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PubMed2004

Global transcriptome analysis of the heat shock response of Shewanella oneidensis.

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. However, the genetic basis and regulatory mechanisms underlying the ability of S. oneidensis to survive and adapt to various environmentally relevant stresses is poorly understood. To define this organism's molecular response to elevated growth temperatures, temporal gene expression profiles were examined in cells subjected to heat stress by using whole-genome DNA microarrays for S. oneidensis. Approximately 15% (n = 711) of the total predicted S. oneidensis genes (n = 4,648) represented on the microarray were significantly up- or downregulated (P < 0.05) over a 25-min period after shift to the heat shock temperature. As expected, the majority of the genes that showed homology to known chaperones and heat shock proteins in other organisms were highly induced. In addition, a number of predicted genes, including those encoding enzymes in glycolysis and the pentose cycle, serine proteases, transcriptional regulators (MerR, LysR, and TetR families), histidine kinases, and hypothetical proteins were induced. Genes encoding membrane proteins were differentially expressed, suggesting that cells possibly alter their membrane composition or structure in response to variations in growth temperature. A substantial number of the genes encoding ribosomal proteins displayed downregulated coexpression patterns in response to heat stress, as did genes encoding prophage and flagellar proteins. Finally, a putative regulatory site with high conservation to the Escherichia coli sigma32-binding consensus sequence was identified upstream of a number of heat-inducible genes.

Bacterial Proteins↗
PubMed2005

Tricksy business: transcriptome analysis reveals the involvement of thioredoxin A in redox homeostasis, oxidative stress, sulfur metabolism, and cellular differentiation in Bacillus subtilis.

Thioredoxins are important thiol-reactive proteins. Most knowledge about this class of proteins is derived from proteome studies, and little is known about the global transcriptional response of cells to various thioredoxin levels. In Bacillus subtilis, thioredoxin A is encoded by trxA and is essential for viability. In this study, we report the effects of minimal induction of a strain carrying an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible trxA gene (ItrxA) on transcription levels, as determined by DNA macroarrays. The effective depletion of thioredoxin A leads to the induction of genes involved in the oxidative stress response (but not those dependent on PerR), phage-related functions, and sulfur utilization. Also, several stationary-phase processes, such as sporulation and competence, are affected. The majority of these phenotypes are rescued by a higher induction level of ItrxA, leading to an approximately wild-type level of thioredoxin A protein. A comparison with other studies shows that the effects of thioredoxin depletion are distinct from, but show some similarity to, oxidative stress and disulfide stress. Some of the transcriptional effects may be linked to thioredoxin-interacting proteins. Finally, thioredoxin-linked processes appear to be conserved between prokaryotes and eukaryotes.

Bacillus subtilis↗
PubMed2005

Transcriptome analysis of Shewanella oneidensis MR-1 in response to elevated salt conditions.

Whole-genomic expression patterns were examined in Shewanella oneidensis cells exposed to elevated sodium chloride. Genes involved in Na(+) extrusion and glutamate biosynthesis were significantly up-regulated, and the majority of chemotaxis/motility-related genes were significantly down-regulated. The data also suggested an important role for metabolic adjustment in salt stress adaptation in S. oneidensis.

Adaptation, Physiological↗
PubMed2005

Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal.

Survival of near-freezing body temperatures and reduced blood flow during hibernation is likely the result of changes in the expression of specific genes. In this study, we described a comprehensive survey of mRNAs in the heart of the thirteen-lined ground squirrel (Spermophilus tridecemlineatus) before and during hibernation. The heart was chosen for this study because it is a contractile organ that must continue to work despite body temperatures of 5 degrees C and the lack of food for periods of 5-6 mo. We used a digital gene expression assay involving high-throughput sequencing of directional cDNA libraries from hearts of active and hibernating ground squirrels to determine the identity and frequency of 3,532 expressed sequence tags (ESTs). Statistical analysis of the active and hibernating heart expression profile indicated the differential regulation of 48 genes based on a P < or = 0.03 threshold. Several of the differentially expressed genes identified in this screen encode proteins that likely account for uninterrupted cardiac function during hibernation, including those involved in metabolism, contractility, Ca2+ handling, and low-temperature catalysis. A sampling of genes showing higher expression during hibernation includes phosphofructokinase, pancreatic triacylglycerol lipase, pyruvate dehydrogenase kinase 4 (PDK4), aldolase A, sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), titin, and four-and-a-half LIM domains protein 2 (FHL2). Genes showing reduced levels of expression during hibernation include cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), troponin C, phospholamban, Ca2+/calmodulin-dependent protein kinase II (CaMKII), calmodulin, and four subunits of cytochrome c oxidase.

Adaptation, Physiological↗
PubMed2003

Cardiac transcriptome analysis in obesity-related hypertension.

Obesity is associated with volumetric arterial hypertension and with early increase in heart rate and decreased heart rate variability. The consequences of obesity-related hypertension on heart gene regulation are poorly known and were investigated in a model of obesity-related hypertension induced by high fat diet in dogs. When compared with control animals (n=6), a 9-week high fat diet (n=6) provoked significant weight gain and increased blood pressure load and heart rate but failed to significantly change left ventricular mass assessed by echocardiography. Subtractive hybridization of dog heart cDNA libraries were used to generate sublibraries containing differentially expressed cDNAs that were in turn spotted onto membranes to create custom microarrays. Hybridizations of these microarrays with complex probes representing mRNAs expressed in right atria and left ventricles from obese hypertensive and control dogs were performed. Thirty-eight differentially expressed genes were identified; altered expression was confirmed by Northern blot analysis in 15. In addition, real-time quantitative polymerase chain reaction confirmed differential expression for 80% of the randomly chosen tested genes. Once identified, transcripts were categorized into groups involved in metabolism, cell signaling, ionic regulation, cell proliferation, protein synthesis, and tissue remodeling. In addition, we found a set of 11 cDNAs encoding proteins with unknown functions. This study clearly shows that obesity-related hypertension, lasting for only 9 weeks, causes marked changes in gene expression in right atrium as well as the left ventricle that may contribute to early functional changes in heart function and to long-term structural changes such as left ventricular hypertrophy and remodeling.

Animals↗
PubMed2006

Transcriptome analysis of Deinagkistrodon acutus venomous gland focusing on cellular structure and functional aspects using expressed sequence tags.

BACKGROUND: The snake venom gland is a specialized organ, which synthesizes and secretes the complex and abundant toxin proteins. Though gene expression in the snake venom gland has been extensively studied, the focus has been on the components of the venom. As far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. Therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components? RESULTS: To examine extensively the transcripts expressed in the venom gland of Deinagkistrodon acutus and unveil the potential of its products on cellular structure and functional aspects, we generated 8696 expressed sequence tags (ESTs) from a non-normalized cDNA library. All ESTs were clustered into 3416 clusters, of which 40.16% of total ESTs belong to recognized toxin-coding sequences; 39.85% are similar to cellular transcripts; and 20.00% have no significant similarity to any known sequences. By analyzing cellular functional transcripts, we found high expression of some venom related genes and gland-specific genes, such as calglandulin EF-hand protein gene and protein disulfide isomerase gene. The transcripts of creatine kinase and NADH dehydrogenase were also identified at high level. Moreover, abundant cellular structural proteins similar to mammalian muscle tissues were also identified. The phylogenetic analysis of two snake venom toxin families of group III metalloproteinase and serine protease in suborder Colubroidea showed an early single recruitment event in the viperids evolutionary process. CONCLUSION: Gene cataloguing and profiling of the venom gland of Deinagkistrodon acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissue. So this study provides a first global view of the genetic programs for the venom gland of Deinagkistrodon acutus described so far and an insight into molecular mechanism of toxin secreting.

Animals↗
PubMed2005

Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method.

BACKGROUND: Neural stem cells (NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. RESULTS: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. CONCLUSIONS: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

Animals↗
PubMed2002

Transcriptome analysis of the retina.

The retina offers unique opportunities to define the molecular and cellular pathways mediating neuronal function and disease because of its morphological complexity, well-defined role in visual transduction and the availability of mutants. These investigations are being greatly facilitated by the ongoing identification of genes expressed in the retina using high-throughput methods.

Animals↗
PubMed2005

Transcriptome analysis of antigenic variation in Plasmodium falciparum--var silencing is not dependent on antisense RNA.

BACKGROUND: Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. To investigate transcriptional features of this mechanism, we conducted a microarray analysis of parasites that were selected to express different var genes by adhesion to chondroitin sulfate A (CSA) or CD36. RESULTS: In addition to oligonucleotides for all predicted protein-coding genes, oligonucleotide probes specific to each known var gene of the FCR3 background were designed and added to the microarray, as well as tiled sense and antisense probes for a subset of var genes. In parasites selected for adhesion to CSA, one full-length var gene (var2csa) was strongly upregulated, as were sense RNA molecules emanating from the 3' end of a limited subset of other var genes. No global relationship between sense and antisense production of var genes was observed, but notably, some var genes had coincident high levels of both antisense and sense transcript. CONCLUSION: Mutually exclusive expression of PfEMP1 proteins results from transcriptional silencing of non-expressed var genes. The distribution of steady-state sense and antisense RNA at var loci are not consistent with a silencing mechanism based on antisense silencing of inactive var genes. Silencing of var loci is also associated with altered regulation of genes distal to var loci.

Animals↗
PubMed2005

Transcriptome analysis and physical mapping of barley genes in wheat-barley chromosome addition lines.

Wheat-barley chromosome addition lines are useful genetic resources for a variety of studies. In this study, transcript accumulation patterns in Betzes barley, Chinese Spring wheat, and Chinese Spring-Betzes chromosome addition lines were examined with the Barley1 Affymetrix GeneChip probe array. Of the 4014 transcripts detected in Betzes but not in Chinese Spring, 365, 271, 265, 323, 194, and 369 were detected in wheat-barley disomic chromosome addition lines 2(2H), 3(3H), 4(4H), 7(5H), 6(6H), and 1(7H), respectively. Thus, 1787 barley transcripts were detected in a wheat genetic background and, by virtue of the addition line in which they were detected, were physically mapped to barley chromosomes. We validated and extended our approach to physically map barley genes to the long and short arms of chromosome 6(6H). Our physical map data exhibited a high level of synteny with homologous sequences on the wheat and/or rice syntenous chromosomes, indicating that our barley physical maps are robust. Our results show that barley transcript detection in wheat-barley chromosome addition lines is an efficient approach for large-scale physical mapping of genes.

Chromosomes, Plant↗
PubMed2006

Evaluation of methods for duration of preservation of RNA quality in rat liver used for transcriptome analysis.

In The Toxicogenomics Project, about 150 chemicals are administered to rats, and gene expression in the liver analyzed by Affymetrix GeneChip and stored in the database. As the quality of RNA greatly influences the accuracy of gene expression data, conditions of the storage of the sample are very important. Recently, an RNA stabilization solution, RNAlater, has become commercially available. In this study, the new storage method was compared with the traditional storage method (stored in freezer or liquid nitrogen) under various conditions by looking at the degradation of RNA assessed by its total yield, OD260/280 ratio, 28S/18S ratio, and quantity of beta-actin. It was confirmed that RNAlater preserved the liver tissue sample by maintaining the quality of RNA for one year (in liquid N(2) or -80 degrees C), for 3 days (4 degrees C), or for 2 hr (room temperature) without degradation of RNA. Quality of RNA samples dissolved in buffer RLT and stored at -20 degrees C tended to decrease, but samples stored at -80 degrees C were almost equivalent to those stored in liquid nitrogen. In conclusion, we recommend the following procedure for preservation of liver tissue for extraction of RNA: 1) tissues removed should be put into chilled RNAlater as soon as possible; 2) samples in RNAlater must be stored overnight or longer at 4 degrees C and can be left for as long as 2 weeks without freezing; 3) samples in RNAlater can be stored for at least one year under less than -20 degrees C and 4) samples dissolved in buffer RLT can be preserved at least for one year under -80 degrees C.

Actins↗
PubMed2001

Transcriptome analysis and crop improvement (a review).

The identification and characterization of differential gene expression from tissues subjected to stress has gained much attention in plant research. The recognition of elements involved in the response to a particular stress enhances the possibility of promoting crop improvement through direct genetic modification. However, the performance of some of the 'first generation' of transgenic plants with the incorporation of a single gene has not always been as expected. These results have stimulated the development of new transgenic constructions introducing more than one gene and capable of modifying complex pathways. Several techniques are available to conduct the analysis of gene regulation, with such information providing the basis for novel constructs specifically designed to modify metabolism. This review deals with techniques that allow the identification and characterization of differentially-expressed genes and the use of molecular pathway information to produce transgenic plants.

DNA Fingerprinting↗
PubMed2002

Extensive oligonucleotide microarray transcriptome analysis of the rat cerebral artery and arachnoid tissue.

Cerebral vessels have certain distinct anatomical and developmental characteristics which are well known, but their characteristic genetic expression profile remains as yet only poorly understood. We investigated gene expression in the rat cerebral artery in comparison with the rat descending aorta, two locations which have obviously different anatomical and developmental characteristics. Since the contamination of cerebral small arteries by arachnoid tissue is to a certain extent inevitable, we also performed a gene expression analysis of arachnoid tissue as a background. In an effort to obtain the necessary quality and quantity of total RNA, a novel freeze-fracture apparatus minimizing the time required for the entire procedure from tissue separation to RNA preparation was used. With the material obtained, a group of genes highly expressed in each tissue was detected by oligonucleotide microarray analysis. In the circle of Willis, peptide-19 (PEP-19), connexin-37 (CXN-37), growth arrest-and DNA damage-inducible gene (GADD45), and the putative G protein coupled receptor RA1c, Notch-1, and jagged-1 were predominantly expressed. In arachnoid tissue, bone morphologic protein (BMP)-7, BMP-6, beta defensin-1, neuroendocrine protein 7B2, thiol-specific antioxidant protein, IL-18, beta-chain clathrin-associated protein complex AP-1, and angiopoietin-2 were highly expressed. In the aorta, most of the abundantly expressed genes related to lipid metabolism. By means of oligonucleotide microarray analysis, the distinct gene expression profiles in the circle of Willis arachnoid tissue, and aorta were made evident. From these findings it is reasonable to conclude that a functional interaction exists between the circle of Willis and arachnoid tissue.

Animals↗
PubMed2001

[Transcriptome analysis in cancerology: bioinformatics aspects].

Recent technological advances (e.g. various DNA arrays and chips) allow the measurement of expression level (mRNA abundance) for thousand of genes simultaneously, over multiple conditions or time. Initially developed and tested on model systems such as yeast or in vitro cell line cultures, these techniques have recently begun to be applied to the analysis of human cancers. Initial results are promising, and large-scale gene expression profiling is now expected to become a clinical tool for better tumour identification, prognosis, and optimal treatment design. It is thus important that clinicians become familiar with the theoretical principles underlying the interpretation of gene expression profiles as used in three different contexts: gene discovery, tumour class prediction, and molecular diagnosis. This is the purpose of the present article.

Forecasting↗
PubMed2003

Transcriptome analysis of HER2 reveals a molecular connection to fatty acid synthesis.

HER2 (erbB2/neu) is a member of the erbB family of receptor tyrosine kinases and is involved in regulating the growth of several types of human carcinomas. HER2 represents a successful therapeutic target of the biotechnology era as exemplified by the drug Herceptin (trastuzumab), which has clinical activity in a subset of breast cancer patients. Using DNA microarrays, we identified a cohort of genes that are differentially regulated by HER2 in breast epithelial cells. One of the HER2-regulated genes discovered was fatty acid synthase (FAS), which has been shown to be overexpressed in breast cancer as well as other cancers. FAS is implicated in tumorigenesis through its role in cell proliferation and membrane lipid incorporation of neoplastic cells. Here, we demonstrate that HER2-mediated induction of FAS is inhibitable by Herceptin and tyrosine kinase inhibitors of HER2. Through a phosphatidylinositol 3'-kinase-dependent pathway, HER2 stimulates the FAS promoter and ultimately mediates increased fatty acid synthesis. Interestingly, pharmacological inhibition of FAS preferentially induced apoptosis of HER2-overexpressing breast epithelial cells relative to matched vector control cells. These studies characterize a molecular connection between two genes individually implicated in tumorigenesis but never linked together.

Antibodies, Monoclonal↗