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Results for “differential expression”

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PubMed2001

[cDNA expression array in the differential expression profiles of p53 regulated genes in nasopharyngeal carcinoma and the human normal nasopharynx].

OBJECTIVE: To compare the gene expression map of nasopharyngeal carcinoma (NPC) tissue with that of the control tissue by cDNA array and to discuss possible reasons of TP53 accumulation in NPC tissue. METHODS: After the hybridization of Atlas human cancer cDNA expression array 7742-1, analysis of Atlas arrays by means of AtlasImage 1.01a was carried out. Then, the results of array were verified by reverse transcription-polymerase chain reaction (RT-PCR). Gene expression alteration on the protein level was verified by immunohistochemistry. RESULTS: 134 of 588 tumor-related genes were upregulated and 88 downregulated. Thirteen of 32 p53-regulated genes showed differential expression with 11 upregulated and 2 downregulated. CONCLUSION: (1) p53 dysfunction exists in NPC tissues, (2) MDM2, p21 and Bax may be involved in the regulation of nasopharyngeal carcinoma cell growth.

DNA, Complementary↗
PubMed2000

The rpl16a gene for ribosomal protein L16A identified from expressed sequence tags is differentially expressed during sexual development of Aspergillus nidulans.

We obtained 305 expressed sequence tags (ESTs), which are from the poly(A) site to the most proximal MboI site, from mycelia at the early sexual developmental (ESD) stage of Aspergillus nidulans. By comparison of these ESTs with those obtained previously from the vegetative stage and from the late sexual developmental stage followed by Northern blot analyses, genes of 17 ESTs were identified as being expressed more abundantly at the ESD stage than at the vegetative stage. Five of 17 genes were expressed more abundantly in the presence of the veA gene or the nsdD gene, suggesting that these 5 genes may be involved in sexual development. In a gene of one EST, appearing three times among 305 ESTs and identified by GenBank, polyadenylation seemed to occur at two sites. Nucleotide sequences of the gene having the EST and its cDNA revealed that the gene can code for a 202-amino-acid polypeptide with an estimated molecular mass of 23 kDa. The deduced amino acid showed 73% identity to Saccharomyces cerevisiae ribosomal protein L16A (RPL16A), and therefore the gene was named rpl16a. A. nidulans RPL16A had a putative leucine zipper motif and a basic leucine zipper motif like those of other organisms. The expression level of the rpl16a gene, present as a single copy in this organism, reached a maximum after 2 h, decreased thereafter, and increased again 30 to 50 h after the end of induction of sexual development. These results clearly indicated that the rpl16a gene is expressed differentially during sexual development.

Amino Acid Sequence↗
PubMed1994

The retinoic acid and retinoid X receptors are differentially expressed during myoblast differentiation.

Retinoids (all trans and 9-cis retinoic acid) are pleiotropic regulators of cell fate, and have been shown to regulate the expression of helix loop helix transcription factors (e.g MyoD, myogenin and Myf-5) that control myogenic differentiation. The effects of retinoids are mediated through the ligand dependent retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We have found that the messenger RNA transcripts encoding RAR are repressed during differentiation of C2C12 myoblasts while, conversely, RXR mRNA transcripts are induced in C2C12 myotubes. These results imply that RXRs, play a major regulatory role in differentiated muscle.

Animals↗
PubMedDate not supplied

[A new method of comparative analysis of gene expression and identification of differentially expressed mRNA].

A novel method of comparative gene expression analysis is proposed which is based on representing a population of expressed mRNA sequences in the form of a set of discrete cDNA restriction fragments. By means of selective isolation of 3' terminal cDNA fragments, each mRNA species is represented by no more than one cDNA fragment of specific length and sequence. Populations of cDNA fragments from different cell types are separated by high-resolution gel electrophoresis, and the separation patterns are compared. Using the proposed approach, fragments of two genes differentially expressed in murine thymus and spleen were identified and cloned. One of the genes was found to encode the terminal deoxynucleotidyltransferase, the there is apparently a heretofore unknown gene.

Animals↗
PubMed1983

Thyrotrophin and the differential expression of proliferation and differentiation in dog thyroid cells in primary culture.

Cell proliferation and the expression of differentiated functions are generally considered to be mutually exclusive states of the cell. Thyrotrophin activates the expression of differentiated functions in dog thyroid cells by means of cyclic AMP. We have recently shown that thyrotrophin also acts through the same intracellular signal molecule to enhance proliferation of dog thyroid cells in primary cultures. In this work we showed that such primary cultures exhibit three successive phases: a latency period during which the expression of differentiated functions (iodide trapping and organification) declined, the cells still being associated with structures derived from the seeded follicles; a cell-proliferation phase with little expression of these functions, the cells being spread in a monolayer; a stationary phase with cell density reaching a plateau but no re-expression of the functions. Thyrotrophin promoted proliferation during the multiplication phase, but induced redifferentiation during the stationary phase. These effects were mimicked by cholera toxin and dibutyryl cyclic AMP, which suggested that they were mediated by cyclic AMP. Iodide uptake was also stimulated by cortisol. Thyrotrophin therefore has a different action in dog thyroid cells depending on the state of the cells. The spontaneous arrest of multiplication appears to make the cells competent to respond to thyrotrophin by the induction of redifferentiation.

Animals↗
PubMed2000

Generation of patterns from gene expression data by assigning confidence to differentially expressed genes.

MOTIVATION: A protocol is described to attach expression patterns to genes represented in a collection of hybridization array experiments. Discrete values are used to provide an easily interpretable description of differential expression. Binning cutoffs for each sample type are chosen automatically, depending on the desired false-positive rate for the predictions of differential expression. Confidence levels are derived for the statement that changes in observed levels represent true changes in expression. We have a novel method for calculating this confidence, which gives better results than the standard methods. Our method reflects the broader change of focus in the field from studying a few genes with many replicates to studying many (possibly thousands) of genes simultaneously, but with relatively few replicates. Our approach differs from standard methods in that it exploits the fact that there are many genes on the arrays. These are used to estimate for each sample type an appropriate distribution that is employed to control the false-positive rate of the predictions made. Satisfactory results can be obtained using this method with as few as two replicates. RESULTS: The method is illustrated through applications to macroarray and microarray datasets. The first is an erythroid development dataset that we have generated using nylon filter arrays. Clones for genes whose expression is known in these cells were assigned expression patterns which are in accordance with what was expected and which are not picked up by the standards methods. Moreover, genes differentially expressed between normal and leukemic cells were identified. These included genes whose expression was altered upon induction of the leukemic cells to differentiate. The second application is to the microarray data by Alizadeh et al. (2000). Our results are in accordance with their major findings and offer confidence measures for the predictions made. They also provide new insights for further analysis.

Algorithms↗
PubMed1998

Identification of genes differentially expressed in nasopharyngeal carcinoma by messenger RNA differential display.

We have applied the mRNA differential display method to compare and analyze mRNAs prepared from five normal nasopharyngeal epithelial cell cultures and five nasopharyngeal carcinoma cell lines. A total of 24 differential display experiments was performed using different combinations of PCR primers. Sixty-nine cDNA fragments differentially expressed in either normal or malignant nasopharyngeal epithelial cells were identified. Subsequent cloning and sequencing of these differentially expressed cDNA fragments resulted in the identification of seventeen distinct sequences. Seven of these sequences were shown to be novel cDNA sequences not previously reported. Ten of the remaining cDNA fragments showed sequence homology to previously reported genes. Differential expression of four of these seventeen cDNA fragments in normal nasopharyngeal epithelial cells was confirmed by reverse Northern hybridization. One of these cloned cDNA fragments is a novel cDNA sequence while the other three matched to previously reported cDNA sequences involved in cell growth and migration. Homologous sequences identified to be differentially expressed in normal nasopharyngeal epithelial cells in this study are: human 26 kDa cell surface protein (TAPA-1) mRNA, NF-E2 like basic leucine zipper transcriptional activator and the human bullous pemphigoid antigen. The mRNA differential display is a useful tool to identify candidate genes involved in the pathogenesis of nasopharyngeal carcinoma.

Base Sequence↗
PubMed1995

ErbB3 and ErbB2/neu mediate the effect of heregulin on acetylcholine receptor gene expression in muscle: differential expression at the endplate.

Motor neurons modulate acetylcholine receptor (AChR) gene expression in skeletal muscle by two signalling pathways: the transmitter-evoked depolarization of muscle membrane inhibits AChR gene transcription throughout the myofibre presumably via activation of a serine/threonine kinase, while the transcription rates of AChR genes in the synaptic region are increased by nerve-derived trophic factors including AChR-inducing activity (ARIA). To gain further insight into these interactions we characterized the receptor for heregulin (HRG)/ARIA in muscle. We showed that HRG increases AChR alpha-subunit mRNA levels via tyrosine phosphorylation of ErbB3 and ErbB2/neu in myotubes. The protein tyrosine phosphatase inhibitor, pervanadate, potentiated the responses to HRG that were in turn blocked by the tyrosine kinase inhibitor erstatin, indicating the relevance of tyrosine phosphorylation to these events. The effects of HRG were inhibited by enhanced cellular serine/threonine phosphorylation which has been implicated in the repression of AChR genes by electrical activity. Immunocytochemical analysis of adult rat muscle revealed that while ErbB2/neu is present throughout the entire surface of the myofibre membrane, ErbB3 expression is exclusively restricted to the endplate suggesting its involvement in synapse-specific transcription of AChR genes by HRG/ARIA.

Animals↗
PubMed2004

Athero-express: differential atherosclerotic plaque expression of mRNA and protein in relation to cardiovascular events and patient characteristics. Rationale and design.

In clinical practice, biological markers are not available to routinely assess the progression of atherosclerotic disease or the development of restenosis following endarterectomy or catheter based interventions. Endarterectomy procedures provide an opportunity to study mechanisms of restenosis and progression of atherosclerotic disease since atherosclerotic tissue is obtained. Athero-Express is an ongoing prospective study, initiated in 2002, with the objective to investigate the etiological value of plaque characteristics for long term outcome. Patients are included who undergo an endarterectomy of the carotid artery. At baseline blood is withdrawn, patients fill in an extensive questionnaire and diagnostic examinations are performed. Atherosclerotic plaques are freshly harvested, immunohistochemically stained and examined for the presence of macrophages, smooth muscle cells, collagen and fat. Parts of the atherosclerotic plaques are freshly frozen to study protease activity and protein and RNA expressions. Patients undergo a duplex follow up to assess procedural restenosis (primary endpoint) at 3 months, 1 year and 2 years. Secondary endpoints encompass major adverse cardiovascular events. In the future, the creation of this biobank with atherosclerotic specimen will allow the design of cross-sectional and follow up studies with the objective to investigate the expression of newly discovered genes and proteins and their interaction with patients and plaque characteristics in the progression of atherosclerotic disease. Objective is to include 1000-1200 patients in 5 years. In January 2004, 289 patients had been included. It is expected that 250 patients will be included yearly.

Arteriosclerosis↗
PubMed1998

The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display.

In many applications, an understanding of differentially expressed genes in different tissues or owing to an applied stimulus is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR [RAP-PCR] and differential display [DDRT-PCR]) has shown that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR, a technique has recently been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application. We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR) is a reliable and useful modification of available differential expression methods.

Cells, Cultured↗
PubMed1998

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR.

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.

Cloning, Molecular↗
PubMed2004

Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages.

We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.

Adjuvants, Immunologic↗
PubMed1998

Recovering and reamplifying of the differentially expressed cDNA bands isolated from mRNA differential display. A modified method.

Methods for retrieving and reamplifying the differentially expressed cDNA bands have been modified. Direct reamplification of differentially expressed bands after cutting from a polyacrylamide gel (PAG) followed by a simple rinse and crush step has proved to be more convenient and effective than the traditional glycogen-precipitation method. Combination of 30 cycles of differential display (DD) polymerase chain reaction (PCR) and 20 cycles of standard PCR reaction also yielded higher reamplification rates.

DNA, Complementary↗
PubMed2002

Classification of genes differentially expressed during water-deficit stress in Arabidopsis thaliana: an analysis using microarray and differential expression data.

Many changes in gene expression occur in response to water-deficit stress. A challenge is to determine which changes support plant adaptation to conditions of reduced soil water content and which occur in response to lesions in metabolic and cellular functions. Microarray methods are being employed to catalogue all of the changes in gene expression that occur in response to specific water-deficit conditions. Although these methods do not measure the amount or activities of specific proteins that function in the water-deficit response, they do target specific biochemical and cellular events that should be detailed in further work. Potential functions of approx. 130 genes of Arabidopsis thaliana that have been shown to be up-regulated are tabulated here. These point to signalling events, detoxification and other functions involved in the cellular response to water-deficit stress. As microarray techniques are refined, plant stress biologists will be able to characterize changes in gene expression within the whole genome in specific organs and tissues subjected to different levels of water-deficit stress.

Adaptation, Physiological↗
PubMed2003

Vesicle-associated protein-A is differentially expressed during intestinal smooth muscle cell differentiation.

Gastrointestinal (GI) smooth muscle diseases represent a major health concern affecting in excess of 2 million people each year. Little is currently known regarding the molecular mechanisms controlling either normal or pathogenic GI smooth muscle development. In an effort to identify the specific gene products responsible for modulating GI smooth muscle cell (SMC) differentiation, we performed differential display on distinct intestinal SMC (ISMC) phenotypes. This analysis identified over 40 unique transcripts that appeared to be differentially expressed in distinct SMC phenotypes. One such transcript that appeared to be preferentially expressed in immature smooth muscle myocytes was identified as vesicle-associated membrane protein, associated protein A (VAP-A). Northern blot analysis confirmed that VAP-A was expressed threefold higher in immature smooth muscle myocytes when compared with both smooth muscle myoblasts and mature smooth muscle myocytes. VAP-A mRNA was differentially expressed during normal rat development and showed peak levels of expression in the intestine during late embryogenesis and early neonatal development. These observations provide the first evidence that VAP-A-mediated membrane trafficking may play an important role in modulating ISMC differentiation.

Amino Acid Sequence↗
PubMed2000

Differential expression of laminin-5 subunits during incisor and molar development in the mouse.

Rodent incisors are continuously growing teeth and enamel deposition is restricted to the labial side. In the present study, the expression of laminin-5 subunits (alpha3, beta3 and gamma2) has been analyzed by in situ hybridization in developing mouse lower incisors and compared to that reported in the molar. At the bud stage (E12), mRNAs for all subunits were detected in the whole epithelial thickening. At E14, when histogenesis had started, transcripts for alpha3 and gamma2 subunits were restricted to the outer dental epithelium (ODE), whereas the beta3 subunit was intensely expressed in the inner dental epithelium (IDE). A transient expression for alpha3 subunit was seen in the enamel knot area and disappeared at E15. Subsequently, all laminin-5 subunit genes were re-expressed in differentiating ameloblasts on the labial side. Similar patterns of transcription were observed in incisor and molar, suggesting that the differential expression of laminin-5 subunits in the IDE might be involved in the histogenesis of the IDE and ameloblast differentiation. At E16.5, cells of the IDE at the anterior extremity of the incisor and in the anterior part of the lingual IDE expressed transcripts for alpha3 and beta3 but not for gamma2 subunit. Similar expression patterns were observed in the enamel-free areas of the E18 molar. This specific expression might thus be related to cells that do not differentiate as functional ameloblasts. Throughout incisor development, intense expression for all laminin-5 subunits was restricted to the labial side of the cervical loop. The asymmetrical expression of laminin-5 might be related to incisor morphogenesis and to the differences in histogenesis and cytodifferentiation of the IDE that exist in the labial versus lingual aspect of the cervical loop.

Animals↗