutant in which the gene for Mlc (Makes Large Colonies) has been replaced by a spectinomycin resistance gene cassette. The cells were grown anaerobically in pairs (wild type paired with mutant). The second half of the study (aerobic samples) was a four biological replicates study using total RNA recovered from four separate aerobic cultures of wild-type Aggregatibacter actinomycetemcomitans strain JP2 and four separate anaerobic cultures of strain AAM155, an isogenic deletion mutant of Mlc. The cells were grown aerobically in pairs (wild type paired with mutant). Each chip measures the expression level of 2,116 genes from A. actinomycetemcomitans strain HK1651. There were 11 different 60-mer probes /gene for 94% of the genes, and the entire probe set (22,537 probes) was replicated (technical replicates) three times on each array. Each probe set is contained on a quarter of a chip and there were also 4,426 random probes per array. A total of four chips were used in these experiments.
Project description:This study examines the changes in genes expression that occur in Drosophila melanogaster during the ecdysone response as well as during RNAi knockdown of the insulator protein, CP190. Analysis was performed in Kc cells after 0, 3, and 48 hours of ecdysone treatment in the presence of either control or CP190 knockdown. Six conditions were analyzed, and each condition was performed for 2 biological replicates making a total of 12 chips. Each chip measures the expression level of 16,637 genes from D.melanogaster with eight, 60-mer probes per gene.
Project description:Investigation of whole genome gene expression level changes in Thermoplasma acidophilum cultured under aerobic and anaerobic conditions. The analysis are further described in Na Sun, Cuiping Pan, Stephan Nickell, Matthias Mann, Wolfgang Baumeister, and István Nagy, Quantitative proteome and transcriptome analysis of the archaeon Thermoplasma acidophilum cultured under aerobic and anaerobic conditions (submitted). Total RNA of T. acidophilum was isolated with the RNeasy Protect Bacteria Kit (Qiagen). The transcriptomics analysis was performed on TI273075 60mer chips of Roche NimbleGen microarrays (NimbleGen Systems of Iceland, LLC). Probes were selected for all protein sequences (1482) and labelled with Cy3. The median number of probes per sequence is 20, and each probe is replicated 5 times on the chip. The probes are randomly distributed over the surface of the array. Unused features are filled with randomly generated probes of comparable GC content. ArrayStar v2.0 software (DNASTAR, Inc.) was used for the data analysis. Three independent biological replicates were processed for aerobic and anaerobic conditions, respectively.
Project description:In this study, we used whole genome comparative oligonucleotide microarrays to investigate the brain transcriptomic response to predator cues using the threespine stickleback, Gasteroteus aculeatus. We showed that exposure to olfactory, visual and tactile cues of a predator (rainbow trout, Oncorhynchus mykiss) for six days resulted in subtle but significant transcriptomic changes in the brain of sticklebacks. Gene functional analysis and gene ontology (GO) enrichment revealed that the majority of the transcripts differentially expressed between the fish exposed to predator cues and the control group are primarily related to antigen processing and presentation (involving primarily the major histocompatibility complex (MHC)), transmission of synaptic signals, brain metabolic processes, gene regulation, or visual perception. Pathway analysis identified synaptic long-term depression, RAN signaling, relaxin signaling and phototransduction as the top four pathways that were over-represented. Adult fish were placed in six different 26L tanks with three fish per tank in a partially recirculating flow-through system. Half of the tanks were assigned to the control group and the other half to the experimental group.10 samples were selected for microarray analysis. The ten samples comprised five biological replicates in the experimental group (fish exposed to predator cues) and five biological replicates in the control group (fish not exposed to predator cues), and were evenly distributed across tanks. The cDNA labeling (single color), hybridization, washing and scanning steps were performed in the NimbleGen microarray gene expression service department.
Project description:Survey of relative gene expression profiles in larvae, pupae, and midguts and fatbodies of teneral, unfed, and fed adult mountain pine beetles from western Nevada, USA. Array prepared on 4-tile chips (4-plex custom arrays), each feature represented by six different oligonucleotides, three replicate blocks/tile, hybridized with total RNA from 11 samples, each replicated four times. Vector (pDONR222) and two plant sequences included as negative controls.