Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.
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The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule. (+info)
Identification of transforming growth factor-beta- regulated genes in caenorhabditis elegans by differential hybridization of arrayed cDNAs.
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Members of the transforming growth factor-beta family play critical roles in body patterning, in both vertebrates and invertebrates. One transforming growth factor-beta-related gene, dbl-1, has been shown to regulate body length and male ray patterning in Caenorhabditis elegans. We screened arrayed cDNAs to identify downstream target genes for the DBL-1 signaling by using differential hybridization. C. elegans cDNAs representing 7,584 independent genes were arrayed on a nylon membrane at high density and hybridized with (33)P-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2, and lon-2 worms. Signals for all the spots representing hybridized DNA were quantified and compared among strains. The screening identified 22 and 2 clones, which were positively and negatively regulated, respectively, by the DBL-1 signal. Northern hybridization confirmed the expression profiles of most of the clones, indicating good reliability of the differential hybridization using arrayed cDNAs. In situ hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed that at least four genes, including the gene for the type I receptor for DBL-1, sma-6, were transcriptionally regulated by the DBL-1 signal. (+info)
Serial microanalysis of renal transcriptomes.
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Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1, 000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods. (+info)
Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays.
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Human immunodeficiency virus type 1 (HIV-1) infection alters the expression of host cell genes at both the mRNA and protein levels. To obtain a more comprehensive view of the global effects of HIV infection of CD4-positive T-cells at the mRNA level, we performed cDNA microarray analysis on approximately 1500 cellular cDNAs at 2 and 3 days postinfection (p.i.) with HIV-1. Host cell gene expression changed little at 2 days p.i., but at 3 days p.i. 20 cellular genes were identified as differentially expressed. Genes involved in T-cell signaling, subcellular trafficking, and transcriptional regulation, as well as several uncharacterized genes, were among those whose mRNAs were differentially regulated. These results support the hypothesis that HIV-1 infection alters expression of a broad array of cellular genes and provides a framework for future functional studies on the differentially expressed mRNA products. (+info)
Prediction of gene function by genome-scale expression analysis: prostate cancer-associated genes.
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We wish to identify genes associated with disease. To do so, we look for novel genes whose expression patterns mimic those of known disease-associated genes, using a method we call Guilt-by-Association (GBA), on the basis of a combinatoric measure of association. Using GBA, we have examined the expression of 40,000 human genes in 522 cDNA libraries, and have discovered several hundred previously unidentified genes associated with cancer, inflammation, steroid-synthesis, insulin-synthesis, neurotransmitter processing, matrix remodeling, and other disease processes. The majority of the genes thus discovered show no sequence similarity to known genes, and thus could not have been identified by homology searches. We present here an example of the discovery of eight genes associated with prostate cancer. Of the 40,000 most-abundant human genes, these 8 are the most closely linked to the known diagnostic genes, and thus are prime targets for pharmaceutical research. (+info)
Expression profiling by iAFLP: A PCR-based method for genome-wide gene expression profiling.
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The availability of comprehensive sets of genes has prompted the researchers to carry out systematic collection of gene expression data. RT-PCR has the highest specificity and sensitivity for transcript detection among all available methods. Low throughput, especially when quantitative data are desired, has precluded RT-PCR from genome-wide application. Here we report a PCR-based expression profiling method, introduced amplified fragment length polymorphism (iAFLP), that has the same specificity and sensitivity as RT-PCR and a throughput level comparable to that of DNA-microarray hybridization. In this method, restricted ends of total cDNAs from six sources were ligated with adaptors having various length of short insertions to a common sequence (polymorphic adaptors). Amplification of a pool of these differentially adapted cDNAs with a gene-specific primer and an adaptor primer allows us to quantitate the abundance of any transcript in six mRNA sources. Using three different primer colors this technique allows quantitation of expression of 864 genes across six different sources per day with a single autosequencer, which is comparable to the throughput of microarray analysis in terms of number of genes x number of sources. (+info)
The genexpress IMAGE knowledge base of the human muscle transcriptome: a resource of structural, functional, and positional candidate genes for muscle physiology and pathologies.
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Sequence, gene mapping, and expression data corresponding to 910 genes transcribed in human skeletal muscle have been integrated to form the muscle module of the Genexpress IMAGE Knowledge Base. Based on cDNA array hybridization, a set of 14 transcripts preferentially or specifically expressed in muscle have been selected and characterized in more detail: Their pattern of expression was confirmed by Northern blot analysis; their structure was further characterized by full-insert cDNA sequencing and cDNA extension; the map location of the corresponding genes was refined by radiation hybrid mapping. Five of the 14 selected genes appear as interesting positional and functional candidate genes to study in relation with muscle physiology and/or specific orphan muscular pathologies. One example is discussed in more detail. The expression profiling data and the associated Genexpress Index2 entries for the 910 genes and the detailed characterization of the 14 selected transcripts are available from a dedicated Web server at. The database has been organized to provide the users with a working space where they can find curated, annotated, integrated data for their genes of interest. Different navigation routes to exploit the resource are discussed. (+info)
The p53 gene family.
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p73 and p63 are two recently discovered p53 homologs. Like p53, these proteins can recognize canonical p53 DNA-binding sites and, when overproduced, can activate p53-responsive target genes and induce apoptosis. Unlike p53, these genes undergo complex alternative splicing which, at least in the case of p63, yields proteins with widely divergent biological properties. In addition p73 and p63 are, in contrast to p53, rarely mutated in human cancer. Furthermore, p73 inactivation is not required for viral transformation. Thus, there is currently no firm evidence that p63 and p73 should be considered tumor suppressors. The early suggestion that monoallelic expression of p73 contributed to carcinogenesis needs to be interpreted cautiously in light of data showing interindividual and intraindividual variation with respect to monoallelic expression of p73 and the finding that p73 mRNA levels are generally increased, rather than decreased, in a host of tumors relative to normal cells. (+info)