scholarly journals Gammaherpesvirus Lytic Gene Expression as Characterized by DNA Array

2002 ◽  
Vol 76 (12) ◽  
pp. 6244-6256 ◽  
Author(s):  
Joo Wook Ahn ◽  
Kenneth L. Powell ◽  
Paul Kellam ◽  
Dagmar G. Alber
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Expression Profiles ◽  
Cdna Array ◽  
Gene Expression Profiles ◽  
Hierarchical Cluster ◽  
Murine Gammaherpesvirus ◽  
Transcription Profiles ◽  
A Genome

ABSTRACT Gammaherpesviruses are associated with a number of diseases including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) constitutes the most amenable animal model for this family of pathogens. However experimental characterization of gammaherpesvirus gene expression, at either the protein or RNA level, lags behind that of other, better-studied alpha- and beta-herpesviruses. We have developed a cDNA array to globally characterize MHV-68 gene expression profiles, thus providing an experimental supplement to a genome that is chiefly annotated by homology. Viral genes started to be transcribed as early as 3 h postinfection (p.i.), and this was followed by a rapid escalation of gene expression that could be seen at 5 h p.i. Individual genes showed their own transcription profiles, and most genes were still being expressed at 18 h p.i. Open reading frames (ORFs) M3 (chemokine-binding protein), 52, and M9 (capsid protein) were particularly noticeable due to their very high levels of expression. Hierarchical cluster analysis of transcription profiles revealed four main groups of genes and allowed functional predictions to be made by comparing expression profiles of uncharacterized genes to those of genes of known function. Each gene was also categorized according to kinetic class by blocking de novo protein synthesis and viral DNA replication in vitro. One gene, ORF 73, was found to be expressed with α-kinetics, 30 genes were found to be expressed with β-kinetics, and 42 genes were found to be expressed with γ-kinetics. This fundamental characterization furthers the development of this model and provides an experimental basis for continued investigation of gammaherpesvirus pathology.

Co-Expression of BAALC with ERG, TCF4, and CD133 Indicates a Shared Pathway in Acute Myeloid and Lymphoblastic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2755-2755 ◽  
Author(s):  
Claudia D. Baldus ◽  
Michael Radmacher ◽  
Guido Marcucci ◽  
Dieter Hoelzer ◽  
Eckhard Thiel ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Newly Diagnosed ◽  
Time Points ◽  
Normal Cytogenetics ◽  
Acute Myeloid

Abstract The human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) is a molecular marker of hematopoietic progenitor cells and is aberrantly expressed in subsets of acute myeloid (AML) and lymphoblastic (ALL) leukemias. High mRNA expression levels of BAALC have been shown to adversely impact outcome in newly diagnosed AML patients (pts) with normal cytogenetics. To gain insight into the functional role of BAALC and its significance to normal hematopoiesis and leukemogenesis we compared gene expression profiles of normal CD34+ progenitors with those of AML and ALL blasts (using oligonucleotide microarrays; HG-U133 plus 2.0, Affymetrix, Santa Clara, CA). First we explored the regulation of BAALC expression during lineage specific maturation of in vitro differentiated human CD34+ bone marrow cells selected from healthy individuals. Microarray analyses were carried out using CD34+ cells stimulated in vitro with EPO, TPO, or G/GM-CSF to induce lineage-specific differentiation. At day 0 of culture and at three different time points during differentiation (days 4, 7, 11) cells were harvested, and if necessary purified by immunomagnetic beads and used for microarray studies. Experiments of all lineages and time points were done in triplicates. A total of 276 genes were identified showing similar changes in expression (with downregulation during differentiation) as BAALC at the three time points in all lineages with a correlation coefficient of R>0.95. This set of 276 BAALC co-expressed genes was investigated in an AML expression dataset generated from 51 adult pts with newly diagnosed de novo AML and normal cytogenetics (Cancer and Leukemia Group B). After exclusion of probesets expressed in fewer than 20% of pt samples, 21 probesets representing 14 named genes 6 of which are known to be involved in AML (BAALC, CD34, CD133, SOX4, ERG, SEPT6) and 4 implicated in lymphoid development (TCF4, SH2D1A, ITM2A, ITM2C) were found to be overexpressed (a significance level of P=0.01 was used) in pts of the highest third compared to pts of the lowest third of BAALC expression values as measured by real-time RT-PCR. We next applied these same 21 BAALC co-expressed probesets to an ALL expression dataset generated from 66 adult pts with newly diagnosed standard risk B-lineage precursor ALL (from the German ALL GMALL study group). A BAALC specific cluster uncovered 7 probesets representing 4 different co-expressed genes: BAALC, CD133, and the transcription factors ERG and TCF4. Thus, applying a BAALC specific expression signature to AML and ALL gene expression profiles revealed 3 genes (CD133, ERG, TCF4), which are highly associated with BAALC in myeloid and lymphoid blasts. Interestingly in non-malignant lymphoid and myeloid cells the oncogeneic ETS transcription factor ERG has shown specificity to immature cells, while its mechanistical role in leukemogenesis remains unknown. ERG and TCF4 may directly regulate BAALC and indicate a specific pathway implicated in leukemogenesis, while co-expression of CD133 and BAALC suggests shared stem cell characteristics. Functional studies are in progress to further explore these findings.

scholarly journals Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Risa Okada ◽  
Shin-ichiro Fujita ◽  
Riku Suzuki ◽  
Takuto Hayashi ◽  
Hirona Tsubouchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Muscle Mass ◽  
Muscle Atrophy ◽  
Transcriptome Analysis ◽  
Fiber Type ◽  
Expression Profiles ◽  
Space Station ◽  
Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

scholarly journals Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e66-e73 ◽  
Author(s):  
Chih-Wen Ni ◽  
Haiwei Qiu ◽  
Amir Rezvan ◽  
Kihwan Kwon ◽  
Douglas Nam ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Expression Profiles ◽  
Disturbed Flow ◽  
A Genome ◽  
Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

scholarly journals Analysis of Expressed Sequence Tag Data and Gene Expression Profiles Involved in Conidial Germination of Fusarium oxysporum

2006 ◽  
Vol 72 (2) ◽  
pp. 1667-1671 ◽  
Author(s):  
Ye Deng ◽  
Haitao Dong ◽  
Qingchao Jin ◽  
Cheng'en Dai ◽  
Yongqi Fang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Fusarium Oxysporum ◽  
Cdna Library ◽  
Expressed Sequence Tag ◽  
Expression Profiles ◽  
Cdna Array ◽  
Gene Expression Profiles ◽  
Conidial Germination ◽  
Expressed Sequence

ABSTRACT We obtained 3,372 tentative unique transcripts (TUTs) from a cDNA library of Fusarium oxysporum. A cDNA array with 3,158 TUTs was produced to analyze gene expression profiles in conidial germination. It seems that ras and other signaling genes, e.g., ccg, cooperatively initiate conidial germination in Fusarium by increasing protein synthesis.

Simulated microgravity using the Random Positioning Machine inhibits differentiation and alters gene expression profiles of 2T3 preosteoblasts

2005 ◽  
Vol 288 (6) ◽  
pp. C1211-C1221 ◽  
Author(s):  
Steven J. Pardo ◽  
Mamta J. Patel ◽  
Michelle C. Sykes ◽  
Manu O. Platt ◽  
Nolan L. Boyd ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Bone Loss ◽  
Simulated Microgravity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Underlying Mechanism ◽  
Bone Biology ◽  
Random Positioning Machine

Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 165
Author(s):  
X. S. Cui ◽  
X. Y. Li ◽  
T. Kim ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Trichostatin A ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Mouse Embryos ◽  
Differentially Expressed ◽  
Gene Expression Patterns ◽  
Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

Gene Expression Profiles in Rat Liver Slices Exposed to Hepatocarcinogenic Enzyme Inducers, Peroxisome Proliferators, and 17α-Ethinylestradiol

2006 ◽  
Vol 25 (5) ◽  
pp. 379-395 ◽  
Author(s):  
Gisela Werle-Schneider ◽  
Andreas Wölfelschneider ◽  
Marie Charlotte von Brevern ◽  
Julia Scheel ◽  
Thorsten Storck ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
Mode Of Action ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Peroxisome Proliferators ◽  
Liver Slices ◽  
Enzyme Inducers

Transcription profiling is used as an in vivo method for predicting the mode-of-action class of nongenotoxic carcinogens. To set up a reliable in vitro short-term test system DNA microarray technology was combined with rat liver slices. Seven compounds known to act as tumor promoters were selected, which included the enzyme inducers phenobarbital, α-hexachlorocyclohexane, and cyproterone acetate; the peroxisome proliferators WY-14,643, dehydroepiandrosterone, and ciprofibrate; and the hormone 17 α-ethinylestradiol. Rat liver slices were exposed to various concentrations of the compounds for 24 h. Toxicology-focused TOXaminer™ DNA microarrays containing approximately 1500 genes were used for generating gene expression profiles for each of the test compound. Hierarchical cluster analysis revealed that (i) gene expression profiles generated in rat liver slices in vitro were specific allowing classification of compounds with similar mode of action and (ii) expression profiles of rat liver slices exposed in vitro correlate with those induced after in vivo treatment (reported previously). Enzyme inducers and peroxisome proliferators formed two separate clusters, confirming that they act through different mechanisms. Expression profiles of the hormone 17 α-ethinylestradiol were not similar to any of the other compounds. In conclusion, gene expression profiles induced by compounds that act via similar mechanisms showed common effects on transcription upon treatment in vivo and in rat liver slices in vitro.

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

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