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Among the metazoan parasites that cause debilitating disease in man, schistosomes are the first group for which near-complete transcriptome complements have been described. This new genomic information will have an enormous impact on all future investigations into the biology, pathogenesis and control of schistosomiasis.
For decades, researchers have focused most of their attention on protein-coding genes and proteins. With the completion of the human and mouse genomes and the accumulation of data on the mammalian transcriptome, the focus now shifts to non-coding DNA sequences, RNA-coding genes and their transcripts. Many non-coding transcribed sequences are proving to have important regulatory roles, but the functions of the majority remain mysterious.
BACKGROUND: Comprehensive, tissue-specific, microarray analysis is a potent tool for the identification of tightly defined expression patterns that might be missed in whole-organism scans. We applied such an analysis to Drosophila melanogaster Malpighian (renal) tubule, a defined differentiated tissue. RESULTS: The transcriptome of the D. melanogaster Malpighian tubule is highly reproducible and significantly different from that obtained from whole-organism arrays. More than 200 genes are more than 10-fold enriched and over 1,000 are significantly enriched. Of the top 200 genes, only 18 have previously been named, and only 45% have even estimates of function. In addition, 30 transcription factors, not previously implicated in tubule development, are shown to be enriched in adult tubule, and their expression patterns respect precisely the domains and cell types previously identified by enhancer trapping. Of Drosophila genes with close human disease homologs, 50 are enriched threefold or more, and eight enriched 10-fold or more, in tubule. Intriguingly, several of these diseases have human renal phenotypes, implying close conservation of renal function across 400 million years of divergent evolution. CONCLUSIONS: From those genes that are identifiable, a radically new view of the function of the tubule, emphasizing solute transport rather than fluid secretion, can be obtained. The results illustrate the phenotype gap: historically, the effort expended on a model organism has tended to concentrate on a relatively small set of processes, rather than on the spread of genes in the genome.
BACKGROUND: The male-specific region of the mouse Y chromosome long arm (MSYq) is comprised largely of repeated DNA, including multiple copies of the spermatid-expressed Ssty gene family. Large deletions of MSYq are associated with sperm head defects for which Ssty deficiency has been presumed to be responsible. RESULTS: In a search for further candidate genes associated with these defects we analyzed changes in the testis transcriptome resulting from MSYq deletions, using testis cDNA microarrays. This approach, aided by accumulating mouse MSYq sequence information, identified transcripts derived from two further spermatid-expressed multicopy MSYq gene families; like Ssty, each of these new MSYq gene families has multicopy relatives on the X chromosome. The Sly family encodes a protein with homology to the chromatin-associated proteins XLR and XMR that are encoded by the X chromosomal relatives. The second MSYq gene family was identified because the transcripts hybridized to a microarrayed X chromosome-encoded testis cDNA. The X loci ('Astx') encoding this cDNA had 92-94% sequence identity to over 100 putative Y loci ('Asty') across exons and introns; only low level Asty transcription was detected. More strongly transcribed recombinant loci were identified that included Asty exons 2-4 preceded by Ssty1 exons 1, 2 and part of exon 3. Transcription from the Ssty1 promotor generated spermatid-specific transcripts that, in addition to the variable inclusion of Ssty1 and Asty exons, included additional exons because of the serendipitous presence of splice sites further downstream. CONCLUSION: We identified further MSYq-encoded transcripts expressed in spermatids and deriving from multicopy Y genes, deficiency of which may underlie the defects in sperm development associated with MSYq deletions.
BACKGROUND: Sequencing and annotation of the genome of rice (Oryza sativa) have generated gene models in numbers that top all other fully sequenced species, with many lacking recognizable sequence homology to known genes. Experimental evaluation of these gene models and identification of new models will facilitate rice genome annotation and the application of this knowledge to other more complex cereal genomes. RESULTS: We report here an analysis of the chromosome 10 transcriptome of the two major rice subspecies, japonica and indica, using oligonucleotide tiling microarrays. This analysis detected expression of approximately three-quarters of the gene models without previous experimental evidence in both subspecies. Cloning and sequence analysis of the previously unsupported models suggests that the predicted gene structure of nearly half of those models needs improvement. Coupled with comparative gene model mapping, the tiling microarray analysis identified 549 new models for the japonica chromosome, representing an 18% increase in the annotated protein-coding capacity. Furthermore, an asymmetric distribution of genome elements along the chromosome was found that coincides with the cytological definition of the heterochromatin and euchromatin domains. The heterochromatin domain appears to associate with distinct chromosome level transcriptional activities under normal and stress conditions. CONCLUSION: These results demonstrated the utility of genome tiling microarray in evaluating annotated rice gene models and in identifying novel transcriptional units. The tiling microarray sanalysis further revealed a chromosome-wide transcription pattern that suggests a role for transposable element-enriched heterochromatin in shaping global transcription in response to environmental changes in rice.
We report the initial sequencing and comparative analysis of the Macaca mulatta transcriptome. Cloned sequences from 11 tissues, nine animals, and three species (M. mulatta, M. fascicularis, and M. nemestrina) were sampled, resulting in the generation of 48,642 sequence reads. These data represent an initial sampling of the putative rhesus orthologs for 6,216 human genes. Mean nucleotide diversity within M. mulatta and sequence divergence among M. fascicularis, M. nemestrina, and M. mulatta are also reported.
BACKGROUND: Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands. RESULTS: Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis. CONCLUSION: This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.
Serial analysis of gene expression (SAGE) was used to identify and quantify all expressed cerebellar genes in the adult (P92) and aged (P810) C57BL/6J mouse cerebellum. A "closest-neighbor" algorithm was used to differentiate low abundance tags from possible sequencing errors in both libraries. Unique tags were categorized into four groups: (1) novel genes; (2) ESTs; (3) RIKEN, KIA, and hypothetical genes; and (4) known genes. Known genes were further subdivided into functional categories based on the gene ontology classification, using a web-based program developed in this laboratory (MmSAGEClass). Comparison of adult and aged cerebellar libraries revealed several genes that were differentially expressed, including growth hormone and prolactin, both of which were markedly decreased in the aged cerebellum. In addition, several tags showing differential expression were not identified in the Unigene database and are likely to represent novel genes. The present SAGE data on the aged cerebellar transcriptome may reveal candidate genes involved in the aging process.
The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.
In central and sub-Saharan Africa, trypanosomosis is a tsetse fly-transmitted disease, which is considered as the most important impediment to livestock production in the region. However, several indigenous West African taurine breeds (Bos taurus) present remarkable tolerance to the infection. This genetic capability, named trypanotolerance, results from numerous biological mechanisms most probably under multigenic dependences, among which are control of the trypanosome infection by limitation of parasitemia and control of severe anemia due to the pathogenic effects. Today, some postgenomic biotechnologies, such as transcriptome analyses, allow characterization of the full expressed genes involved in the majority of animal diseases under genetic control. One of them is serial analysis of gene expression (SAGE) technology, which consists of the construction of mRNA transcript libraries for qualitative and quantitative analysis of the entire genes expressed or inactivated at a particular step of cellular activation. We developed four different mRNA transcript libraries from white blood cells on a N'Dama trypanotolerant animal during an experimental Trypanosoma congolense (T. congolense) infection: one before experimental infection (ND0), one at the parasitemia peak (NDm), one at the minimal packed cell volume (NDa), and the last one at the end of the experiment after normalization (NDf). Bioinformatic comparisons in bovine genomic databases allowed us to obtain more than 75,000 sequences, among which are several known genes, some others are already described as expressed sequence tags (ESTs), and the last are completely new, but probably functional in trypanotolerance. The knowledge of all identified named or unnamed genes involved in trypanotolerance characteristics will allow us to use them in a field marker-assisted selections strategy and in microarrays prediction sets for bovine trypanotolerance.
PURPOSE: Insight into clinical response to platinum-based chemotherapy (PBC) in non-small-cell lung cancer (NSCLC). METHODS: Matched tumor and nontumor lung tissues from PBC-treated NSCLC patients (four nonresponders and four responders) and tumor tissue from an independent test set (four nonresponders and four responders), were profiled using microarrays. Lysosomal protease inhibitors SerpinB3 and cystatin C were highly correlated with clinical response and were further evaluated by immunohistochemistry in PBC-treated patients (36 prechemotherapy and 13 postchemotherapy). Investigation of the pathogenic and prognostic significance of SerpinB3 was performed in 251 primary tumors, with 64 regional lymph node pairs, from chemotherapy-naïve NSCLC patients using immunohistochemistry. RESULTS: Bioinformatic analyses of gene expression in the training set identified a gene set (n = 17) that separated all patients in the training and test sets (n = 16) according to response in hierarchical clustering. Transcriptome profiling revealed that SerpinB3 mRNA was highly correlated with degree of response (r = -0.978; P < .0001) and was a clear outlier (nonresponders:responders > 50-fold). SerpinB3 protein expression was correlated with clinical response in PBC-treated NSCLC patients (P = .045). Expression of SerpinB3 and cystatin C, relative to the target, protease cathepsin B, was independently predictive of response (odds ratio, 17.8; 95% CI, 2.0 to 162.4; P = .01), with an accuracy of 72%. High SerpinB3 expression levels, invariably associated with chemoresistance, had contrasting prognostic impact in untreated squamous cell carcinomas (hazard ratio [HR], 0.43; 95% CI, 0.18 to 0.93) or adenocarcinomas (HR, 2.09; 95% CI, 1.03 to 4.72). CONCLUSION: This provides the first comprehensive molecular characterization of clinical responsiveness to PBC in NSCLC and reveals the predictive and prognostic impact of two lysosomal protease inhibitors, potentially representing novel targets for NSCLC therapeutics.
CONTEXT: Primary aldosteronism (PAL) is the most frequent cause of secondary arterial hypertension. In PAL, aldosterone production is chronic, excessive, and autonomous. OBJECTIVE: The objective of this study was to identify the angiotensin-II independent alterations of steroidogenesis responsible for PAL. DESIGN: Genomewide gene expression was compared in two tissues differentiated for aldosterone production, both nonstimulated by circulating angiotensin II and differing in their autonomy to produce aldosterone: aldosterone-producing adenoma (APA) and its adjacent dissected zona glomerulosa (ZG). SETTING: The setting of this study was the Comete Network. PATIENTS: Patients with APA were studied. INTERVENTION: Transcriptome comparison was made of one APA and its adjacent ZG by serial analysis of gene expression; validation by in situ hybridization was performed for 19 genes in 11 samples. OUTCOME: The study outcome was genes differentially expressed in APA and adjacent ZG. RESULTS: Activation of steroidogenesis in PAL is restricted to the overexpression of the enzymes producing aldosterone-specific steroids, aldosterone synthase and also 21-hydroxylase, suggesting that upstream precursor production is not limiting. Increased expression of high-density lipoprotein receptor, adrenodoxin and P450 oxidoreductase suggests that these systems provide cholesterol and electrons to the mitochondrial steroidogenic enzymes. As for acute stimulation of aldosterone production, an activation of calcium signaling is suggested by concordant overexpression of calcium-binding proteins or effectors. Calcium activation may result from an abnormal activity of G(q) protein-coupled receptors. This calcium activation may be the starting point of the other gene expression changes observed in APA. Finally, other differentially expressed genes include three genes encoding unidentified proteins. CONCLUSION: This work provides an original and integrated view of the mechanisms of aldosterone production in PAL.
The transcription factor Gata3 is crucially involved in epidermis and hair follicle differentiation. Yet, little is known about how Gata3 co-ordinates stem cell lineage determination in skin, what pathways are involved and how Gata3 differentially regulates distinct cell populations within the hair follicle. Here, we describe a conditional Gata3-/- mouse (K14-Gata3-/-) in which Gata3 is specifically deleted in epidermis and hair follicles. K14-Gata3-/- mice show aberrant postnatal growth and development, delayed hair growth and maintenance, abnormal hair follicle organization and irregular pigmentation. After the first hair cycle, the germinative layer surrounding the dermal papilla was not restored; instead, proliferation was pronounced in basal epidermal cells. Transcriptome analysis of laser-dissected K14-Gata3-/- hair follicles revealed mitosis, epithelial differentiation and the Notch, Wnt and BMP signaling pathways to be significantly overrepresented. Elucidation of these pathways at the RNA and protein levels and physiologic endpoints suggests that Gata3 integrates diverse signaling networks to regulate the balance between hair follicle and epidermal cell fates.
Predicting who will develop cancer and how the cancer will behave and respond to therapy after diagnosis are some of the potential benefits of the ongoing genetic revolution that can be envisioned within the next decade. Translational applications of genomic-based research efforts may actually precede the development of effective therapeutic agents that can exploit the vast amounts of data derived from these efforts. In the future, understanding the wealth of information generated by high-throughput molecular efforts and how it can be applied to clinical problems will likely be critical to the surgeon who guides the multidisciplinary care of the cancer patient. This review will discuss the advances in our understanding of the human genome (DNA), its derived transcriptome (RNA), and its translated proteome (proteins) and will focus on the translation of this information into routine clinical practice. In particular, we will focus on the potential for clinical application of microarray-based gene-expression profiling to the diagnosis, prognosis, and therapy of malignancies.
Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production.
Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.
RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence that many more should exist. Here we identify intramolecular pairs of Alu elements as a major target for editing in the human transcriptome. An experimental demonstration in 43 genes was extended by a broader computational analysis of more than 100,000 human mRNAs. We find that 1,445 human mRNAs (1.4%) are subject to RNA editing at more than 14,500 sites, and our data further suggest that the vast majority of pre-mRNAs (greater than 85%) are targeted in introns by the editing machinery. The editing levels of Alu-containing mRNAs correlate with distance and homology between inverted repeats and vary in different tissues. Alu-mediated RNA duplexes targeted by RNA editing are formed intramolecularly, whereas editing due to intermolecular base-pairing appears to be negligible. We present evidence that these editing events can lead to the posttranscriptional creation or elimination of splice signals affecting alternatively spliced Alu-derived exons. The analysis suggests that modification of repetitive elements is a predominant activity for RNA editing with significant implications for cellular gene expression.
Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html).