Showing papers on "Gene expression published in 2000"

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

Abstract: The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Abstract: Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of ~21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila , at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Systematic variation in gene expression patterns in human cancer cell lines.

Abstract: We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

Genomics, gene expression and DNA arrays.

Abstract: Experimental genomics in combination with the growing body of sequence information promise to revolutionize the way cells and cellular processes are studied. Information on genomic sequence can be used experimentally with high-density DNA arrays that allow complex mixtures of RNA and DNA to be interrogated in a parallel and quantitative fashion. DNA arrays can be used for many different purposes, most prominently to measure levels of gene expression (messenger RNA abundance) for tens of thousands of genes simultaneously. Measurements of gene expression and other applications of arrays embody much of what is implied by the term ‘genomics’; they are broad in scope, large in scale, and take advantage of all available sequence information for experimental design and data interpretation in pursuit of biological understanding.

Synthetic DNA delivery systems.

Abstract: The ability to safely and efficiently transfer foreign DNA into cells is a fundamental goal in biotechnology. Toward this end, rapid advances have recently been made in our understanding of mechanisms for DNA stability and transport within cells. Current synthetic DNA delivery systems are versatile and safe, but substantially less efficient than viruses. Indeed, most current systems address only one of the obstacles to DNA delivery by enhancing DNA uptake. In fact, the effectiveness of gene expression is also dependent on several additional factors, including the release of intracellular DNA, stability of DNA in the cytoplasm, unpackaging of the DNA-vector complex, and the targeting of DNA to the nucleus. Delivery systems of the future must fully accommodate all these processes to effectively shepherd DNA across the plasma membrane, through the hostile intracellular environment, and into the nucleus.

Human UDP-Glucuronosyltransferases: Metabolism, Expression, and Disease

Abstract: In vertebrates, the glucuronidation of small lipophilic agents is catalyzed by the endoplasmic reticulum UDP-glucuronosyltransferases (UGTs). This metabolic pathway leads to the formation of water-soluble metabolites originating from normal dietary processes, cellular catabolism, or exposure to drugs and xenobiotics. This classic detoxification process, which led to the discovery nearly 50 years ago of the cosubstrate UDP-glucuronic acid (19), is now known to be carried out by 15 human UGTs. Characterization of the individual gene products using cDNA expression experiments has led to the identification of over 350 individual compounds that serve as substrates for this superfamily of proteins. This data, coupled with the introduction of sophisticated RNA detection techniques designed to elucidate patterns of gene expression of the UGT superfamily in human liver and extrahepatic tissues of the gastrointestinal tract, has aided in understanding the contribution of glucuronidation toward epithelial first-pass metabolism. In addition, characterization of the UGT1A locus and genetic studies directed at understanding the role of bilirubin glucuronidation and the biochemical basis of the clinical symptoms found in unconjugated hyperbilirubinemia have uncovered the structural gene polymorphisms associated with Crigler-Najjar's and Gilbert's syndrome. The role of the UGTs in metabolism and different disease states in humans is the topic of this review.

A gene expression database for the molecular pharmacology of cancer

Abstract: We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

Distinct Roles of CONSTANS Target Genes in Reproductive Development of Arabidopsis

Abstract: In plants, flowering is triggered by endogenous and environmental signals. CONSTANS (CO) promotes flowering of Arabidopsis in response to day length. Four early target genes of CO were identified using a steroid-inducible version of the protein. Two of these genes,SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) andFLOWERING LOCUS T (FT), are required for CO to promote flowering; the others are involved in proline or ethylene biosynthesis. The SOC1 and FT genes are also regulated by a second flowering-time pathway that acts independently of CO. Thus, early target genes of CO define common components of distinct flowering-time pathways.

Hypoxia-inducible Expression of Tumor-associated Carbonic Anhydrases

Abstract: The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis

Abstract: Wounding in multicellular eukaryotes results in marked changes in gene expression that contribute to tissue defense and repair. Using a cDNA microarray technique, we analyzed the timing, dynamics, and regulation of the expression of 150 genes in mechanically wounded leaves of Arabidopsis. Temporal accumulation of a group of transcripts was correlated with the appearance of oxylipin signals of the jasmonate family. Analysis of the coronatine-insensitive coi1-1 Arabidopsis mutant that is also insensitive to jasmonate allowed us to identify a large number of COI1-dependent and COI1-independent wound-inducible genes. Water stress was found to contribute to the regulation of an unexpectedly large fraction of these genes. Comparing the results of mechanical wounding with damage by feeding larvae of the cabbage butterfly (Pieris rapae) resulted in very different transcript profiles. One gene was specifically induced by insect feeding but not by wounding; moreover, there was a relative lack of water stress-induced gene expression during insect feeding. These results help reveal a feeding strategy of P. rapae that may minimize the activation of a subset of water stress-inducible, defense-related genes.

An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants

Abstract: We have developed an estrogen receptor-based chemical-inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol-activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the -46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating transgene expression in plants.

The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor.

Abstract: The Arabidopsis abscisic acid (ABA)-insensitive abi5 mutants have pleiotropic defects in ABA response, including decreased sensitivity to ABA inhibition of germination and altered expression of some ABA-regulated genes. We isolated the ABI5 gene by using a positional cloning approach and found that it encodes a member of the basic leucine zipper transcription factor family. The previously characterized abi5-1 allele encodes a protein that lacks the DNA binding and dimerization domains required for ABI5 function. Analyses of ABI5 expression provide evidence for ABA regulation, cross-regulation by other ABI genes, and possibly autoregulation. Comparison of seed and ABA-inducible vegetative gene expression in wild-type and abi5-1 plants indicates that ABI5 regulates a subset of late embryogenesis-abundant genes during both developmental stages.

Transcriptional silencing and promoter methylation triggered by double-stranded RNA

Abstract: Double‐stranded RNA induces a post‐transcriptional gene silencing process, termed RNAi, in diverse organisms. It is shown here that transcriptional gene silencing accompanied by de novo methylation of a target promoter in plants can be triggered by a double‐stranded RNA containing promoter sequences. Similar to the double‐stranded RNA involved in RNAi, this promoter double‐stranded RNA, which is synthesized in the nucleus, is partially cleaved into small RNAs ∼23 nucleotides in length. Both transcriptional and post‐transcriptional gene silencing can thus be initiated by double‐stranded RNAs that enter the same degradation pathway. The results also implicate double‐stranded RNA in directing DNA methylation. Different constructs designed to produce double‐stranded promoter RNA in various ways were evaluated for their ability to induce gene silencing in tobacco and Arabidopsis . RNA hairpins transcribed from inverted DNA repeats were the most effective trans ‐acting silencing signals. This strategy could be useful for transcriptionally downregulating genes in a variety of plants.

Signaling and Circuitry of Multiple MAPK Pathways Revealed by a Matrix of Global Gene Expression Profiles

Abstract: Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Specific interference with gene function by double-stranded RNA in early mouse development

Abstract: The use of double-stranded (ds) RNA is a powerful way of interfering with gene expression in a range of organisms, but doubts have been raised about whether it could be successful in mammals. Here, we show that dsRNA is effective as a specific inhibitor of the function of three genes in the mouse, namely maternally expressed c-mos in the oocyte and zygotically expressed E-cadherin or a GFP transgene in the preimplantation embryo. The phenotypes observed are the same as those reported for null mutants of the endogenous genes. These findings offer the opportunity to study development and gene regulation in normal and diseased cells.

Control selection for RNA quantitation.

Abstract: The study of mammalian gene expression is often carried out at the level of mRNA. In such analyses, one usually measures the amount of an mRNA of interest under different conditions such as stress,...

Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.

Abstract: We investigated the potential of double-stranded RNA interference (RNAi) with gene activity in Arabidopsis thaliana. To construct transformation vectors that produce RNAs capable of duplex formation, gene-specific sequences in the sense and antisense orientations were linked and placed under the control of a strong viral promoter. When introduced into the genome of A. thaliana by Agrobacterium-mediated transformation, double-stranded RNA-expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference. The severity of phenotypes varied between transgenic lines. In situ hybridization revealed a correlation between a declining AG mRNA accumulation and increasingly severe phenotypes in AG (RNAi) mutants, suggesting that endogenous mRNA is the target of double-stranded RNA-mediated genetic interference. The ability to generate stably heritable RNAi and the resultant specific phenotypes allows us to selectively reduce gene function in A. thaliana.

Decreased expression of striatal signaling genes in a mouse model of Huntington’s disease

Abstract: To understand gene expression changes mediated by a polyglutamine repeat expansion in the human huntingtin protein, we used oligonucleotide DNA arrays to profile approximately 6000 striatal mRNAs in the R6/2 mouse, a transgenic Huntington's disease (HD) model. We found diminished levels of mRNAs encoding components of the neurotransmitter, calcium and retinoid signaling pathways at both early and late symptomatic time points (6 and 12 weeks of age). We observed similar changes in gene expression in another HD mouse model (N171-82Q). These results demonstrate that mutant huntingtin directly or indirectly reduces the expression of a distinct set of genes involved in signaling pathways known to be critical to striatal neuron function.

Protein–protein interactions define specificity in signal transduction

Abstract: Virtually every aspect of cellular function within a metazoan organism, including proliferative status, metabolism, gene expression, cytoskeletal organization, and indeed the cell’s very survival, is dependent on external signaling molecules, either in the form of soluble hormones or proteins anchored to the surface of an adjacent cell or the extracellular matrix (ECM). These factors exert their effects either by binding receptors displayed on the surface of the cell or, in the case of compounds such as steroids, by traversing the plasma membrane and directly engaging intracellular receptors. In addition, these external signals can be linked to intrinsic cues that regulate events such as polarity and asymmetric cell division, and that monitor the molecular composition of the cell, and therefore determine whether suitable conditions prevail for cell growth and division. Over the last two decades, we have achieved considerable understanding of the mechanisms by which signals are conveyed from receptors at the plasma membrane to their targets in the cytoplasm and nucleus. At heart, this is a problem of molecular recognition. Hormones must bind selectively to their receptors and these in turn must interact with specific cytoplasmic targets. To understand signal transduction in a general sense, it is important to know whether different biochemical pathways use related molecular devices to control cellular behavior. To understand specificity in signaling, we need to know how receptors interact with particular targets and how the proteins of one pathway can be insulated from related signaling components. At the same time, it is important to learn how distinct signaling pathways communicate with one another, since the entire cell must ultimately function as a single unit whose different elements respond in an organized fashion to external signals. A cell in the body will be exposed to many different stimuli, which it must integrate into a coherent response. Furthermore, although a rather large fraction of genes within nucleated cells appear to function in the processes of signal transduction and cellular organization (Plowman et al. 1999), it is still remarkable that only a few thousand gene products can control the sophisticated behaviors of many different cell types. This immediately suggests that signaling proteins must act in a combinatorial fashion, since there are insufficient proteins for each to have a single biological role. For example, there are billions of neurons in the human brain, each of which must project its axon to the appropriate target, let alone undertake the complex biochemical events associated with neurotransmission and synaptic plasticity. Clearly, the signaling molecules that function in the process of axon guidance must act in a combinatorial way to generate the extreme complexity of the human nervous system. Here we will address some of the underlying biochemical mechanisms through which specificity is generated during signal transduction, and pursue the means by which signaling molecules may act in combination to generate complex biological responses.

Differential Effects of Culture on Imprinted H19 Expression in the Preimplantation Mouse Embryo

Abstract: The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten’s medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM1AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten’s medium but not in embryos cultured in KSOM1AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten’s medium, while the response of H19 to culture in KSOM1AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten’s medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.

High-fidelity mRNA amplification for gene profiling.

Abstract: The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

Regulation of B lymphocyte and macrophage development by graded expression of PU.1

Abstract: The ets family transcription factor PU.1 is required for the development of multiple lineages of the immune system. Using retroviral transduction of PU.1 complementary DNA into mutant hematopoietic progenitors, we demonstrate that differing concentrations of the protein regulate the development of B lymphocytes as compared with macrophages. A low concentration of PU. 1 protein induces the B cell fate, whereas a high concentration promotes macrophage differentiation and blocks B cell development. Conversely, a transcriptionally weakened mutant protein preferentially induces B cell generation. Our results suggest that graded expression of a transcription factor can be used to specify distinct cell fates in the hematopoietic system.

Plaid models for gene expression data

Abstract: Motivated by genetic expression data, we introduce plaid models. These are a form of two-sided cluster analysis that allows clusters to overlap. Plaid models also incorporate additive two way ANOVA models within the two-sided clusters. Using these models we find interpretable structure in some yeast expression data, as well as in some nutrition data and some foreign exchange data.