Analysis Of Gene Expression Libraries Research Articles

De Novo Assembled Wheat Transcriptomes Delineate Differentially Expressed Host Genes in Response to Leaf Rust Infection.

Pathogens like Puccinia triticina, the causal organism for leaf rust, extensively damages wheat production. The interaction at molecular level between wheat and the pathogen is complex and less explored. The pathogen induced response was characterized using mock- or pathogen inoculated near-isogenic wheat lines (with or without seedling leaf rust resistance gene Lr28). Four Serial Analysis of Gene Expression libraries were prepared from mock- and pathogen inoculated plants and were subjected to Sequencing by Oligonucleotide Ligation and Detection, which generated a total of 165,767,777 reads, each 35 bases long. The reads were processed and multiple k-mers were attempted for de novo transcript assembly; 22 k-mers showed the best results. Altogether 21,345 contigs were generated and functionally characterized by gene ontology annotation, mining for transcription factors and resistance genes. Expression analysis among the four libraries showed extensive alterations in the transcriptome in response to pathogen infection, reflecting reorganizations in major biological processes and metabolic pathways. Role of auxin in determining pathogenesis in susceptible and resistant lines were imperative. The qPCR expression study of four LRR-RLK (Leucine-rich repeat receptor-like protein kinases) genes showed higher expression at 24 hrs after inoculation with pathogen. In summary, the conceptual model of induced resistance in wheat contributes insights on defense responses and imparts knowledge of Puccinia triticina-induced defense transcripts in wheat plants.

A novel acrosomal protein, IQCF1, involved in sperm capacitation and the acrosome reaction.

On the basis of the unknown tags in the mature human sperm serial analysis of gene expression library constructed by our laboratory, some transcripts were cloned, including Iqcf1 (IQ motif containing F1). To investigate the function of sperm-retained Iqcf1 in spermatogenesis and fertilization of mice, we investigated the spatial and temporal expression of IQCF1. By using the (transcription activator-like effector nuclease) strategy, Iqcf1-knockout mice were produced, and the phenotypes of the Iqcf1(-/-) mice were analyzed. The results showed that IQCF1 was localized in the acrosome of spermatozoa and spermatids; the expression of IQCF1 in testes was associated with spermatogenic capacity. The Iqcf1(-/-) mice were significantly less fertile than the wild-type mice (p = 0.0057) because of reduced sperm motility (p = 0.0094) and the acrosome reaction (AR) (p = 0.0093). In spermatozoa, IQCF1 interacted with calmodulin (CaM) and possibly participated in the tyrosine phosphorylation of sperm proteins during capacitation. In conclusion, a newly identified acrosomal protein, IQCF1, is closely related to sperm capacitation and AR; in particular, it is involved in tyrosine phosphorylation of sperm proteins through interaction with CaM. Research into the function of IQCF1 during fertilization could facilitate the investigation of the molecular mechanism of capacitation, which is unclear.

Changes in hippocampal gene expression by 7‐nitroindazole in rats submitted to forced swimming stress

Nitric oxide (NO) is an atypical neurotransmitter that has been related to the pathophysiology of major depression disorder. Increased plasma NO levels have been reported in depressed and suicidal patients. Inhibition of neuronial nitric oxide synthase (nNOS), on the other hand, induces antidepressant effects in clinical and pre-clinical trials. The mechanisms responsible for the antidepressant-like effects of nNOS inhibitors, however, are not completely understood. In this study, genomic and proteomic analyses were used to investigate the effects of the preferential nNOS inhibitor 7-nitroindazole (7-NI) on changes in global gene and protein expression in the hippocampus of rats submitted to forced swimming test (FST). Chronic treatment (14 days, i.p.) with imipramine (15 mg/kg daily) or 7-NI (60 mg/kg daily) significantly reduced immobility in the FST. Saturation curves for Serial analysis of gene expression libraries showed that the hippocampus of animals submitted to FST presented a lower number of expressed genes compared to non-FST stressed groups. Imipramine, but not 7-NI, reverted this effect. GeneGo analyses revealed that genes related to oxidative phosphorylation, apoptosis and survival controlled by HTR1A signaling and cytoskeleton remodeling controlled by Rho GTPases were significantly changed by FST. 7-NI prevented this effect. In addition, 7-NI treatment changed the expression of genes related to transcription in the cAMP response element-binding pathway. Therefore, this study suggests that changes in oxidative stress and neuroplastic processes could be involved in the antidepressant-like effects induced by nNOS inhibition.

Identification of a secretory protein c19orf10 activated in hepatocellular carcinoma

The identification of genes involved in tumor growth is crucial for the development of inventive anticancer treatments. Here, we have cloned a 17-kDa secretory protein encoded by c19orf10 from hepatocellular carcinoma (HCC) serial analysis of gene expression libraries. Gene expression analysis indicated that c19orf10 was overexpressed in approximately two-thirds of HCC tissues compared to the adjacent noncancerous liver tissues, and its expression was significantly positively correlated with that of alpha-fetoprotein (AFP). Overexpression of c19orf10 enhanced cell proliferation of AFP-negative HLE cells, whereas knockdown of c19orf10 inhibited cell proliferation of AFP-positive Hep3B and HuH7 cells along with G1 cell cycle arrest. Supplementation of recombinant c19orf10 protein in culture media enhanced cell proliferation in HLE cells, and this effect was abolished by the addition of antibodies developed against c19orf10. Intriguingly, c19orf10 could regulate cell proliferation through the activation of Akt/mitogen-activated protein kinase pathways. Taken together, these data suggest that c19orf10 might be one of the growth factors and potential molecular targets activated in HCC.

Genomic analysis distinguishes phases of early development of the mouse atrio-ventricular canal

Valve formation during embryonic heart development involves a complex interplay of regional specification, cell transformations, and remodeling events. While many studies have addressed the role of specific genes during this process, a global understanding of the genetic basis for the regional specification and development of the heart valves is incomplete. We have undertaken genome-wide transcriptional profiling of the developing heart valves in the mouse. Four Serial Analysis of Gene Expression libraries were generated and analyzed from the mouse atrio-ventricular canal (AVC) at embryonic days 9.5-12.5, covering the stages from initiation of endothelial to mesenchymal transition (EMT) through to the beginning of endocardial cushion remodeling. We identified 14 distinct temporal patterns of gene expression during AVC development. These were associated with specific functions and signaling pathway members. We defined the temporal distribution of mesenchyme genes during the EMT process and of specific Notch and transforming growth factor-beta targets. This work provides the first comprehensive temporal dataset during the formation of heart valves. These results identify molecular signatures that distinguish different phases of early heart valve formation allowing gene expression and function to be further investigated.

A Gene Expression Classifier of Node-Positive Colorectal Cancer

We used digital long serial analysis of gene expression to discover gene expression differences between node-negative and node-positive colorectal tumors and developed a multigene classifier able to discriminate between these two tumor types. We prepared and sequenced long serial analysis of gene expression libraries from one node-negative and one node-positive colorectal tumor, sequenced to a depth of 26,060 unique tags, and identified 262 tags significantly differentially expressed between these two tumors (P < 2 x 10-6). We confirmed the tag-to-gene assignments and differential expression of 31 genes by quantitative real-time polymerase chain reaction, 12 of which were elevated in the node-positive tumor. We analyzed the expression levels of these 12 upregulated genes in a validation panel of 23 additional tumors and developed an optimized seven-gene logistic regression classifier. The classifier discriminated between node-negative and node-positive tumors with 86% sensitivity and 80% specificity. Receiver operating characteristic analysis of the classifier revealed an area under the curve of 0.86. Experimental manipulation of the function of one classification gene, Fibronectin, caused profound effects on invasion and migration of colorectal cancer cells in vitro. These results suggest that the development of node-positive colorectal cancer occurs in part through elevated epithelial FN1 expression and suggest novel strategies for the diagnosis and treatment of advanced disease.

Gene Expression Profiling of Papillary Thyroid Carcinoma Identifies Transcripts Correlated with BRAF Mutational Status and Lymph Node Metastasis

To identify papillary thyroid carcinoma (PTC)-associated transcripts, we compared the gene expression profiles of three Serial Analysis of Gene Expression libraries generated from thyroid tumors and a normal thyroid tissue. Selected transcripts were validated in a panel of 57 thyroid tumors using quantitative PCR (qPCR). An independent set of 71 paraffin-embedded sections was used for validation using immunohistochemical analysis. To determine if PTC-associated gene expression could predict lymph node involvement, a separate cohort of 130 primary PTC (54 metastatic and 76 nonmetastatic) was investigated. The BRAF(V600E) mutational status was compared with qPCR data to identify genes that might be regulated by abnormal BRAF/MEK/extracellular signal-regulated kinase signaling. We identified and validated new PTC-associated transcripts. Three genes (CST6, CXCL14, and DHRS3) are strongly associated with PTC. Immunohistochemical analysis of CXCL14 confirmed the qPCR data and showed protein expression in PTC epithelial cells. We also observed that CST6, CXCL14, DHRS3, and SPP1 were associated with PTC lymph node metastasis, with CST6, CXCL14, and SPP1 being positively correlated with metastasis and DHRS3 being negatively correlated. Finally, we found a strong correlation between CST6 and CXCL14 expression and BRAF(V600E) mutational status, suggesting that these genes may be induced subsequently to BRAF activation and therefore may be downstream in the BRAF/MEK/extracellular signal-regulated kinase signaling pathway. CST6, CXCL14, DHRS3, and SPP1 may play a role in PTC pathogenesis and progression and are possible molecular targets for PTC therapy.

Identification of candidate cancer genes involved in human retinoblastoma by data mining

The objective of this study was to discover potential cancer-related genes involved in retinoblastoma (RB) tumorigenesis. Using a data-mining tool called cDNA Digital Gene Expression Displayer (DGED) and serial analysis of gene expression DGED from the Cancer Genome Anatomy Project (CGAP) database, eight cDNA libraries and five serial analysis of gene expression libraries from retinoblastoma (RB) solid tumors and normal retina tissues were analyzed. The deregulated genes were classified into major families using information from Gene Ontology. Several candidate cancer-related genes were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) on tissue microarrays (TMA) of RB and human normal retina samples. A total of 260 genes with deregulated expression emerged when examined by DGED from the CGAP database. Functional classification of these genes not only provided an interesting insight into RB tumorigenesis but also facilitated target identification for RB therapeutics. Several candidate genes were confirmed by real-time RT-PCR and IHC analysis on TMA and were found to be associated with RB genesis through text-mining in Information Hyperlinked over Proteins. The results also implicated MCM7 and WIF1 as promising therapeutic targets for RB, but further validation is needed.

In Silico analysis of Gastric carcinoma Serial Analysis of Gene Expression libraries reveals different profiles associated with ethnicity

Worldwide gastric carcinoma has marked geographical variations and worse outcome in patients from the West compared to the East. Although these differences has been explained by better diagnostic criteria, improved staging methods and more radical surgery, emerging evidence supports the concept that gene expression differences associated to ethnicity might contribute to this disparate outcome. Here, we collected datasets from 4 normal and 11 gastric carcinoma Serial Gene Expression Analysis (SAGE) libraries from two different ethnicities. All normal SAGE libraries as well as 7 tumor libraries were from the West and 4 tumor libraries were from the East. These datasets we compare by Correspondence Analysis and Support Tree analysis and specific differences in tags expression were identified by Significance Analysis for Microarray. Tags to gene assignments were performed by CGAP-SAGE Genie or TAGmapper. The analysis of global transcriptome shows a clear separation between normal and tumor libraries with 90 tags differentially expressed. A clear separation was also found between the West and the East tumor libraries with 54 tags differentially expressed. Tags to gene assignments identified 15 genes, 5 of them with significant higher expression in the West libraries in comparison to the East libraries. qRT-PCR in cell lines from west and east origin confirmed these differences. Interestingly, two of these genes have been associated to aggressiveness (COL1A1 and KLK10). In conclusion we found that in silico analysis of SAGE libraries from two different ethnicities reveal differences in gene expression profile. These expression differences might contribute to explain the disparate outcome between the West and the East.

SAGE library screening reveals ILT7 as a specific plasmacytoid dendritic cell marker that regulates type I IFN production

Plasmacytoid dendritic cells (pDCs) link innate to acquired immune responses by producing high levels of type I IFN upon infection. In order to identify the specific genes that control pDC, we compared serial analysis of gene expression libraries from human pDCs, herpes simplex virus-stimulated pDCs and monocytes. We found that Ig-like transcript ILT7 is specifically expressed on pDC cell surfaces and is down-regulated when pDC mature in response to viral or bacterial stimulation. ILT7 expression on the cell surface required association with the Fc epsilon RI gamma adaptor molecule. Although treatment with one anti-ILT7-specific mAb suppressed type I IFN production in response to cytosine-phosphate-guanosice (CpG) stimulation, another anti-ILT7 mAb up-regulated type I IFN production. We conclude that ILT7 is a key regulator of human pDC function.

A Comprehensive Proteomics and Genomics Analysis Reveals Novel Transmembrane Proteins in Human Platelets and Mouse Megakaryocytes Including G6b-B, a Novel Immunoreceptor Tyrosine-based Inhibitory Motif Protein

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.

Gene profiling during development and after a peripheral nerve traumatism reveals genes specifically induced by injury in dorsal root ganglia

In order to shed light on transcriptional networks involved in adult peripheral nerve repair program, we propose for the first time an organization of the transcriptional dynamics of the mouse dorsal root ganglia (DRG) following a sciatic nerve lesion. This was done by a non-hierarchical bioinformatical clustering of four Serial Analysis of Gene Expression libraries performed on DRG at embryonic day E13, neonatal day P0, adult and adult 3 days post-sciatic nerve section. Grouping genes according to their expression profiles shows that a combination of down-regulation of genes expressed at the adult stages, re-expression of embryonic genes and induction of a set of de novo genes takes place in injured neurons. Focusing on this latter event highlights Ddit3, Timm8b and Oazin as potential new injury-induced molecular actors involved in a stress response pathway. Their association with the traumatic state was confirmed by real-time PCR and in situ hybridization investigations. Clustering analysis allows us to distinguish developmental re-programming events from nerve-injury-induced processes and thus provides a basis for molecular understanding of transcriptional alterations taking place in the DRG after a sciatic nerve lesion.

Serial Analysis of Gene Expression Study of a Hybrid Rice Strain (LYP9) and Its Parental Cultivars

Using the serial analysis of gene expression technique, we surveyed transcriptomes of three major tissues (panicles, leaves, and roots) of a super-hybrid rice (Oryza sativa) strain, LYP9, in comparison to its parental cultivars, 93-11 (indica) and PA64s (japonica). We acquired 465,679 tags from the serial analysis of gene expression libraries, which were consolidated into 68,483 unique tags. Focusing our initial functional analyses on a subset of the data that are supported by full-length cDNAs and the tags (genes) differentially expressed in the hybrid at a significant level (P<0.01), we identified 595 up-regulated (22 tags in panicles, 228 in leaves, and 345 in roots) and 25 down-regulated (seven tags in panicles, 15 in leaves, and three in roots) in LYP9. Most of the tag-identified and up-regulated genes were found related to enhancing carbon- and nitrogen-assimilation, including photosynthesis in leaves, nitrogen uptake in roots, and rapid growth in both roots and panicles. Among the down-regulated genes in LYP9, there is an essential enzyme in photorespiration, alanine:glyoxylate aminotransferase 1. Our study adds a new set of data crucial for the understanding of molecular mechanisms of heterosis and gene regulation networks of the cultivated rice.

Genome-wide transcriptome mapping analysis identifies organ-specific gene expression patterns along human chromosomes

The Human Genome Project has revealed that there about 32,000 protein-encoding genes, which are distributed throughout the genome. It is unclear, however, whether genes are distributed on the chromosomes according to patterns linked to organ specificity. To explore the relationship between genes actively transcribed in normal tissues and their chromosomal locations, we analyzed serial analysis of gene expression libraries of normal human liver, brain, breast, and colon tissues. Transcriptome mapping analysis revealed that transcriptional activity in each tissue varied according to the chromosomal domains, and a weak positive correlation was observed between transcription density and gene density. We identified six liver-related and five colon-related chromosomal domains highly transcribed in each tissue, whereas no brain-related or breast-related chromosomal domains were identified. Representative genes located on these chromosomal domains were associated with the function of each organ and were highly conserved in both mouse and rat genomes. These data revealed that the transcriptional activities of normal human tissues are well orchestrated at chromosomal levels, suggesting that highly expressed genes may share physical proximity.

Saci-1, -2, and -3 and Perere, four novel retrotransposons with high transcriptional activities from the human parasite Schistosoma mansoni.

Using the data set of 180,000 expressed sequence tags (ESTs) of the blood fluke Schistosoma mansoni generated recently by our group, we identified three novel long-terminal-repeat (LTR)- and one novel non-LTR-expressed retrotransposon, named Saci-1, -2, and -3 and Perere, respectively. Full-length sequences were reconstructed from ESTs and have deduced open reading frames (ORFs) with several uncorrupted features, characterizing them as possible active retrotransposons of different known transposon families. Alignment of reconstructed sequences to available preliminary genome sequence data confirmed the overall structure of the transposons. The frequency of sequenced transposon transcripts in cercariae was 14% of all transcripts from that stage, twofold higher than that in schistosomula and three- to fourfold higher than that in adults, eggs, miracidia, and germ balls. We show by Southern blot analysis, by EST annotation and tallying, and by counting transposon tags from a Serial Analysis of Gene Expression library, that the four novel retrotransposons exhibit a 10- to 30-fold lower copy number in the genome and a 4- to 200-fold-higher transcriptional rate per copy than the four previously described S. mansoni retrotransposons [corrected]. Such differences lead us to hypothesize that there are two different populations of retrotransposons in S. mansoni genome, occupying different niches in its ecology. Examples of retrotransposon fragment inserts were found into the 5' and 3' untranslated regions of four different S. mansoni target gene transcripts. The data presented here suggest a role for these elements in the dynamics of this complex human parasite genome.

A Serial Analysis of Gene Expression in Sun-Damaged Human Skin

To study the phenotypic changes in human skin associated with repeated sun exposure at the transcription level, we have undertaken a comparative serial analysis of gene expression of sun-damaged preauricular skin and sun-protected postauricular skin as well as sun-protected epidermis. Serial analyses of gene expression libraries, containing multiple mRNA-derived tag recombinants, were made to poly(A+)RNA isolated from human postauricular skin and preauricular skin, as well as epidermal nick biopsy samples. 5330 mRNA-derived cDNA tags from the postauricular serial analysis of gene expression library were sequenced and these tag sequences were compared to cDNA sequences identified from 5105 tags analyzed from a preauricular serial analysis of gene expression library. Of the total of 4742 different tags represented in both libraries we found 34 tags with at least a 4-fold difference of tag abundance between the libraries. Among the mRNAs with altered steady-state(1) levels in sun-damaged skin, we detected those encoding keratin 1, macrophage inhibitory factor, and calmodulin-like skin protein. In addition, a comparison of cDNA sequences identified in the serial analysis of gene expression libraries obtained from the epidermal biopsy samples (5257 cDNA tags) and from both full-thickness skin samples indicated that many genes with altered steady-state transcript levels upon sun exposure were expressed in epidermal keratinocytes. These results suggest a major role for the epidermis in the pathomechanism of largely dermal changes in chronically sun-exposed skin.