Showing papers on "Gene expression published in 1999"

The transcriptional program in the response of human fibroblasts to serum.

Abstract: The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor

Abstract: Plant productivity is greatly affected by environmental stresses such as drought, salt loading, and freezing. We reported previously that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA

Abstract: Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.

Distinctive gene expression patterns in human mammary epithelial cells and breast cancers

Abstract: cDNA microarrays and a clustering algorithm were used to identify patterns of gene expression in human mammary epithelial cells growing in culture and in primary human breast tumors. Clusters of coexpressed genes identified through manipulations of mammary epithelial cells in vitro also showed consistent patterns of variation in expression among breast tumor samples. By using immunohistochemistry with antibodies against proteins encoded by a particular gene in a cluster, the identity of the cell type within the tumor specimen that contributed the observed gene expression pattern could be determined. Clusters of genes with coherent expression patterns in cultured cells and in the breast tumors samples could be related to specific features of biological variation among the samples. Two such clusters were found to have patterns that correlated with variation in cell proliferation rates and with activation of the IFN-regulated signal transduction pathway, respectively. Clusters of genes expressed by stromal cells and lymphocytes in the breast tumors also were identified in this analysis. These results support the feasibility and usefulness of this systematic approach to studying variation in gene expression patterns in human cancers as a means to dissect and classify solid tumors.

Gene Expression Profile of Aging and Its Retardation by Caloric Restriction

Abstract: The gene expression profile of the aging process was analyzed in skeletal muscle of mice. Use of high-density oligonucleotide arrays representing 6347 genes revealed that aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were either completely or partially prevented by caloric restriction, the only intervention known to retard aging in mammals. Transcriptional patterns of calorie-restricted animals suggest that caloric restriction retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage.

Regulation of Intestinal α-Defensin Activation by the Metalloproteinase Matrilysin in Innate Host Defense

Abstract: Precursors of α-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with α-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT−/−) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT−/− mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT−/− mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.

Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor β

Abstract: We had previously identified the cDNA for a novel protein called osteoblast-specific factor 2 (OSF-2) from an MC3T3-E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF-2, the protein was renamed "periostin." Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events. Reverse transcription- polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin-Fc recombinant protein supported attachment and spreading of MC3T3-E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to betaig-h3, a molecule induced by transforming growth factor beta (TGF-beta) that promotes the adhesion and spreading of fibroblasts. Because TGF-beta has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF-beta increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.

Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle.

Abstract: Poly(ethylenimine) (PEI) samples of various molecular weights and pHs were used to transfect endothelial cells to achieve levels of gene expression for comparison. PEIs with nominal molecular weights of 600, 1200, 1800, 10,000, and 70,000 Da were examined at pHs of 5. 0, 6.0, 7.0, and 8.0, and the results were recorded in terms of transfection efficiencies at 24, 48, 68, 92, and 120 h post-transfection. Trials were performed on the human endothelial cell-derived cell line EA.hy 926. We found that, for the polymers tested, transfection efficiency increased as the molecular weight of PEI increased. Representative values of PEIs at pH 6 and molecular weight 70,000 produced average transfection efficiencies of 25.6 +/- 7.9% (n = 8) at the greatest average expression levels, while PEI of molecular weight 10,000 yielded efficiencies of only 11.4 +/- 1.7% (n = 6). Transfection efficiencies for molecular weight 1,800 PEI were essentially zero, and PEIs of lower molecular weights produced no transfection at all. In contrast, the pH of the PEI solutions had no discernible effect on transfection. Optimal expression of the green fluorescent protein reporter occurred between 2 and 3 days post-transfection. The amount of reporter expression also was noted, as determined by the brightness of fluorescing cells under UV. The data obtained demonstrate that the molecular weight of the PEI carrier has an effect on transfection efficiency while the pH of the PEI solutions prior to DNA complexation has no such effect.

Oxidative Stress as a Regulator of Gene Expression in the Vasculature

Abstract: Oxidative stress and the production of intracellular reactive oxygen species (ROS) have been implicated in the pathogenesis of a variety of diseases. In excess, ROS and their byproducts that are capable of causing oxidative damage may be cytotoxic to cells. However, it is now well established that moderate amounts of ROS play a role in signal transduction processes such as cell growth and posttranslational modification of proteins. Oxidants, antioxidants, and other determinants of the intracellular reduction-oxidation (redox) state play an important role in the regulation of gene expression. Recent insights into the etiology and pathogenesis of atherosclerosis suggest that this disease may be viewed as an inflammatory disease linked to an abnormality in oxidation-mediated signals in the vasculature. In this review, we summarize the evidence supporting the notion that oxidative stress and the production of ROS function as physiological regulators of vascular gene expression mediated via specific redox-sensitive signal transduction pathways and transcriptional regulatory networks. Elucidating, at the molecular level, the regulatory processes involved in redox-sensitive vascular gene expression represents a foundation not only for understanding the pathogenesis of atherosclerosis and other inflammatory diseases but also for the development of novel therapeutic treatment strategies.

The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans

Abstract: Longevity is regulated by the daf-2 gene network in Caenorhabditis elegans. Mutations in the daf-2 gene, which encodes a member of the insulin receptor family, confer the life extension (Age) phenotype and the constitutive dauer (a growth-arrested larval form specialized for dispersal) formation phenotype. The Age phenotype is mutually potentiated by two life extension mutations in the daf-2 gene and the clk-1 gene, a homologue of yeast CAT5/COQ7 known to regulate ubiquinone biosynthesis. In this study, we demonstrated that the daf-2 mutation also conferred an oxidative stress resistance (Oxr) phenotype, which was also enhanced by the clk-1 mutation. Similar to the Age phenotype, the Oxr phenotype was regulated by the genetic pathway of insulin-like signaling from daf-2 to the daf-16 gene, a homologue of the HNF-3/forkhead transcription factor. These findings led us to examine whether the insulin-like signaling pathway regulates the gene expression of antioxidant defense enzymes. We found that the mRNA le...

Gene expression profiles of laser-captured adjacent neuronal subtypes.

Abstract: , as well as in mammalian cell lines 6 , primary cells 7 and tissues 8 . However, present applications of microarray technology do not include the study of gene expression from individual cell types residing in a given tissue/organ (that is, in situ). Such studies would greatly facilitate our understanding of the complex interactions that exist in vivo between neighboring cell types in normal and disease states. We demonstrate here that gene expression profiles from adjacent cell types can be successfully obtained by integrating the technologies of laser capture microdissection 9 (LCM) and T7-based RNA amplification 10 with cDNA microarrays 11

Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase.

Abstract: In plants and fungi, the introduction of transgenes can lead to post-transcriptional gene silencing. This phenomenon, in which expression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance. Transgene-induced gene silencing has been termed quelling in Neurospora crassa and co-suppression in plants. Quelling-defective (qde) mutants of N. crassa, in which transgene-induced gene silencing is impaired, have been isolated. Here we report the cloning of qde-1, the first cellular component of the gene-silencing mechanism to be isolated, which defines a new gene family conserved among different species including plants, animals and fungi. The qde-1 gene product is similar to an RNA-dependent RNA polymerase found in the tomato. The identification of qde-1 strongly supports models that implicate an RNA-dependent RNA polymerase in the post-transcriptional gene-silencing mechanism. The presence of qde-1 homologues in a variety of species of plants and fungi indicates that a conserved gene-silencing mechanism may exist, which could have evolved to preserve genome integrity and to protect the genome against naturally occurring transposons and viruses.

The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-κB that blocks TNFα-induced apoptosis

Abstract: Bcl-2-family proteins are key regulators of the apoptotic response. Here, we demonstrate that the pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kB. We show that bfl-1 gene expression is dependent on NF-kB activity and that it can substitute for NF-kB to suppress TNFa-induced apoptosis. bfl-1 promoter analysis identified an NF-kB site responsible for its Rel/NF-kB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kB in the immune system. The activation of Bfl-1 may be the means by which NF-kB functions in oncogenesis and promotes cell resistance to anti-cancer therapy.

Reciprocal Positive Regulation of Hypoxia-inducible Factor 1α and Insulin-like Growth Factor 2

Abstract: Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other proteins involved in O 2 homeostasis and tumor progression. The expression and transcriptional activity of the HIF-1α subunit is regulated by the cellular O 2 concentration. We demonstrate that insulin, insulin-like growth factor (IGF)-1, and IGF-2 induce expression of HIF-1α, which is required for expression of genes encoding IGF-2, IGF-binding protein (IGFBP)-2 and IGFBP-3. These data provide a novel mechanism by which HIF-1α overexpression may occur in tumor cells and contribute to an autocrine growth factor loop.

Ploidy Regulation of Gene Expression

Abstract: Microarray-based gene expression analysis identified genes showing ploidy-dependent expression in isogenic Saccharomyces cerevisiae strains that varied in ploidy from haploid to tetraploid. These genes were induced or repressed in proportion to the number of chromosome sets, regardless of the mating type. Ploidy-dependent repression of some G1 cyclins can explain the greater cell size associated with higher ploidies, and suggests ploidy-dependent modifications of cell cycle progression. Moreover, ploidy regulation of the FLO11 gene had direct consequences for yeast development.

Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic acid signaling.

Abstract: The Arabidopsis PAD4 gene previously was found to be required for expression of multiple defense responses including camalexin synthesis and PR-1 gene expression in response to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola. This report describes the isolation of PAD4. The predicted PAD4 protein sequence displays similarity to triacyl glycerol lipases and other esterases. The PAD4 transcript was found to accumulate after P. syringae infection or treatment with salicylic acid (SA). PAD4 transcript levels were very low in infected pad4 mutants. Treatment with SA induced expression of PAD4 mRNA in pad4–1, pad4–3, and pad4–4 plants but not in pad4–2 plants. Induction of PAD4 expression by P. syringae was independent of the regulatory factor NPR1 but induction by SA was NPR1-dependent. Taken together with the previous observation that pad4 mutants have a defect in accumulation of SA upon pathogen infection, these results suggest that PAD4 participates in a positive regulatory loop that increases SA levels, thereby activating SA-dependent defense responses.

Genome-wide expression profiling in Escherichia coli K-12

Abstract: We have established high resolution methods for global monitoring of gene expression in Escherichia coli. Hybridization of radiolabeled cDNA to spot blots on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to glass microarrays for efficiency and reproducibility. A complete set of PCR primers was created for all 4290 annotated open reading frames (ORFs) from the complete genome sequence of E.coli K-12 (MG1655). Glass- and nylonbased arrays of PCR products were prepared and used to assess global changes in gene expression. Full-length coding sequences for array printing were generated by two-step PCR amplification. In this study we measured changes in RNA levels after exposure to heat shock and following treatment with isopropyl-β-D-thiogalactopyranoside (IPTG). Both radioactive and fluorescence-based methods showed comparable results. Treatment with IPTG resulted in high level induction of the lacZYA and melAB operons. Following heat shock treatment 119 genes were shown to have significantly altered expression levels, including 35 previously uncharacterized ORFs and most genes of the heat shock stimulon. Analysis of spot intensities from hybridization to replicate arrays identified sets of genes with signals consistently above background suggesting that at least 25% of genes were expressed at detectable levels during growth in rich media.

Cloning of Human Telomerase Catalytic Subunit (hTERT) Gene Promoter and Identification of Proximal Core Promoter Sequences Essential for Transcriptional Activation in Immortalized and Cancer Cells

Abstract: Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. Of the three major subunits comprising human telomerase, human telomerase catalytic subunit (hTERT) has been shown to be a rate-limiting determinant of the enzymatic activity of human telomerase. However, little is known concerning how expression of hTERT is regulated in human cells. To identify the regulatory elements controlling hTERT gene expression, approximately 3.5 kb of the 5'-flanking sequence of hTERT was cloned and characterized. The promoter of hTERT was GC rich and lacked both TATA and CAAT boxes. The CapSite Hunting method identified transcription start site 19 bp upstream of the first nucleotide of the published cDNA sequence. Transient expression assays revealed that transcription of hTERT was significantly activated in cancer cell lines but repressed in normal primary cells. Using the fibroblast lineage at various stages of transformation, we found that transcription occurred in strains that had overcome replicative senescence and expressed telomerase activity. Deletion analysis of hTERT promoter identified the 181-bp core promoter region upstream of the transcription start site. Gel shift analysis revealed two major factors binding to core promoter, an E box (CACGTG) binding factor and Sp1. Overexpression of c-Myc resulted in a significant increase in transcriptional activity of the core promoter. These findings suggest that hTERT expression is strictly regulated at the transcription machinery, and that the proximal core promoter containing an E box and Sp1 sites is required for transactivation of hTERT.

Transcriptional Cross Talk between NF-κB and p53

Abstract: Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF-κB. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF-κB has been shown to promote resistance to programmed cell death. These observations suggest that a regulatory mechanism must exist to integrate these opposing outcomes and coordinate this critical cellular decision-making event. Here we show that both p53 and NF-κB inhibit each other’s ability to stimulate gene expression and that this process is controlled by the relative levels of each transcription factor. Expression of either wild-type p53 or the RelA(p65) NF-κB subunit suppresses stimulation of transcription by the other factor from a reporter plasmid in vivo. Moreover, endogenous, tumor necrosis factor alpha-activated NF-κB will inhibit endogenous wild-type p53 transactivation. Following exposure to UV light, however, the converse is observed, with p53 downregulating NF-κB-mediated transcriptional activation. Both p53 and RelA(p65) interact with the transcriptional coactivator proteins p300 and CREB-binding protein (CBP), and we demonstrate that these results are consistent with competition for a limiting pool of p300/CBP complexes in vivo. These observations have many implications for regulation of the transcriptional decision-making mechanisms that govern cellular processes such as apoptosis. Furthermore, they suggest a previously unrealized mechanism through which dysregulated NF-κB can contribute to tumorigenesis and disease.

Nrf2 is essential for protection against acute pulmonary injury in mice

Abstract: Nrf2 is a member of the "cap 'n' collar" family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2(-/-) mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2(-/-) mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2(-/-) mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2(-/-) mice. The Nrf2(-/-) mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.

The plant-growth-promoting rhizobacterium Paenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression: a possible connection between biotic and abiotic stress responses.

Abstract: This paper addresses changes in plant gene expression induced by inoculation with plant-growth-promoting rhizobacteria (PGPR). A gnotobiotic system was established with Arabidopsis thaliana as model plant, and isolates of Paenibacillus polymyxa as PGPR. Subsequent challenge by either the pathogen Erwinia carotovora (biotic stress) or induction of drought (abiotic stress) indicated that inoculated plants were more resistant than control plants. With RNA differential display on parallel RNA preparations from P. polymyxa-treated or untreated plants, changes in gene expression were investigated. From a small number of candidate sequences obtained by this approach, one mRNA segment showed a strong inoculation-dependent increase in abundance. The corresponding gene was identified as ERD15, previously identified to be drought stress responsive. Quantification of mRNA levels of several stress-responsive genes indicated that P. polymyxa induced mild biotic stress. This suggests that genes and/or gene classes associated with plant defenses against abiotic and biotic stress may be co-regulated. Implications of the effects of PGPR on the induction of plant defense pathways are discussed.

ADD1/SREBP-1c Is Required in the Activation of Hepatic Lipogenic Gene Expression by Glucose

Abstract: The transcription of genes encoding proteins involved in the hepatic synthesis of lipids from glucose is strongly stimulated by carbohydrate feeding. It is now well established that in the liver, glucose is the main activator of the expression of this group of genes, with insulin having only a permissive role. While ADD1/SREBP-1 has been implicated in lipogenic gene expression through temporal association with food intake and ectopic gain-of-function experiments, no genetic evidence for a requirement for this factor in glucose-mediated gene expression has been established. We show here that the transcription of ADD1/SREBP-1c in primary cultures of hepatocytes is controlled positively by insulin and negatively by glucagon and cyclic AMP, establishing a link between this transcription factor and carbohydrate availability. Using adenovirus-mediated transfection of a powerful dominant negative form of ADD1/SREBP-1c in rat hepatocytes, we demonstrate that this factor is absolutely necessary for the stimulation by glucose of l-pyruvate kinase, fatty acid synthase, S14, and acetyl coenzyme A carboxylase gene expression. These results demonstrate that ADD1/SREBP-1c plays a crucial role in mediating the expression of lipogenic genes induced by glucose and insulin.