Downregulation Of Specific Genes Research Articles

Global Analysis of Genes Regulated by Low Temperature and Photoperiod in Peach Bark

In response to environmental cues plants undergo changes in gene expression that result in the up- or down-regulation of specific genes. To identify genes in peach [Prunus persica (L.) Batsch.] trees whose transcript levels are specifically affected by low temperature (LT) or short day photoperiod (SD), we have created suppression subtractive hybridization (SSH) libraries from bark tissues sampled from trees kept at 5 °C and 25 °C under short day (SD) photoperiod or exposed to a night break (NB) interruption during the dark period of the SD cycle to simulate a long day (LD) photoperiod. Sequences expressed in forward and reverse subtractions using various subtracted combinations of temperature and photoperiod treatments were cloned, sequenced, and identified by BLAST and ClustalW analysis. Low temperature treatment resulted in the up-regulation of a number of cold-responsive and stress-related genes and suppression of genes involved in “housekeeping” functions (e.g., cell division and photosynthesis). Some stress-related genes not observed to be up-regulated under LT were increased in response to SD photoperiod treatments. Comparison of the patterns of expression as a consequence of different temperature and photoperiod treatments allowed us to determine the qualitative contribution of each treatment to the regulation of specific genes.

Monitoring the Dynamics of Transcription and Translation Within the Time Course of Recombinant E. coli Cultivations

Background Among the key objectives for the optimization of recombinant protein production on industrial scale high yield of the product is one of the most important issues. To achieve high quantities of recombinant protein strong host/vector systems are utilized, generally leading to an overburden of the host cell metabolism. To cope with different types of stress cells have evolved complex regulatory entities acting on the highest level of metabolic regulation, such as the stringent response network. Highly specific signal molecules of these regulatory networks, like sigma factors and ppGpp, as well as global regulators like ArcA, Crp and Fis enable accurate up-and downregulation of specific genes [1,2]. The thereby altered expression profile confers increased resistance to adverse conditions. As the exploitation of the cell factory will always approach the physiological limits of the cellular machinery the shortas well as the long-term impact of high recombinant gene expression rates have to be investigated. Therefore key variables of metabolic stress to define and thus quantify the actual metabolic load of host cell metabolism can be inferred from variations of genome and proteome patterns. Due to the availability of whole genome E. coli microarrays this technology provides an efficient tool for screening overall transcriptional changes in the course of fermentation processes. 2D-Fluorescence Differencel Gel Electrophoresis (DIGE, Amersham Biosciences) and MALDI-MS are established to identify changes on proteome level. A combination of these three methods (microarrays, DIGE and MS) paired with the application of time series experiments during a recombinant fermentation process provide an insight into the black box of cell metabolism and improve the understanding of cellular physiology.

Decreased expression of hypothalamic neuropeptides in Huntington disease transgenic mice with expanded polyglutamine‐EGFP fluorescent aggregates

Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the down-regulation of specific genes in this region of the brain.

Transcriptional Profiling Identifies Genes Differentially Regulated by the BCR/ABL Fusion Oncogene.

The translocation t(9;22) is associated with chronic myeloid leukemia (CML) and also occurs in 30% of adult acute lymphoblastic leukemia (ALL). In this study, we analyzed differential gene expression using microarrays to determine if upregulation or downregulation of specific genes may explain the distinct phenotypes. Enriched monoculear cells from 218 adult patients were hybridized to Affymetrix U133 set (A+B) microarrays (discovery set). Resulting lists of differentially expressed genes were further analyzed in an independent set of 110 patients hybridized to U133 Plus 2.0 microarrays (validation set). In a first analysis ALL with t(9;22) (n=34 discovery, n=6 validation) and CML (n=75 discovery, n=49 validation) were included. Various unsupervised data analysis algorithms, e.g. hierarchical clustering and principal component analysis, clearly separated both types of t(9;22) leukemias from each other. A supervised approach, i.e. t-test statistics followed by false discovery rate estimation, identified genes that were significantly differentially expressed. Using the top differentially expressed genes in a classification algorithm (SVM) >97% of the samples were correctly assigned to their classes, both in the discovery and the validation cohort. This set of genes was further examined by pathway analysis (Ingenuity software). Numerous networks point at clear biological differences between both t(9;22) types. Higher expressed genes in CML were connected to networks related to leukotriene metabolism, immune response, integrin signaling, non-selective vesicle transport, or humoral defense mechanisms. This reflects the underlying transcriptional profile of granulation of promyelocytes in CML in contrast to the non-granulated immature ALL blasts. The aggressiveness of acute leukemic blasts is visualized by several pathways where genes with higher expression in t(9;22) positive ALL were aggregated to networks with cellular functions of DNA metabolism and replication, cell cycle progression, and protein biosynthesis. Next analyses were performed to mine for common t(9;22) target genes. CML samples were compared against an equal number of AML with normal karyotype, and t(9;22) ALL against an equal number of c-ALL/Pre-B-ALL without t(9;22). Then both lists of differentially expressed genes were compared for overlapping probe sets. Here, no statistically significant differentially expressed genes were identified as consistently associated with the presence of t(9;22) across the two lineages. In contrast, using a similar strategy where ALL and AML with t(11q23)/MLL were grouped together and were analyzed against various non-MLL positive leukemia subtypes it is possible to identify common t(11q23)/MLL target genes, e.g. a overexpressed HOXA cluster gene signature. This leads to the hypothesis that both types of t(9;22) leukemias, despite an identical underlying chromosomal aberration, trigger different genes involved in BCR/ABL-dependent leukemogenesis. Thus, depending on the cellular background, i.e. myeloid or lymphoid, translocation t(9;22) results in two types of leukemias with fundamental differences in gene expression, clinical course, and the time and quality of response to therapy which is demonstrated also if a BCR/ABL-specific tyrosine kinase inhibitor (e.g. imatinib mesylate) is administered.

Molecular pathogenesis of uterine smooth muscle tumors from transcriptional profiling

Uterine smooth muscle tumors range from the very common benign leiomyoma to the uncommon, but frequently lethal, leiomyosarcoma. Morphological and clinical differences between these tumors are presumed to result from differences in gene expression. To test this hypothesis, RNAs from four normal uterine myometria, seven uterine leiomyomas, and nine uterine leiomyosarcomas were profiled using microarrays of oligonucleotides representing about 7,000 unique probe sets. RNAs whose levels distinguished any of the three sample types were selected by analysis of variance (ANOVA). The 153 (2.2% of the total) probe sets representing 146 unique genes with the highest test statistic selected for further analysis met minimum ratio and range thresholds between groups. Cluster analysis distinguished benign and malignant samples at the first node, and myometrium and leiomyoma were resolved in a secondary node. Downregulation of specific genes in uterine leiomyosarcoma was the most common pattern of differential gene expression selected by the three-way ANOVA. Four extrauterine leiomyosarcomas had profiles most similar to that of the uterine leiomyosarcomas. Functional analysis of the 146 genes did not reveal any strong biological theme. These genes were distributed throughout the genome, but there was slight overrepresentation of genes on 1p and 2q. These genes define a tumor signature for uterine smooth muscle neoplasia, and they suggest that the molecular pathways in leiomyoma and leiomyosarcoma are distinct.

Elevated expression of a subset of interferon inducible genes in primary bone marrow cells expressing p185 Bcr-Abl versus p210 Bcr-Abl by DNA microarray analysis

p185 Bcr-Abl has a more aggressive biological/clinical leukemia phenotype than p210 Bcr-Abl. In this study, we examined differential gene expression using microarrays to determine if upregulation or downregulation of specific genes may explain the distinct phenotypes produced by the two Bcr-Abl forms. RNA was collected from mouse bone marrow mononuclear cells expressing equivalent levels of p185 or p210, and the RNAs were subjected to microarray analysis. Significant differences in gene expression were observed on hierarchical clustering. A group of interferon-γ-inducible genes, including those encoding a family of 47 kDa GTPases, were significantly increased in p185 versus p210. This family of GTPases has previously been implicated in interferon-γ-induced resistance against intracellular pathogens, however their exact cellular functions are unknown. Our data suggest that their increased expression may contribute to the biological/clinical phenotype associated with p185.

Transcription factors and cancer: an overview

Cancer represents the out-of-control proliferation of a particular cell type, which originates with an unwanted mutation, followed by an accumulation of defects in many classes of genes. The two well-known types of primary genes that govern cell division and are responsible for cancer include: protooncogenes (gain-of-function) that serve as accelerators to activate the cell cycle, and tumor suppressor genes (loss-of-function) that serve as brakes to slow the growth of cells. There are now 17 known signal transduction pathways, plus at least two stress-response pathways ; all of these appear to be highly conserved in nematodes, flies and all vertebrates. Ultimately, transcription factors participate at the ends of all 19 pathways—by causing the up- or down-regulation of specific genes. All primary and modifier genes leading to cancer participate in one or another of these pathways. Innumerable exogenous and (autocrine and paracrine) endogenous signals bombard our cells each day and all are channeled through these 19 pathways, leading to the cell's response to these signals. Tumor progression represents a loss of normal cross-talk between cells, breakdown in communication between classes of genes, DNA methylation abnormalities, genetic instability, and hypermutability. Cancer is thus a multiplex phenotype: a crescendo of defects in hundreds if not thousands of genes, as a function of time, leading to an invasive and lethal disease.

In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels.

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.

Structural modifications of Methanococcus jannaschii flagellin proteins revealed by proteome analysis.

Methanococcus jannaschii is an autotrophic archaeon originally isolated from an oceanic thermal vent. The primary metabolic pathway for energy production in this hyperthermophilic microbe is methanogenesis from H2 and CO2. As an autotroph, M. jannaschii requires only CO2 as a carbon source for synthesizing all necessary biomolecules. Changes in the environmental availability of these molecules can be expected to activate regulatory mechanisms manifested as the up and down regulation of specific genes and the concomitant increase and decrease in abundance of the corresponding proteins. In our analysis of the proteome of M. jannaschii, we have observed significant changes in the abundance of a common subset of predominant proteins in response to reduced H2 concentration, limited ammonium availability, and the stage of cell growth (exponential compared with stationary). The masses of tryptic peptides from these proteins match those predicted by M. jannaschii genome open reading frames annotated as flagellin B1 (MJ0891) and flagellin B2 (MJ0892). Multiple proteins with different isoelectric points and molecular weights match each of these proteins, and the abundance of these protein variants changes with growth conditions. These data indicate that structural modifications altering both the isoelectric point and size of the M. jannaschii flagellin B1 and B2 proteins occur in response to growth conditions and growth stage of M. jannaschii and further suggest the regulation of M. jannaschii motility through structural modifications of the building blocks of the flagella.

Structural modifications of Methanococcus jannaschii flagellin proteins revealed by proteome analysis

Methanococcus jannaschii is an autotrophic archaeon originally isolated from an oceanic thermal vent. The primary metabolic pathway for energy production in this hyperthermophilic microbe is methanogenesis from H2 and CO2. As an autotroph, M. jannaschii requires only CO2 as a carbon source for synthesizing all necessary biomolecules. Changes in the environmental availability of these molecules can be expected to activate regulatory mechanisms manifested as the up and down regulation of specific genes and the concomitant increase and decrease in abundance of the corresponding proteins. In our analysis of the proteome of M. jannaschii, we have observed significant changes in the abundance of a common subset of predominant proteins in response to reduced H2 concentration, limited ammonium availability, and the stage of cell growth (exponential compared with stationary). The masses of tryptic peptides from these proteins match those predicted by M. jannaschii genome open reading frames annotated as flagellin B1 (MJ0891) and flagellin B2 (MJ0892). Multiple proteins with different isoelectric points and molecular weights match each of these proteins, and the abundance of these protein variants changes with growth conditions. These data indicate that structural modifications altering both the isoelectric point and size of the M. jannaschii flagellin B1 and B2 proteins occur in response to growth conditions and growth stage of M. jannaschii and further suggest the regulation of M. jannaschii motility through structural modifications of the building blocks of the flagella.

Structural modifications ofMethanococcus jannaschii flagellin proteins revealed by proteome analysis

Methanococcus jannaschii is an autotrophic archaeon originally isolated from an oceanic thermal vent. The primary metabolic pathway for energy production in this hyperthermophilic microbe is methanogenesis from H2 and CO2. As an autotroph, M. jannaschii requires only CO2 as a carbon source for synthesizing all necessary biomolecules. Changes in the environmental availability of these molecules can be expected to activate regulatory mechanisms manifested as the up and down regulation of specific genes and the concomitant increase and decrease in abundance of the corresponding proteins. In our analysis of the proteome of M. jannaschii, we have observed significant changes in the abundance of a common subset of predominant proteins in response to reduced H2 concentration, limited ammonium availability, and the stage of cell growth (exponential compared with stationary). The masses of tryptic peptides from these proteins match those predicted by M. jannaschii genome open reading frames annotated as flagellin B1 (MJ0891) and flagellin B2 (MJ0892). Multiple proteins with different isoelectric points and molecular weights match each of these proteins, and the abundance of these protein variants changes with growth conditions. These data indicate that structural modifications altering both the isoelectric point and size of the M. jannaschii flagellin B1 and B2 proteins occur in response to growth conditions and growth stage of M. jannaschii and further suggest the regulation of M. jannaschii motility through structural modifications of the building blocks of the flagella.

Structural Modifications of Methanococcus Jannaschii Flagellin Proteins Revealed by Proteome Analysis

Methanococcus jannaschii is an autotrophic archaeon originally isolated from an oceanic thermal vent. The primary metabolic pathway for energy production in this hyperthermophilic microbe is methanogenesis from H2 and CO2. As an autotroph, M. jannaschii requires only CO2 as a carbon source for synthesizing all necessary biomolecules. Changes in the environmental availability of these molecules can be expected to activate regulatory mechanisms manifested as the up and down regulation of specific genes and the concomitant increase and decrease in abundance of the corresponding proteins. In our analysis of the proteome of M. jannaschii, we have observed significant changes in the abundance of a common subset of predominant proteins in response to reduced H2 concentration, limited ammonium availability and the stage of cell growth (exponential compared with stationary). The masses of tryptic peptides from these proteins match those predicted by M. jannaschii genome open reading frames annotated as flagellin B1 (MJ0891) and flagellin B2 (MJ0892). Multiple proteins with different isoelectric points and molecular weights match each of these proteins and the abundance of these protein variants changes with growth conditions. These data indicate that structural modifications altering both the isoelectric point and size of the M. jannaschii flagellin B1 and B2 proteins occur in response to growth conditions and growth stage of M. jannaschii and further suggest the regulation of M. jannaschii motility through structural modifications of the building blocks of the flagella.

Inhibition of Neomycin Phosphorotransferase Expression in Entamoeba histolytica with Antisense Peptide Nucleic Acid (PNA) Oligomers

PNA are DNA analogs in which the entire phosphate-sugar backbone has been replaced by an isomorphic pseudopeptidic chain to which the four bases are linked. They have been shown to bind complementary natural polynucleotide sequences with higher affinity than their natural counterparts, partially due to the high flexibility and absence of charge of the artificial backbone. Furthermore, their artificial structure is not recognized by proteases and nucleases, making PNA exceptionally stable in biological environments (1). This consideration is of particular relevance to Entamoeba histolytica , as it possesses numerous hydrolases, some membrane bound. PNA oligomers have been used in the last few years as antisense and antigene agents in a variety of cell-free and cellular systems, showing that they can effectively and selectively downregulate gene expression at the translational or transcriptional levels when used at micromolar concentrations (2). The difficulties inherent in the cultivation of E. histolytica trophozoites and the lack of procedures for deletion of genes have made it very difficult to perform classical genetic studies aimed at the characterization of phenotypes resulting from gene inactivation. Only recently, antisense strategies using plasmids transcribing antisense mRNAs have been successfully used, allowing the downregulation of specific genes and the analysis of resulting phenotypes (3). In this study, we report the use of PNA oligomers as antisense agents for effective and specific downregulation of expression of the bacterial gene neomycin phosphorotransferase (NPT) in E. histolytica in culture, with results comparable to antisense strategies recently reported. In contrast to other cellular types tested, notably mammalian cells, E. histolytica is permeable to unmodified PNA, and inhibition of gene expression is significant and easily achieved in a dose-dependent manner, saving the work of cloning, transfection, and selection procedures needed for plasmid-mediated antisense inhibition.