Differential Display Reverse Transcription-polymerase Chain Research Articles

Embryotrophic factor-3 from human oviductal cells affects the messenger RNA expression of mouse blastocyst.

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 x BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03-3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2beta, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

Search for genes potentially related to germ tube formation in Candida albicans by differential-display reverse transcription polymerase chain reaction.

Candida albicans exhibits the ability to grow in either a yeast or a mycelia form in response to different environmental factors. The mycelia form, found in infected tissues, is important as a virulence factor in the adherence of the organism to the host epithelium. In vitro, the morphological transition can be induced by environmental shifts in the growing conditions, or by a variety of exogenous factors, including ambient pH, nutritional status and temperature. The differential-display reverse transcription polymerase chain reaction (DDRT-PCR) is a powerful technique for comparing gene expression between cell types, stages of development or differentiation. Hyphae related genes were identified and characterized using a PCR-based differential display. Candida albicans formed a germ tube when cultured in rabbit serum, RPMI 1640 medium or 39 degrees C-YPD medium. We gained 21 cDNA bands showing a different expression pattern from that of the uninduced culture. DNA was extracted from the same location of the isolated bands, and PCR was performed under the same conditions, which reamplified the PCR product, showing the specific expression patterns according to the culture conditions. We cloned 18 germ tube-related cDNA clones (inserts average size is 80 - 700 bp) and sequenced them. The nucleotide sequences of the 18 clones were identified through in the present study from GenBank, and were found to have the accession number (AF405213-AF405230). We could not find any nucleotide sequence having a high homology with these clones. This study could form a part of the projects in the search for genes related to the germ tube formation of C. albicans.

Identification and characterization of cytopathogenic Mycoplasma hyorhinis from swine farms with a history of abortions.

A virus-like cytopathic agent isolated from swine farms with a history of recurrent abortion episodes was investigated. We employed a differential display reverse transcription-polymerase chain reaction (ddRT-PCR) to obtain genetic information of the cytopathic agent. Partial nucleotide sequence (527 bp) obtained from differentially displayed PCR fragments showed 88.7% similarity with the 23S rRNA gene of Mycoplasma hyopneumoniae. Unexpectedly, the 5' portion (1-333 bp) of the sequence shared 96.1% similarity with 5' untranslated region (UTR) of human prostate tumor inducing gene 1 (PTI-1). Cytopathic effects and extranuclear DNA fluorescence were no longer observed when BM-cyclin was added in the culture medium, suggesting that BM-cyclin sensitive mycoplasma-like organisms caused the cell death. Further evidence supporting the cytopathic agent as a mycoplasma-like organism was obtained by the capability of (3)H-thymidine and (3)H-uridine incorporation, a single peak in buoyant density gradient profile (1.20-1.24 g/ml), and ultrastructural morphology. Unlike M. hyopneumoniae, the organism was not propagated in Friis medium. Nucleotide sequence of 16S rRNA obtained from the cytopathic agent showed 0.8-1.0% divergences with other M. hyorhinis strains, suggesting that the newly isolated cytopathogenic swine mycoplasma was a variant form of M. hyorhinis. Striking homology between a portion of the 23S rRNA gene of M. hyorhinis and 5' UTR of human PTI-1 implicated that M. hyorhinis might potentially be related to the evolution of human PTI-1.

Mss4 gene is up-regulated in rat brain after chronic treatment with antidepressant and down-regulated when rats are anhedonic.

Differential display reverse transcription-polymerase chain reaction was used to identify mRNAs that are differentially expressed in the brain of rats treated chronically with the reference tricyclic antidepressant, imipramine, in comparison with control rats. The gene encoding for a mutation suppressor for Sec4-8 yeast (Mss4) transcript is overexpressed in the amygdala of treated rats after 3 weeks of daily administration. This overexpression is also found in the hippocampus of rats treated chronically with either tianeptine or fluoxetine. Mss4 protein has the properties of a guanine nucleotide exchange factor, interacting with several members of the Rab family implicated in Ca(2+)-dependent exocytosis of neurotransmitters. Mss4 was also overexpressed in other brain structures as judged by in situ hybridization. The kinetics of the up-regulation of Mss4 gene expression measured by Northern blot during the imipramine, tianeptine, or fluoxetine treatments are consistent with an antidepressant effect that occurs after 3 weeks. In rats in which anhedonia was induced by chronic mild stress during 3 weeks, Mss4 transcripts were specifically down-regulated in hippocampus and amygdala compared with control rats. It is proposed that Mss4 protein, which stimulates exocytosis in vivo, participates in the potentiation of the activity of neurotransmitter pathways implicated in the action of several antidepressants and constitutes one of the common functional molecules induced after chronic antidepressant treatment.

Amino Acid Deficiency Up-regulates Specific mRNAs in Murine Embryonic Cells

The flow of amino acids to both protein and DNA synthesis is particularly important during periods of rapid cell proliferation such as the fetal stages of life. The changes in mRNA levels caused by the different types of growth arrest were studied in F9 embryonal carcinoma cells. The cells were grown in medium deficient in the amino acid lysine or in one containing phosphonoacetyl L-aspartic acid (PALA), which inhibits the incorporation of aspartic acid into pyrimidine nucleotides. A number of mRNAs known to be elevated in growth arrested cells (gas and gadd) were studied by Northern blotting. Samples of RNA from the cells were also compared by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The results showed that lysine deficiency increased the steady-state levels of a number of mRNAs by 5- to 40-fold. In contrast, the changes in cells treated with PALA were much smaller and less pronounced. Amino acid deficiency induced the mRNAs coding for gadd153 (CHOP-10), gas5, the mouse doublesex-related gene (Dmrt1) and the polyamine modulated factor (PA-1) as well as a number of unidentified expressed sequence tags (EST). These mRNAs were all induced within 24 h of exposure to amino acid deficiency. These very different transcriptional responses may be important in understanding the interactions between protein quantity and quality in different physiologic situations.

Paraquat-induced gene expression in rat lung tissues using a differential display reverse transcription-polymerase chain reaction.

Increased formation of reactive oxygen species is a cause of paraquat (PQ)-induced injury and also provides a link between the signaling pathways and transcriptional events that regulate the expression of a large number of genes. However, the molecular mechanisms involved in PQ-induced injury remain unclear. To investigate the changes in gene expression at the onset of PQ injury, we used the differential display-polymerase chain reaction (PCR) method. Rats were treated intraperitoneally with 20 mg/kg PQ, and after 3 h the lungs were immediately excised. Samples of mRNA from normal and treated rats were used to prepare radiolabeled cDNAs, which were electrophoresed. Then the transcription levels were compared. We isolated 26 fragments of cDNA that were potentially affected by PQ, and determined their nucleotide sequences. Six clones of interest were selected and analyzed further. The reverse transcript-PCR based on their sequence information confirmed the differential expression for five clones: four clones were up-regulated and one was down-regulated. We were particularly interested in two genes that had homology with the known gene: TATA box-binding protein-associated factor, RNA polymerase II, B, 150 kDa (TAFIIB), and a candidate gene for lipodystrophy, Lpin2. Both genes were significantly up-regulated within 3 h of PQ intake and the stimulation continued during our 24-h observation period. In addition, up-regulation of Lpin2 was observed in the lungs, but not in the liver and kidneys. In situ hybridization using lung sections showed that the expression of both genes was strongly visualized in Clara cells and in alveolar macrophages. These findings suggest a stimulation of transcription levels and changes in lipid metabolism in Clara cells and in macrophages in the lungs, which result in their playing a crucial role at the onset of PQ-driven pulmonary injury.

Rab7 gene is up-regulated by cholesterol-rich diet in the liver and artery

To identify genes responding to the cholesterol-rich diet, differentially expressed hepatic genes have been searched from a diet-induced hypercholesterolemic rabbit by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Among the many screened genes, Rab7 gene was shown to be distinctively up-regulated in response to the cholesterol-loading into the rabbit. To visualize the location of elevated Rab7 expression in tissues, patterns of the gene expression were monitored within hepatic and aortic tissues by in situ hybridization and immunohistochemistry. The expression of Rab7 was obviously increased in the hepatic tissues, especially in the endothelial cells and hepatocytes around central veins of the high cholesterol-fed rabbit, compared to the tissues from rabbit fed a normal diet. To find out a potential relationship between the Rab7 and the atherogenesis, the same experiments were conducted with the atherosclerotic plaques obtained from rabbit and human. The elevated expression of Rab7 gene was clearly evident in both tissues, suggesting that the Rab7 may be involved in the process of atherogenesis.

Activity-dependent expression of calbindin in rabbit floccular Purkinje cells modulated by optokinetic stimulation

Optokinetic stimulation activates visual climbing fiber pathways that synapse upon contralateral floccular Purkinje cells. Long-term horizontal optokinetic stimulation causes a progressive decrease in gain of the optokinetic reflex and leads to the subsequent genesis of a prolonged negative optokinetic afternystagmus. Since the flocculus is involved in adaptation to optokinetic stimulation, we used the technique of differential display reverse transcription-polymerase chain reaction to explore transcriptional changes in the flocculus evoked by long-term optokinetically evoked climbing fiber discharge. Several differentially transcribed gene products were isolated and sequenced. One of these, calbindin mRNA, was expressed in relatively decreased abundance in the flocculus that received increased climbing fiber input. Decreased transcription of calbindin mRNA was confirmed by northern blots. Hybridization histochemistry was used to localize calbindin mRNA to Purkinje cells and confirmed decreased transcription of calbindin mRNA in Purkinje cells located in folium 1 of the flocculus. Western blots and immunohistochemistry localized the climbing fiber-evoked decreased expression of calbindin to Purkinje cells in folia 1 of the flocculus. The expression of four other calcium-binding proteins in the flocculus was not influenced by optokinetic stimulation. Changes in expression of calbindin could be evoked by decreases in intracellular calcium associated with climbing fiber-evoked decreases in Purkinje cell simple spike activity. The application of differential display reverse transcription-polymerase chain reaction has provided a positive screen for several molecules in addition to calbindin whose expression is affected by naturally evoked activity in a major synaptic pathway to the cerebellum. Further experiments will be required to specify the functional role of each of these molecules.

Interferon β Secreted from Human Hair Dermal Papilla Cells Inhibits the Growth of Outer Root Sheath Cells Cultured in Vitro

Hair growth is tightly regulated by the epithelial–mesenchymal interaction of hair follicle cells. To investigate the molecular interaction between epithelial and mesenchymal cells of the hair follicle, the outer root sheath (ORS) cells were cultured with the dermal papilla (DP)-conditioned medium. Using the techniques of differential display reverse transcription–polymerase chain reaction (DD RT–PCR) and Northern blot analysis, we identified that an interferon-inducible gene 6–16 is regulated in the ORS cells when cultured with the DP-conditioned medium. We further studied the effect of the DP-conditioned medium and found that interferon β, a predominant interferon secreted by the fibroblasts, is expressed by the DP cells. Moreover, the anti-interferon β neutralized DP-conditioned medium dramatically inhibited the 6–16 mRNA expression. Interferon β or DP-conditioned medium suppressed the growth of the in vitro cultured ORS cells, suggesting that interferon β secreted from the DP cells negatively regulates the growth of ORS cells. Together these results reveal the potential importance of the interferon β as a negative regulator of hair growth.

Novel feto‐specific mRNA species suitable for identification of fetal cells from the maternal circulation

Feto-specific markers are necessary for genetic diagnostics on fetal cells isolated from maternal blood. Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to identify mRNA species preferentially expressed in trophoblast-enriched primary cell cultures compared to female peripheral mononuclear blood cells (FPMBCs). We obtained 15 different cDNA clones expressed predominantly in placenta cells using a commercial kit with oligo-(dT) anchor primers and arbitrary upstream primers for differential display analysis of total RNA. Reverse Northern dot-blot analysis and semi-quantitative RT-PCR confirmed the different expression levels. Seven mRNA species were exclusively expressed in the placenta: the mRNAs of epsilon-globin, a renowned marker of fetal erythroblasts, early placenta insulin-like peptide (EPIL), Yes-associated protein (YAP65) and osteopontin were expressed at high levels, whereas the mRNAs of PP14, JM27 protein, and an unidentified expressed sequence tag (EST) were moderately expressed. A further eight mRNAs with low expression in FPMBCs and higher expression in first trimester placenta were identified. Some mRNAs were expressed in a trophoblast cell line, e.g. YAP65, whereas others were not, e.g. EPIL. The new mRNA markers may be used to construct DNA/RNA probes to identify fetal cells in maternal circulation by hybridization techniques, or to identify proteins selectively expressed in fetal cells and amenable to immunocytochemical detection.

Evidence for Direct Involvement of β‐Catenin in Phorbol Ester‐Induced Neurite Outgrowth in GT1‐1 Hypothalamic Neurones

Gonadotropin-releasing hormone (GnRH) is a pivotal neuroendocrine regulator controlling reproductive functions. However, the scattered distribution of GnRH neurones in the mammalian brain has hindered studies on the development and differentiation of GnRH neurones. In the present study, we used the immortalized GnRH-producing GT1-1 cells to examine whether activation of protein kinase C (PKC) pathway with 12-O-tetradecanoyl-13-acetate (TPA) induces morphological and functional differentiation of GnRH neurones. TPA induced neurite outgrowth and inhibited proliferation of GT1-1 cells that were specifically antagonized by cotreatment of PKC inhibitor, calphostin C. The functional significance of TPA-induced differentiation of GT1-1 cells was manifested in part by the changes in the effects of gamma-aminobutyric acid (GABA) on intracellular Ca2+ levels. In untreated GT1-1 cells, activation of GABA-A receptor with 10 microM muscimol increased intracellular Ca2+ levels, whereas such stimulatory effects disappeared in GT1-1 cells bearing neurites. Accordingly, muscimol could not stimulate GnRH release in TPA-treated GT1-1 cells. To elucidate the molecular mechanism underlying TPA-induced neurite outgrowth, we performed differential display reverse transcription-polymerase chain reaction. Among several genes that are affected by TPA treatment, we found a significant induction of beta-catenin mRNA expression. Along with the rapid induction of beta-catenin protein levels, we observed that beta-catenin was reallocated from cell-cell adhesion sites to the growth cones within 3 h of TPA treatment. Transient transfection studies with green fluorescent protein as a reporter gene demonstrated that beta-catenin overexpression alone can promote neurite outgrowth in GT1-1 cells. Moreover, TPA was found to increase the transcription-activational roles of beta-catenin. Together, these data provide evidence that beta-catenin is involved in the TPA-induced functional differentiation of immortalized GnRH neurones.

Gene expression in epithelial cells in response to pneumovirus infection

Respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM) are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner.

Identification of differentially expressed genes in human melanoma cells with acquired resistance to various antineoplastic drugs

Malignant melanoma displays strong resistance against various antineoplastic drugs. The mechanisms conferring this intrinsic resistance are unclear. To better understand the molecular events associated with drug resistance in melanoma, a panel of human melanoma cell variants exhibiting low and high levels of resistance to 4 commonly used drugs in melanoma treatment, i.e., vindesine, etoposide, fotemustine and cisplatin, was characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Of 269 mRNA fragments found to be altered in expression level by DDRT-PCR, a total of 11 cDNA clones was characterized after confirmation of a differential expression pattern by Northern blot analyses. These clones include 3 genes (DSM-1, DSM-3 and DSM-5) of known function, 4 previously sequenced genes (DSM-2, DSM-4, DSM-6 and DSM-7) of uncharacterized function and 4 novel genes (DSM-8–DSM-11) without match in GenBank. All of these genes exhibited altered mRNA expression in high level etoposide-resistant cells, whereby 7 genes (DSM-1–DSM-6 and DSM-8) were found to be decreased in the transcription rate in these etoposide-resistant cells. The mRNA synthesis of the remaining genes (DSM-7 and DSM-9–DSM11) was enhanced in high level etoposide-resistant melanoma cells. The expression of 5 (DSM-5 and DSM-7–DSM-10) of the cloned cDNA encoding mRNAs was modulated in various independently established drug-resistant melanoma cells, indicating to be associated with drug resistance. Further characterization of these genes may yield inside into the biology and development of drug resistance in malignant melanoma. Int. J. Cancer 88:535–546, 2000. © 2000 Wiley-Liss, Inc.

Profiling of changes in gene expression during raspberry (Rubus idaeus) fruit ripening by application of RNA fingerprinting techniques.

Processes that contribute to the overall phenomenon of fruit ripening are not well understood for many soft fruit species, including raspberry (Rubus idaeus L.) Recent biochemical data implicate ethylene and a range of cell wall hydrolases in ripening processes. However, the genes encoding these activities, and others related to ripening, remain to be characterised. With the advent of high-throughput RNA-fingerprinting techniques it is possible to characterise rapidly the changes in gene expression during developmental processes. This paper describes the application of two RNA-fingerprinting techniques (cDNA amplified fragment length polymorphism and differential display reverse transcription-polymerase chain reaction) to the ripening fruit of Rubus idaeus. Copy-DNA tags were isolated representing 34 genes, up-regulated during fruit ripening. The expression profiles of these genes were determined by RNA-blot analysis and their sequences were compared with those in public databases. Potential roles for some of these gene products are considered, providing valuable insights into the processes that underpin fruit ripening. Many of the cDNAs isolated in this study provide tools for the biotechnological improvement of fruit quality.

Increased expression of insulin-like growth factor binding protein-1 during induced regression of bovine corpora lutea.

Three experiments were conducted to examine gene expression during induced luteal regression in the cow; the initial purpose was the identification of potential embryotoxins. In experiment 1, changes in gene expression in the corpus luteum (CL) were identified by differential display reverse transcription-polymerase chain reaction (DD-PCR) during the first 72 h of luteal regression in cows treated with prostaglandin F(2alpha) (PGF(2alpha)) on Days 4-7 after estrus. Expression of insulin-like growth factor-binding protein-1 (IGFBP-1) was up-regulated, with greatest expression at 24 h (P < 0.05) after treatment with PGF(2alpha) began. In experiment. 2, IGFBP-1 and its mRNA were quantified in CL collected 24 or 48 h after treatment with PGF(2alpha) on Day 4 or 10 after estrus. Because local mechanisms for exchange of hormones between the ovary and uterus are known in ruminants, uterine flushings were assayed for IGFBP-1 to seek evidence of local transfer of luteal IGFBP-1 to the uterus. IGFBP-1 mRNA was increased (P < 0.05) in CL 24 h after treatment when PGF(2alpha) that began on Day 10, and by 48 h after treatment that began on Day 4. Concentrations of IGFBP-1 increased (P < 0.05) in a pattern similar to mRNA, by 24 h on Day 10, and by 48 h on Day 4. Concentrations of IGFBP-1 in uterine flushings did not change on either day. Concentrations of progesterone decreased (P < 0.05) by 8 h after treatment with PGF(2alpha) that began on Day 10, but not until 24 h after treatment that began on Day 4. In experiment 3, cows received either saline or PGF(2alpha) and CL were collected 2 or 10 h after a single treatment, or 2 h after a second treatment that was given 8 h after the first. Expression of IGFBP-1 was increased by 2 h after treatment with PGF(2alpha) on both Days 4 and 10 after estrus. In conclusion, secretion of IGFBP-1 is increased during luteolysis, and may inhibit the steroidogenic effects of insulin-like growth factor-I (IGF-I), but no evidence was found to implicate IGFBP-1 in the embryotoxic effect of regressing CL.

Multiple Growth Factor Induction of a Murine Early Response Gene That Complements a Lethal Defect in Yeast Ribosome Biogenesis

Identification of the transcriptionally activated targets of receptor tyrosine kinases is critical to understanding biologic programs directing both normal and neoplastic growth. To elucidate these molecular processes, we identified genes induced by a potent mesenchymal mitogen, platelet-derived growth factor (PDGF). Using differential display reverse transcription-polymerase chain reaction technology, we isolated a novel growth factor-induced cDNA, San5. San5 transcript induction occurred within 60 min in NIH 3T3 fibroblasts and proceeded in the presence of cycloheximide. Maximal induction of the San5 transcript occurred between 8 and 16 h, concurrent with passage of fibroblasts through G(1). San5 message was potently induced by PDGF AA and BB and acidic and basic fibroblast growth factors, all strong activators of fibroblast proliferation, but not by epidermal growth factor and interleukin-4. In a murine hematopoietic progenitor cell line, San5 transcript induction strictly correlated with [(3)H]thymidine uptake. Isolation and sequencing of the murine San5 cDNA revealed amino acid sequence homology to yeast Nop5p, a nucleolar protein required for pre-rRNA processing and ribosome assembly. Strikingly, SAN5 was able to rescue a nop5 null mutant, implicating SAN5 in the process of ribosome biogenesis. Consistent with this result, SAN5 was localized to the nucleolus in both yeast and mouse. Thus, San5 may provide a link between growth factor receptor activation and the cellular translational machinery.

Mechanical stress induces the expression of high molecular mass heat shock protein in human chondrocytic cell line CS-OKB

Objective Mechanical stress is an important regulator of chondrocyte function, but it is unknown how chondrocytes respond to mechanical stress. This study was performed to clarify the underlying mechanisms in human chondrocytes.Design Using a Flexercell strain unit (25% maximal elongation, 0.05 Hz-cyclic manner, and 48h), mechanical stimulation was applied to confluent CS-OKB cells, human chondrocytic cells. To analyze transcriptional changes in response to mechanical stress, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) and Northern blot analysis were performed.Results Among several differentially displayed fragments, one fragment (927bp) tentatively named as SIC (Stress-Induced Chondrocytic) 1 was isolated from the human chondrocytic cell line and identified as one of the high molecular mass heat shock proteins.Conclusion Mechanical stress induces the expression of a high molecular mass heat shock protein corresponding to SIC 1 in human chondrocytic cells. SIC 1 may play an important role in the mechanical stress-responded metabolism of human chondrocytes.

Expression of serum- and glucocorticoid-regulated kinase (sgk) mRNA is up-regulated by GM-CSF and other proinflammatory mediators in human granulocytes.

Stimulation of human peripheral blood granulocytes with the proinflammatory cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), increases incorporation of [3H]uridine into RNA. We investigated the nature of the RNA synthesized under these conditions. Using transcription inhibitors, gel electrophoresis, and high-salt precipitation, it was concluded that as much as 90% of this radiolabeled RNA represents polymerase II transcripts. Differential display reverse transcription-polymerase chain reaction was used to identify and clone GM-CSF-responsive mRNAs. Serum- and glucocorticoid-regulated kinase (sgk) mRNA was identified that could be up-regulated 10- to 20-fold by > or =0. 1 ng/mL recombinant human GM-CSF. The 2.6-kb sgk mRNA was induced rapidly (within 30 min) by GM-CSF and remained at high levels for at least 12 h. Up-regulation was blocked completely by the transcription inhibitor, actinomycin D, but not by the translation inhibitor, cycloheximide, nor by the tyrosine kinase inhibitor, genistein. Up-regulation did not appear to be caused by enhanced mRNA stability. Other inflammatory mediators could also increase sgk mRNA levels (GM-CSF > > lipopolysaccharide > fMLP = tumor necrosis factor alpha). The function of sgk in granulocytes remains unknown.

Refinements of the Differential Display Reverse Transcription–Polymerase Chain Reaction Technique: Use of Oligo(dT)-Based Anchored Primers with Escherichia coli Messenger RNA Identifies a Salt-Induced Promoter in the dcw Gene Cluster

Differential display reverse transcription PCR (DDRT–PCR)2 is a popular technique used to identify differences in messenger RNA species expressed under different conditions (1–4). The use of this technique in prokaryotic systems has been greatly hampered by the lack of a poly(A) tail at the 39 end of these messages, resulting in the use of random primers to define both ends of the PCR products, in turn leading to lower probability of identifying any differentially expressed gene.

Identification of regulated genes in rat heart after myocardial infarction by means of differential mRNA display.

In order to testify the hypothesis that unknown mechanisms are involved in the process of cardiac remodeling after myocardial infarction (MI), we employed differential display reverse transcription-polymerase chain reaction (DDRT-PCR) as our primary inspection tool. An animal model of MI was established by ligation of the left anterior descending coronary artery (LAD) in rat. Fifty upregulated candidate cDNA fragments were obtained in the right ventricle (RV) of the heart six weeks after MI. Eight cDNA fragments isolated from DD denaturing gel were extracted and reamplified, cloned into pCR II vector and sequenced. A Genbank search of these clones showed that three of them have a high homology with known genes not previously associated with cardiac remodeling, i.e., mouse interleukin-4 receptor gene, rat ferritin mRNA, and T-cell receptor beta chain V beta 5. The remaining clones have no similarity to known sequences. These data suggest that certain genes which were not previously being associated with cardiac hypertrophy are turned on during the process of cardiac remodeling after MI.