Serial Analysis Of Gene Expression Research Articles

The effects of mild‐exercise training cessation on physiological adaptations and gene expressions in human skeletal muscle

Stoppage of endurance exercise training leads to complete loss of V̇O2 max gain but not submaximal and maximal exercise blood lactate concentrations. However, the detailed mechanisms are still unknown because the transcriptome of skeletal muscle after detraining has never been characterized. Thus, we investigated the effects of exercise-training cessation at lactate threshold (LT) intensity on physiological adaptations and skeletal muscle transcriptome. Twelve weeks of detraining abolished the effect of 12-weeks LT training on V̇O2 max. The detraining also reversed V̇O2 at LT intensity, however, the value tended to be higher than the pre-training level (p = 0.07). Moreover, the training cessation did not affect the number of capillaries around type I fiber which was increased by the LT training. The global gene expression profile measured by serial analysis of gene expression (SAGE) revealed that the training and detraining modulated 249 and 245 characterized transcripts, respectively. The majority of training-responsive transcripts (86%) showed a significant reversible effect of detraining. However, the most-induced transcripts by the training were still elevated after the same period of detraining. These genes are involved in oxygen transport, creatine and mitochondrial energy metabolisms, contractile apparatus, and protein synthesis. Incomplete reversal of these transcripts as well as sustained capillary distribution by the detraining may contribute to the partial reversal of V̇O2 at LT intensity.

Transcriptional profiling of inductive mesenchyme to identify molecules involved in prostate development and disease

Comparison of SAGE libraries for prostatic inductive mesenchyme and the complete prostatic rudiment revealed 219 transcripts that were enriched in, or specific to, inductive mesenchyme. Further analysis suggested that Scube1 is a novel stromal molecule involved in prostate development and tumorigenesis.

Large-scale analysis by SAGE reveals new mechanisms of v-erbA oncogene action

Background:The v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAα proto-oncogene that encodes the nuclear receptor for triiodothyronine (T3R). v-ErbA transforms erythroid progenitors in vitro by blocking their differentiation, supposedly by interference with T3R and RAR (Retinoic Acid Receptor). However, v-ErbA target genes involved in its transforming activity still remain to be identified.Results:By using Serial Analysis of Gene Expression (SAGE), we identified 110 genes deregulated by v-ErbA and potentially implicated in the transformation process. Bioinformatic analysis of promoter sequence and transcriptional assays point out a potential role of c-Myb in the v-ErbA effect. Furthermore, grouping of newly identified target genes by function revealed both expected (chromatin/transcription) and unexpected (protein metabolism) functions potentially deregulated by v-ErbA. We then focused our study on 15 of the new v-ErbA target genes and demonstrated by real time PCR that in majority their expression was activated neither by T3, nor RA, nor during differentiation. This was unexpected based upon the previously known role of v-ErbA.Conclusion:This paper suggests the involvement of a wealth of new unanticipated mechanisms of v-ErbA action.

Inflammation and Genes

Inflammation is a protective immune response to infection, trauma, or injury; however, only a subset of patients develops inflammation, suggesting other contributing factors involved, such as the environment and genes. Inflammation-associated genes involving those with pro- and anti-inflammatory effect should be properly balanced and regulated; the protein products of these genes ultimately determine the outcome of inflammation. Apart from gene mutations, gene polymorphisms related to some inflammatory markers also appear to correlate with the incidence and/or outcome of serious inflammatory events. Some genes recently recognized to be associated with inflammation are briefly reviewed. Modern genomic approaches, such as DNA micro-arrays and serial analysis of gene expression, allow for determining the extremely complex profile of inflammatory genes.

Advances in Swine Transcriptomics

The past five years have seen a tremendous rise in porcine transcriptomic data. Available porcine Expressed Sequence Tags (ESTs) have expanded greatly, with over 623,000 ESTs deposited in Genbank. ESTs have been used to expand the pig-human comparative maps, but such data has also been used in many ways to understand pig gene expression. Several methods have been used to identify genes differentially expressed (DE) in specific tissues or cell types under different treatments. These include open screening methods such as suppression subtractive hybridization, differential display, serial analysis of gene expression, and EST sequence frequency, as well as closed methods that measure expression of a defined set of sequences such as hybridization to membrane arrays and microarrays. The use of microarrays to begin large-scale transcriptome analysis has been recently reported, using either specialized or broad-coverage arrays. This review covers published results using the above techniques in the pig, as well as unpublished data provided by the research community, and reports on unpublished Affymetrix data from our group. Published and unpublished bioinformatics efforts are discussed, including recent work by our group to integrate two broad-coverage microarray platforms. We conclude by predicting experiments that will become possible with new anticipated tools and data, including the porcine genome sequence. We emphasize that the need for bioinformatics infrastructure to efficiently store and analyze the expanding amounts of gene expression data is critical, and that this deficit has emerged as a limiting factor for acceleration of genomic understanding in the pig.

ZOOMING IN: a new high‐resolution gene expression atlas of the brain

Mol Syst Biol. 3: 75 If you walk through a laboratory at the Salk Institute, all the people working in it look fairly similar: two legs, a head and so on. However, it is only through a more detailed analysis that one could reveal the differences between the first year graduate student (nervously pipetting) and the well‐respected principal investigator (PI) (editing manuscripts). Analogous to this, how can we discriminate diverse neuronal populations (e.g. graduate student versus PI) and even understand their function in the adult brain? Genome‐wide, unbiased gene expression studies using DNA microarrays or serial analyses of gene expression have created a tremendous amount of data defining tissue‐specific and even region‐specific gene expression in mammals (Lockhart et al , 1996; Datson et al , 2001; Lewandowski and Small, 2005). In other words, the ‘laboratory’ (in analogy to organs, e.g., the brain) and maybe even the ‘job description’ (e.g., the substructure of the brain) of many genes have been already identified. In a study recently published in Nature , Lein et al (2007) went one step further and have provided a genome‐wide gene expression atlas of the adult mouse brain (the Allen Brain …

LongSAGE analysis of skeletal muscle at three prenatal stages in Tongcheng and Landrace pigs

Transcriptional profiling of Tongcheng and Landrace pigs using long serial analysis of gene expression provides insight into the molecular mechanism underlying differences in muscle growth.

Functional genomics in chickpea: an emerging frontier for molecular-assisted breeding.

Chickpea is a valuable and important agricultural crop, but yield potential is limited by a series of biotic and abiotic stresses, including Ascochyta blight, Fusarium wilt, drought, cold and salinity. To accelerate molecular breeding efforts for the discovery and introgression of stress tolerance genes into cultivated chickpea, functional genomics approaches are rapidly growing. Recently a series of genetic tools for chickpea have become available that have allowed high-powered functional genomics studies to proceed, including a dense genetic map, large insert genome libraries, expressed sequence tag libraries, microarrays, serial analysis of gene expression, transgenics and reverse genetics. This review summarises the development of these genomic tools and the achievements made in initial and emerging functional genomics studies. Much of the initial research focused on Ascochyta blight resistance, and a resistance model has been synthesised based on the results of various studies. Use of the rich comparative genomics resources from the model legumes Medicago truncatula and Lotus japonicus is also discussed. Finally, perspectives on the future directions for chickpea functional genomics, with the goal of developing elite chickpea cultivars, are discussed.

The biologic basis of in vivo angiogenesis imaging

Knowledge of the different physiology and endothelial markers present in tumor vessels is essential to enable both the development of new anti-angiogenic chemotherapeutic agents and of more specific imaging techniques. Tumor blood vessels are disorganized, irregular in caliber, tortuous, and do not have specialized features of normal arterioles, capillaries or venules. Neo-angiogenic tumor vessels have large gaps between or through cells, loose pericytes, and discontinuities or redundant layers within the basement membrane, rendering these vessels hyper-permeable. Furthermore, the endothelia of tumor vessels may express unique markers on their surface. Imaging is becoming increasingly important in the evaluation of angiogenesis. Clinical imaging is minimally invasive and enables sampling of the whole tumor in a nondestructive manner. The patterns of increased permeability seen on Dynamic contrast-enhanced Magnetic Resonance Imaging (DCE-MRI) mirror the known ultrastructural defects associated with angiogenic vessels. Conventional low-molecular weight contrast agents are currently in clinical use for DCE-MRI studies and have proven successful in detecting changes related to novel angiogenic inhibitors. However, they are relatively non-specific. Macromolecular contrast media may be more suitable for imaging tumor vessels. It is hoped that imaging modalities can be adapted to specifically target markers expressed on the endothelium of tumor vessels. The number of cell surface markers of angiogenesis is relatively low, and only small amounts of contrast agents can bind to these receptors; currently only Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) tracers have sufficient sensitivity to allow detection at this low level. Despite limitations in their spatial resolution, PET and SPECT imaging are more likely to enter the clinic as targeted angiogenesis imaging methods. The quest for selective targets on the tumor vasculature continues, currently the integrin family of receptors offer the most promise but other targets are being pursued by investigators. Serial analysis of gene expression or in vivo phage display may help identify new, more selective, markers that can be utilized for the targeted imaging and treatment of angiogenesis.

Serial Analysis of Gene Expression in Plasmodium berghei salivary gland sporozoites

BackgroundThe invasion of Anopheles salivary glands by Plasmodium sporozoites is an essential step for transmission of the parasite to the vertebrate host. Salivary gland sporozoites undergo a developmental programme to express genes required for their journey from the site of the mosquito bite to the liver and subsequent invasion of, and development within, hepatocytes. A Serial Analysis of Gene Expression was performed on Anopheles gambiae salivary glands infected or not with Plasmodium berghei and we report here the analysis of the Plasmodium sporozoite transcriptome.ResultsAnnotation of 530 tag sequences homologous to Plasmodium berghei genomic sequences identified 123 genes expressed in salivary gland sporozoites and these genes were classified according to their transcript abundance. A subset of these genes was further studied by quantitative PCR to determine their expression profiles. This revealed that sporozoites modulate their RNA amounts not only between the midgut and salivary glands, but also during their storage within the latter. Among the 123 genes, the expression of 66 is described for the first time in sporozoites of rodent Plasmodium species.ConclusionThese novel sporozoite expressed genes, especially those expressed at high levels in salivary gland sporozoites, are likely to play a role in Plasmodium infectivity in the mammalian host.

Comprehensive serial analysis of gene expression of the cervical transcriptome

BackgroundMore than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE).ResultsWe have sequenced 691,390 tags from four L-SAGE libraries increasing the existing gene expression data on cervical tissue by 20 fold. One-hundred and eighteen unique tags were highly expressed in normal cervical tissue and 107 of them mapped to unique genes, most belong to the ribosomal, calcium-binding and keratinizing gene families. We assessed these genes for aberrant expression in CIN III and five genes showed altered expression. In addition, we have identified twelve unique HPV 16 SAGE tags in the CIN III libraries absent in the normal libraries.ConclusionEstablishing a baseline of gene expression in normal cervical tissue is key for identifying changes in cancer. We demonstrate the utility of this baseline data by identifying genes with aberrant expression in CIN III when compared to normal tissue.

Genes involved in oxidative phosphorylation are coordinately upregulated with fasting hyperglycaemia in livers of patients with type 2 diabetes

Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in the pathophysiology of type 2 diabetes. Genes involved in OXPHOS have been reported to be down-regulated in skeletal muscle from patients with type 2 diabetes; however, hepatic regulation is unknown. We analysed expression of genes involved in OXPHOS from the livers of 14 patients with type 2 diabetes and 14 subjects with NGT using serial analysis of gene expression (SAGE) and DNA chip analysis. We evaluated the correlation between expression levels of genes involved in OXPHOS and the clinical parameters of individuals with type 2 diabetes and NGT. Both gene analyses showed that genes involved in OXPHOS were significantly upregulated in the type 2 diabetic liver. In the SAGE analysis, tag count comparisons of mitochondrial transcripts showed that ribosomal RNAs (rRNA) were 3.5-fold over-expressed, and mRNAs were 1.2-fold over-expressed in the type 2 diabetes library. DNA chip analysis revealed that expression of genes involved in OXPHOS, which correlated with several nuclear factors, including estrogen-related receptor-alpha or peroxisome proliferator-activated receptor-gamma, was a predictor of fasting plasma glucose levels, independently of age, BMI, insulin resistance and fasting insulin levels (p = 0.04). Surprisingly, genes involved in OXPHOS did not correlate with peroxisome proliferator-activated receptor-gamma coactivator-1alpha or nuclear respiratory factor 1. Our results indicate that upregulation of genes involved in OXPHOS in the liver, which are regulated by different mechanisms from genes in the skeletal muscle, is associated with fasting hyperglycaemia in patients with type 2 diabetes.

A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein

Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, “HCNCp” (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many “CxxC” tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple “CxC” motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing ≥20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine candidates, and subjects for further research into Giardia biology.

Identification of corticosteroid-regulated genes in cardiomyocytes by serial analysis of gene expression

Corticosteroids (aldosterone, cortisol/corticosterone) exert direct functional effects on cardiomyocytes. However, gene networks activated by corticosteroids in cardiomyocytes, as well as the involvement of the mineralocorticoid receptor (MR) vs the glucocorticoid receptor (GR) in these effects, remain largely unknown. Here we characterized the corticosteroid-dependent transcriptome in primary culture of neonatal mouse cardiomyocytes treated with 10 − 6 M aldosterone, a concentration predicted to occupy both MR and GR. Serial analysis of gene expression revealed 101 aldosterone-regulated genes. The MR/GR specificity was characterized for one regulated transcript, namely ecto-ADP-ribosyltransferase-3 (Art3). Using cardiomyocytes from GR(null/null) or MR(null/null) mice we demonstrate that in GR(null/null) cardiomyocytes the response is abrogated, but it is fully maintained in MR(null/null) cardiomyocytes. We conclude that Art3 expression is regulated exclusively via the GR. Our study identifies a new set of corticosteroid-regulated genes in cardiomyocytes and demonstrates a new approach to studying the selectivity of MR- vs GR-dependent effects.

Cell Type-Specific Gene Expression Profiling in Plants by Using a Combination of Laser Microdissection and High-Throughput Technologies

Laser microdissection (LM) allows for the isolation of specific cells of interest from heterogeneous tissues under direct microscopic visualization with the assistance of a laser beam. By permitting global analyses of gene expression and metabolites in the selected cells, it is a powerful tool for understanding the biological processes in individual cell types during development or in response to various stimuli. Recently, LM technology has been successfully applied to the separation of individual plant cell types. Here, we provide an overview of applications of LM combined with high-throughput technologies including transcript analyses [microarrays, serial analysis of gene expression (SAGE) and 454-sequencing], proteomic analyses and metabolomic profiling, for cell type-specific gene expression analyses in plants.

Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

Gene expression profiles of erythroid precursors characterise several mechanisms of the action of hydroxycarbamide in sickle cell anaemia

Hydroxycarbamide (HC) (or hydroxyurea) has been reported to increase fetal haemoglobin levels and improve clinical symptoms in sickle cell anaemia (SCA) patients. However, the complete pathway by which HC acts remains unclear. To study the mechanisms involved in the action of HC, global gene expression profiles were obtained from the bone marrow cells of a SCA patient before and after HC treatment using serial analysis of gene expression. In the comparison of both profiles, 147 differentially expressed transcripts were identified. The functional classification of these transcripts revealed a group of gene categories associated with transcriptional and translational regulation, e.g. EGR-1, CENTB1, ARHGAP4 and RIN3, suggesting a possible role for these pathways in the improvement of clinical symptoms of SCA patients. The genes involved in these mechanisms may represent potential tools for the identification of new targets for SCA therapy.

A BTB/POZ protein, NAC-1, is related to tumor recurrence and is essential for tumor growth and survival

Recent studies have suggested an oncogenic role of the BTB/POZ-domain genes in hematopoietic malignancy. The aim of this study is to identify and characterize BTB/POZ-domain genes in the development of human epithelial cancers, i.e., carcinomas. In this study, we focused on ovarian carcinoma and analyzed gene expression levels using the serial analysis of gene expression (SAGE) data in all 130 deduced BTB/POZ genes. Our analysis reveals that NAC-1 is significantly overexpressed in ovarian serous carcinomas and several other types of carcinomas. Immunohistochemistry studies in ovarian serous carcinomas demonstrate that NAC-1 is localized in discrete nuclear bodies (tentatively named NAC-1 bodies), and the levels of NAC-1 expression correlate with tumor recurrence. Furthermore, intense NAC-1 immunoreactivity in primary tumors predicts early recurrence in ovarian cancer. Both coimmunoprecipitation and double immunofluorescence staining demonstrate that NAC-1 molecules homooligomerize through the BTB/POZ domain. Induced expression of the NAC-1 mutant containing only the BTB/POZ domain disrupts NAC-1 bodies, prevents tumor formation, and promotes tumor cell apoptosis in established tumors in a mouse xenograft model. Overexpression of full-length NAC-1 enhanced tumorigenicity of ovarian surface epithelial cells and NIH 3T3 cells in athymic nu/nu mice. In summary, NAC-1 is a tumor recurrence-associated gene with oncogenic potential, and the interaction between BTB/POZ domains of NAC-1 proteins is critical to form the discrete NAC-1 nuclear bodies and essential for tumor cell proliferation and survival.

Pim‐1 induced polyploidy but did not affect megakaryocytic differentiation of K562 cells and CD34+ cells from cord blood

In a previous study, we determined the gene expression profile of both megakaryocytic and non-megakaryocytic lineage cells via serial analysis of gene expression and microarray methods, and demonstrated that Pim-1 was expressed more abundantly in megakaryocytic lineage cells. In this study, we knocked down Pim-1 in K562 cells, as well as in CD34+ cells from cord blood, via RNA interference, in order to analyze the effects of Pim-1 expression on the megakaryocytic differentiation of these cells. We then additionally overexpressed the Pim-1 genes in K562 cells, and conducted a comparison of these effects with those of RNAi cells on the course of megakaryocytic differentiation. The results of this study revealed that Pim-1 knockdown exerted no effects on commitment or differentiation toward megakaryocytic lineage, as evidenced by the detected CD41+ or CD61+ cells, or on the number of megakaryocytic colony forming units. However, Pim-1 knockdown was found to elicit a reduction in CD41+ cells with >4n DNA content, and a concomitant increase in the fraction of cells achieving a ploidy of >4n in the Pim-1 overexpressing population of K562 cells. Collectively, the findings of these studies indicate that the expression of Pim-1 expression is both necessary and sufficient for polyploidization, but is not critical to cytoplasmic differentiation on megakaryopoiesis.

The expression of phosphatidic acid phosphatase 2a, which hydrolyzes lipids to generate diacylglycerol, is regulated by p73, a member of the p53 family

p73, a p53-related gene, is essential for a development of animals, while p53 is important for tumor formation. And little is known about the target genes specifically regulated by p73. Identifying the specific targets of p73 is important to understand the physiological roles of p73. To identify the genes specifically regulated by p73, we conducted serial analysis of gene expression to quantitatively evaluate messenger RNA populations. We found that the gene for phosphatidic acid phosphatase 2a (PAP2a), an enzyme that hydrolyzes lipids to generate diacylglycerol, was specifically upregulated by ectopic production of p73beta. The promoter region of this gene contains an element that is functionally responsive to p73beta. And the quantity of PAP2a protein was upregulated by ectopic production of p73beta. These results suggest that the expression of PAP2a is directly regulated by p73.