Use Of mRNA Differential Display Research Articles

Pancreatic cholesterol esterase, ES-10, and fatty acid ethyl ester synthase III gene expression are increased in the pancreas and liver but not in the brain or heart with long-term ethanol feeding in rats.

Chronic alcohol consumption predisposes susceptible individuals to both acute and chronic pancreatitis. Our hypothesis was that alcohol increases the risk of pancreatitis by disrupting defense mechanisms and/or enhancing injury-associated pathways through altered gene expression. Hence, we studied the expression of pancreatic genes in rats chronically exposed to ethanol. Male Wistar rats were pair-fed liquid diets without and with ethanol for 4 weeks. Total RNA was extracted from rat pancreas and other organs. The mRNA expression patterns among pancreatic samples from ethanol-fed rats and controls were compared with use of mRNA differential display. The differentially expressed cDNA tags were isolated, cloned, and sequenced. One cDNA tag that was overexpressed in the pancreas showed 99% sequence homology to a rat pancreatic cholesterol esterase mRNA (CEL; Enzyme Commission number [EC] 3.1.1.13). The differential expression was confirmed by realtime PCR. Gene expression was also increased in the liver but not in the heart or brain of the alcohol-fed rats. Because CEL has fatty acid ethyl ester (FAEE)-generating activity and FAEEs play a major role in acute alcoholic pancreatitis, we determined the expression of other genes encoding for FAEE-generating enzymes and showed similar organ-specific expression patterns. Our results demonstrate that chronic ethanol consumption induced expression of FAEE-related genes in the pancreas and liver. This upregulation may be a central mechanism leading to acinar cell injury.

Use of mRNA differential display to study the action of lymphocyte subsets in vivo and application to a murine model of herpes simplex virus infection

Depletion of lymphocyte subsets in vivo using monoclonal antibodies against cell surface markers has helped to define the roles for these subsets in many immune processes. However, in some cases the mechanisms through which these lymphocytes act remain partially elucidated or completely unknown. A new approach to these biological problems is the use of transcriptional analyses to find mRNAs whose abundance in tissues is altered by depletion of lymphocyte subsets. We have verified the use mRNA differential display (DD) for this purpose and applied it in a study of CD8 + lymphocyte mediated clearance of herpes simplex virus (HSV) from the nervous systems of experimentally infected mice. The results of the differential displays and characterisation of a large mRNA identified using this strategy are presented.

Use of mRNA differential display to identify 3 genes involved in adaptation to injury in the stomach

Use of mRNA differential display to identify 3 genes involved in adaptation to injury in the stomach

Down-regulation of DPH2L gene during cellular differentiation /apoptosis: Use of mRNA differential display

To characterize the genes associated with differentiation/apoptosis induced by all-trans retinoic acid (ATRA) in human lung cancer cells, mRNA differential display was employed. Six cDNA fragnents have been isolated, and one of them corresponds to a sequence-known gene DPH2L with unknown function, which was first isolated from the critical region of deletion on chromosome 17p13.3 in human ovarian carcinoma, and regarded as a candidate tumor suppressor gene. Results show that DPH2L is a wide expressed gene, and has another major transcript besides the previously reported 2.3 kb transcript. It is proved that the DPH2L gene is down-regulated during differentiation or apoptosis in several kinds of cancer cells induced by all-trans retinoic acid and N-(4-hydroxyphenyl) retinamide (4HPR). This may suggest that DPH2L does not play a role as a tumor suppressor gene, on the contrary, its down-regulation may be functionally involved in the transition from cell growth to differentiation or apoptosis.

Molecular cloning of a novel heat induced/chilling tolerance related cDNA in tomato fruit by use of mRNA differential display.

Chilling injury was circumvented by heat-treating mature green tomatoes (Lycopersicon esculentum, cv. Mountain Springs) at 42 degrees C for two days prior to storing them at 2 degrees C for one or two weeks, whereas fruits stored at 2 degrees C without preheating developed typical chilling injury symptoms and failed to ripen at 20 degrees C. Using mRNA differential display and screening of the cDNA libraries, we have cloned from tomato fruit a full-length HCT1 cDNA (heat induced/chilling tolerance related). The protein ( 17.6 kDa) predicted from coding region of HCT1 cDNA has high identity with class II cytosolic small HSPs. The gene corresponding to HCT1 cDNA was termed as LeHSP 17.6. Southern-blot hybridization indicates that LeHSP 17.6 belongs to a two-member gene family. Northern blot analysis indicates the heat-induced transcript of the LeHSP 17.6 remains up-regulated during subsequent exposure of the fruit to chilling temperatures for at least one week and upon transfer to ripening temperatures for one day. Fruits which were only chilled show a low level of expression of the LeHSP 17.6 transcript. We hypothesize that LeHSP 17.6 may be involved in protecting the cell from metabolic dysfunctions leading to ripening failure caused by chilling injury. This is the first report of a class II cytosolic smHSPs encoding gene in tomato.

Analysis of gene expression during hepatocarcinogenesis: use of mRNA differential display

Analysis of gene expression during hepatocarcinogenesis: use of mRNA differential display

The use of mRNA differential display for discovery of novel therapeutic targets in cardiovascular disease.

Recent advances in molecular biology techniques have provided powerful tools for discovery of novel genes relevant to both biological and pathological processes. mRNA differential display is an emerging technique for novel gene discovery and it has been successfully applied to many physiological and pathological conditions including normal development, cell differentiation, cancer, cardiovascular disease, inflammation and CNS disorders. In the present work, we briefly illustrate the critical procedure and highlight most recent technical improvements and modifications of this technology. Based upon the successful applications of this technique in cardiovascular research, it may provide a valuable and powerful investigational tool for the identification of novel therapeutic targets in cardiovascular diseases.

A novel gene (drad-1) expressed in hematopoiesis-supporting stromal cell lines, ST2, PA6 and A54 preadipocytes: use of mRNA differential display.

We established a differentiation-inducible preadipocyte cell line, designated A54 preadipocytes, from C3H10T1/2 (10T1/2) mouse embryo fibroblasts. A54 preadipocytes had marked hematopoiesis-supporting ability in vitro but this ability was lost after terminal differentiation to adipocytes. In this study, to identify molecules that contribute to the hematopoiesis-supporting ability of A54 preadipocytes, we screened genes that were differentially expressed in A54 preadipocytes and isolated seven novel genes by reverse transcriptase polymerase chain reaction mRNA differential display. An RNase protection assay confirmed that one of these genes was expressed at high levels in parent 10T1/2 cells and A54 preadipocytes but to a much lesser extent in fully differentiated A54 adipocytes. This gene was defined as a gene that was downregulated during adipocyte differentiation-1 (drad-1). The size of drad-1 mRNA was 8.2 kb, and the gene was expressed in other mouse preadipocytes, namely, ST2 and PA6 cells, that have hematopoiesis-supporting ability. Moreover, drad-1 was also found to be expressed in bone marrow in vivo. The function of the protein encoded by drad-1 is unknown, but the expression of the gene may be useful as a molecular marker of adipocyte differentiation.

T11 2:15 Use of mRNA differential display to identify genes involved in oyster immunity

T11 2:15 Use of mRNA differential display to identify genes involved in oyster immunity

Analysis of upregulated cellular genes in pseudorabies virus infection: use of mRNA differential display.

Virus infection usually alters the host cell and shuts off the synthesis of cellular macromolecules. In order to screen the upregulated cellular transcripts during pseudorabies virus (PRV) infection, we employed the mRNA differential display technique. The screen is based on positive selection at the mRNA level for genes expressed in normal cells but increased in corresponding PRV-infected cells. Over 14000 species of mRNA, isolated from mock-infected and PRV-infected Madin-Darby bovine kidney cell at 1 h post infection, were screened, and 40 candidate clones were recovered. Southern blot analysis revealed that 17 out of 40 candidate clones, were enhanced in PRV-infected cells. Partial DNA sequences demonstrated that 17 clones were distinct cellular genes, including those encoding the modulators of signal transduction (saposin, 14-3-3, adenylate kinase, adenylyl cyclase, protein kinase C-alpha), those encoding the components of translation (fau, ribosomal proteins S11, L31, L36), other cellular genes (peptidase, cyclin E, rch1, oligo-C-rich single-stranded nucleic acid binding protein, rap, arginyl-tRNA synthetase), and two unknown genes. Thus, this study identifies successfully the transcriptionally regulated cellular genes which are associated with PRV infection. Furthermore, this study provides support for the use of mRNA differential display as a method to rapidly isolate differentially expressed genes in virus infection.

Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus.

Persistent infection of the chestnut blight fungus Cryphonectria parasitica with the prototypic hypovirus CHVI-713 results in attenuation of fungal virulence (hypo-virulence) and reduced accumulation of the GTP-binding (G) protein a subunit CPG-1. Transgenic cosuppression of CPG-1 accumulation in the absence of virus infection also confers hypovirulence. We now report the use of mRNA differential display to examine the extent to which virus infection alters fungal gene transcript accumulation and to assess the degree to which modification of CPG-1 signal transduction contributes to this alteration. More than 400 PCR products were identified that either increased (296 products) or decreased (127 products) in abundance as a result of virus infection. Significantly, 65% of these products exhibited similar changes as a result of CPG-1 cosuppression in the absence of virus infection. We also report that both virus infection and CPG-1 cosuppression elevate cAMP levels 3- to 5-fold. Additionally, it was possible to mimic the effect of virus infection and CPG-1 cosuppression on transcript accumulation for representative fungal genes by drug-induced elevation of cAMP levels. These results strengthen and extend previous indications that hypovirus infection causes a significant and persistent alteration of fungal gene expression/transcript accumulation. They further show that this alteration is primarily mediated through modification of the CPG-1 signaling pathway and suggest that, similar to mammalian Gi alpha subunits, CPG-1 functions as a negative modulator of adenylyl cyclase. Finally, these results suggest a role for G-protein-regulated cAMP accumulation in hypovirus-mediated alteration of fungal gene expression.

Analysis of gene expression in the preimplantation mouse embryo: Use of mRNA differential display

Analysis of gene expression in the preimplantation mouse embryo: Use of mRNA differential display

Analysis of gene expression in the preimplantation mouse embryo: use of mRNA differential display.

The analysis of differential gene expression during preimplantation embryogenesis has been hindered by the paucity of biological material. We report modifications of the recently described mRNA differential display method (Liang, P. & Pardee, A. B. (1992) Science 257, 967-971) to analyze differential gene expression during mouse preimplantation development. The method detects the appropriate changes in the temporal pattern of expression of an amplicon that by DNA sequence analysis is the cytokeratin endo A, a gene whose temporal pattern of expression has been previously determined by S1 nuclease digestion. In addition, this method identifies amplicons that likely represent genes (i) that encode maternal mRNAs, (ii) that are products of early and late zygotic gene activation, (iii) whose expression is greatest during the eight-cell stage (i.e., expressed in a stage-specific manner), and (iv) whose expression is greatest in the blastocyst. In addition to endo A, sequence analysis of these amplicons reveals that an amplicon that displays a temporal pattern of expression consistent with it being a maternal mRNA is the alpha subunit of the mitochondrial F1 ATP synthase.