Restriction Fragment Differential Display Research Articles

Molecular Regulation of the Metabolic Pathways of the Medicinal Plants: <i>Phyla dulcis</i>

Phyla (Lippia) dulcis contains hernundulcin sesquiterpene zero-caloric sweetener that is about a thousand times sweeter than sucrose, and also bitter constituents including camphor and limonene. There is yet no simple method to remove the undesirable constituents. The yield of sweetener hernundulcin is very low, and there is no simple method to maximize its composition. The aim of the project was to characterize the mRNA targets that regulate the primary and terpenoid metabolic enzymes of P. dulcis. Restriction fragment differential display polymerase chain reaction of P. dulcis glutamate dehydrogenase-synthesized RNA showed that many mRNAs encoding β-caryophyllene, (+)-epi-α-bisabolol, bicyclogermacrene, bifunctional sesquiterpene, and geraniol synthases shared sequence homologies with ribulose-1,5-bisphophatase carboxylase, granule-bound starch synthase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and phosphoenol pyruvate carboxylase. Sequence similarities between mRNAs encoding primary metabolic enzymes and terpene synthases suggested that photosynthesis could regulate terpenoid metabolism in order to increase the yield of sweetener hernundulcin.

High intensity training induces alteration of the ubiquitin-proteasome system gene expression profile and structural changes in the ovaries

The female reproductive system is highly sensitive to physiological stress. However, the mechanism(s) involved in dysfunction of the ovaries induced by high intensity exercise training remain unknown. In the present study, we established a rat model of high intensity exercise training, and investigated the morphological and functional changes in ovaries. Furthermore, we profiled the differential gene expression in the ovaries between high intensity exercise training rats and control rats by restriction fragment differential display PCR. Differentially expressed genes were validated by real-time PCR. The results showed that the weight and microstructure of the ovaries were significantly altered in the exercise training group rats. In addition, the levels of estradiol, progesterone, luteinizing hormone and follicle-stimulating hormone were significantly decreased in the exercise training group rats compared with the control group rats. Immunohistochemistry revealed weak expression of estradiol, estrogen receptor and progesterone receptor in ovarian tissues from the high intensity exercise training group compared with the control group. Furthermore, the gene expression profiles of the exercise training and control group rats were analyzed, and differentially expressed genes, such as those involved in the ubiquitin-proteasome system, were identified. Our results indicate that high intensity exercise training can suppress the cellular function of the ovary glands. The mechanism may involve a series of alterations in the expression of genes particularly associated with the ubiquitin-proteasome system.

The Imprinted Gene Neuronatin Is Regulated by Metabolic Status and Associated With Obesity

Using restriction fragment differential display (RFDD) technology, we have identified the imprinted gene neuronatin (Nnat) as a hypothalamic target under the influence of leptin. Nnat mRNA expression is decreased in several key appetite regulatory hypothalamic nuclei in rodents with impaired leptin signaling and during fasting conditions. Furthermore, peripheral administration of leptin to ob/ob mice normalizes hypothalamic Nnat expression. Comparative immunohistochemical analysis of human and rat hypothalami demonstrates that NNAT protein is present in anatomically equivalent nuclei, suggesting human physiological relevance of the gene product(s). A putative role of Nnat in human energy homeostasis is further emphasized by a consistent association between single nucleotide polymorphisms (SNPs) in the human Nnat gene and severe childhood and adult obesity.

The influence of triethylene glycol derived from dental composite resins on the regulation of Streptococcus mutans gene expression

Bacterial microleakage along the tooth/composite resin dental restoration interface contributes to post-operative sensitivity, recurrent caries, pulp inflammation and necrosis. Studies have confirmed that saliva can catalyze the degradation of constitutive monomers in dental restorative composites, forming biodegradation by-products (BBPs) such as methacrylic acid (MA), and triethylene glycol (TEG). TEG accelerates the growth of Streptococcus mutans, a major etiological agent of dental caries. Restriction fragment differential display polymerase chain reaction (RFDD-PCR) in conjunction with single strand conformation polymorphism (SSCP) was used to identify S. mutans genes with differential expression when grown in the presence of TEG at pH levels 5.5 and 7.0. Quantitative real-time PCR (q-RT PCR) was utilized to study specific gene expression patterns. TEG modulated the expression levels of glucosyltransferase B ( gtfB) (involved in biofilm formation) and yfiV (a putative transcription regulator) in S. mutans. The expression patterns were dependent on the bacterial growth mode (planktonic vs. biofilm) as well as pH (5.5 vs. 7.0). The findings describe the effect of composite resin-derived BBPs on important physiological functions of S. mutans (at BBP concentration levels found in vivo), and indicate the potential influence of BBPs in biofilm formation and microbial survival on surfaces in the oral cavity.

Identification of differentially expressed root genes upon rhizomania disease.

Rhizomania is one of the most devastating sugar beet diseases. It is caused by Beet necrotic yellow vein virus (BNYVV), which induces abnormal rootlet proliferation. To understand better the physiological and molecular basis of the disorder, transcriptome analysis was performed by restriction fragment differential display polymerase chain reaction (RFDD-PCR), which provided differential gene expression profiles between non-infected and infected sugar beet roots. Two distinct viral isolates were used to detect specific or general virus-induced genes. Differentially expressed genes were selected and identified by sequence analysis, followed by reverse Northern and reverse transcriptase PCR experiments. These latter analyses of different plants (Beta vulgaris and Beta macrocarpa) infected under distinct standardized conditions revealed specific and variable expressions. Candidate genes were linked to cell development, metabolism, defence signalling and oxidative stress. In addition, the expression of already characterized genes linked to defence response (pathogenesis-related protein genes), auxin signalling and cell elongation was also studied to further examine some aspects of the disease. Differential expression was retrieved in both B. vulgaris and B. macrocarpa. However, some candidate genes were found to be deregulated in only one plant species, suggesting differential response to BNYVV or specific responses to the BNYVV vector.

Gene Expression Profiling in Brodmann's Area 46 from Subjects with Schizophrenia

To identify altered gene expression in the dorsolateral prefrontal cortex obtained after death from subjects with schizophrenia. Restriction fragment differential display (RFDD) was used to measure levels of mRNA in Brodmann area (BA) 46 from schizophrenia and control subjects. Findings on specific mRNA identified with RFDD were further investigated using real-time polymerase chain reaction (real-time PCR), PCR and western blotting. Levels of mRNA for 63 of approximately 12,500 genes differed in BA 46 in schizophrenia. Subsequent real-time PCR has shown that mRNA for muscleblind protein 1 (MBNL1) and protocadherin 17 (PCDH17) are increased in BA 46 from subjects with schizophrenia of short, but not long, duration. Altered levels of mRNA for neither gene were present in BA 9 from subjects with schizophrenia or in either cortical area from subjects with bipolar 1 disorder. By contrast, both RFDD and real-time PCR failed to show altered expression of the schizophrenia candidate gene disrupted in schizophrenia 1 (DISC1) BA46 from any diagnostic cohort. The present study has identified genes that are differentially expressed in BA 46 in schizophrenia. Initial studies have shown that there is a need for a careful validation of genes shown to be affected in schizophrenia using high-throughput technologies. In addition the present study has shown that gene expression may vary considerably depending on the duration of schizophrenia. This raises the hypothesis that changing gene expression may be underlying the change in symptom profile that occurs with disease progression in some subjects with schizophrenia.

An increase of cytochrome C oxidase mediated disruption of gemcitabine incorporation into DNA in a resistant KB clone

Mechanistic aberrations leading to Gemcitabine (2′,2′-dFdCyd,2,2-difluorodeoxycytidine, Gem) resistance may include alteration in its transport, metabolism and incorporation into DNA. To explore the mechanism of Gem resistance, the restriction fragment differential display PCR (RFDD-PCR) was employed to compare the mRNA expression patterns of KBGem (Gem resistant), KBHURs (hydroxyurea resistant) and KBwt (parental KB cell). Nine gene fragments were overexpressed specifically in the KBGem clone. Sequencing and BLAST results showed that three fragments represent cytochrome C oxidase (CCOX, respiration complex IV) subunit III (CCOX3). The cDNA microarray confirmed that the mRNAs of CCOX and ATP synthase subunits were upregulated in KBGem as compared to KBwt and KBHURs. The increase in CCOX1 protein and activity led to the increase of free ATP concentration, which is consistent with the gene expression profile of KBGem. Furthermore, the sensitivity to Gem could be reversed by sodium azide, a CCOX inhibitor. Following the treatment of sodium azide, the cellular accumulation of [ 3H]-Gem increased in a dose (of azide)-dependent manner, which is associated with increase of [ 3H]-Gem incorporation into DNA in KBGem. In summary, an increase of CCOX activity and free ATP level may reduce the transport, metabolism and DNA incorporation of Gem, resulting in Gem resistance.

Over-expression of genes and proteins of ubiquitin specific peptidases (USPs) and proteasome subunits (PSs) in breast cancer tissue observed by the methods of RFDD-PCR and proteomics

The ubiquitin-proteasome system facilitates the degradation of damaged proteins and regulators of growth and stress response. Alterations in this proteolytic system are associated with a variety of human pathologies. By restriction fragment differential display polymerase chain reaction (RFDD-PCR) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) based on two-dimensional polyacrylamide gel electrophoresis (2-DE), differentially expressed genes and proteins of ubiquitin specific proteases (USPs), proteasome subuinits (PSs) and ubiquitin protein ligase E3A (UBE3A) were analyzed between breast cancer and adjacent normal tissues. Some of them were further verified as over-expression by immunohistochemical stain. Five genes of proteasome subunits (PSs), including PSMB5, PSMD1, PSMD2, PSMD8 and PSMD11, four genes of USPs, including USP9X, USP9Y, USP10 and USP25, and ubiquitin protein ligase E3A (UBE3A) were over-expressed (>3-fold) in breast cancer tissue compared to adjacent normal tissue, and over-expression (>4-fold) of proteins of PSMA1 and SMT3A were observed in breast cancer tissue. PSMD8, PSMD11 and UBE3A were further verified as over-expression by immunohistochemical stain. The action of ubiquitin-proteasome system were obviously enhanced in breast cancer, and selectively intervention in action of ubiquitin-proteasome system may be a useful method of treating human breast cancer.

Physiological oxygenation status is required for fully differentiated phenotype in kidney cortex proximal tubules

Hypoxia has been suspected to trigger transdifferentiation of renal tubular cells into myofibroblasts in an epithelial-to-mesenchymal transition (EMT) process. To determine the functional networks potentially altered by hypoxia, rat renal tubule suspensions were incubated under three conditions of oxygenation ranging from normoxia (lactate uptake) to severe hypoxia (lactate production). Transcriptome changes after 4 h were analyzed on a high scale by restriction fragment differential display. Among 1,533 transcripts found, 42% were maximally expressed under severe hypoxia and 8% under mild hypoxia (Po(2) = 48 mmHg), suggesting two different levels of oxygen sensing. Normoxia was required for full expression of the proximal tubule-specific transcripts 25-hydroxyvitamin D 1-hydroxylase (Cyp27b1) and l-pyruvate kinase (Pklr), transcripts involved in tissue cohesion such as fibronectin (Fn1) and N-cadherin (Cdh2), and non-muscle-type myosin transcripts. Mild hypoxia increased myogenin transcript level. Conversely, severe hypoxia increased transcripts involved in extracellular matrix remodeling, those of muscle-type myosins, and others involved in creatine phosphate synthesis and lactate transport (Slc16a7). Accordingly, microscopy showed loss of tubule aggregation under hypoxia, without tubular disruption. Hypoxia also increased the levels of kidney-specific transcripts normally restricted to the less oxygenated medullary zone and others specific for the distal part of the nephron. We conclude that extensive oxygen supply to the kidney tubule favors expression of its differentiated functions specifically in the proximal tubule, whose embryonic origin is mesenchymal. The phenotype changes could potentially permit transient adaptation to hypoxia but also favor pathological processes such as tissue invasion.

Differential display analysis of Porphyromonas gingivalis gene activation response to heat and oxidative stress

The etiologic relationship between periodontitis and Porphyromonas gingivalis is attributed to the ability of the organism to express a variety of virulence factors, many of which are cell surface components including lipopolysaccharide and arginine-specific cysteine proteases (Arg-gingipains, RgpA, and RgpB). P. gingivalis responds to the stress of rapid elevation in temperature by activating a set of genes to produce heat shock proteins that mediate the effects of sudden changes in environmental temperatures by repairing or eliminating cellular proteins denatured by that stress. We used restriction fragment differential display (RFDD) to identify and measure the genes expressed by surrogates of environmental stresses, heat and oxidative stress. The results were then confirmed using quantitative reverse-transcription polymerase chain reaction. We selected 16 genes differentially induced from over 800 total expression fragments on the RFDD gels for further characterization. With primers designed from those fragments we found that a + 5 degrees C heat shock caused a statistically significant increase in expression compared 12 of 18 untreated genes tested. The exposure of P. gingivalis to atmospheric oxygen resulted in statistically significant increases in five of the target genes. These genes are likely involved in transport and synthesis of components of the lipopolysaccharide biosynthetic pathway important in anchoring the Arg-gingipains required for virulence-related activities. These results emphasize the need for studies to measure the coordinated responses of bacteria like P. gingivalis which use a multitude of interrelated metabolic activities to survive the environmental hazards of the infection process.

RNA synthetic activity of glutamate dehydrogenase

The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology for assay of the enzyme's activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.

Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display.

To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells.

Cytokines are important humoral mediators of beta cell destruction in autoimmune diabetes. The aim of this study was to identify novel cytokine-induced genes in insulin-producing RINm5F cells, which may contribute to beta cell death or survival. A global gene expression profile in cytokine-exposed insulin-producing RINm5F cells was achieved by automated restriction fragment differential display PCR. The expression of selected candidate genes was confirmed by real-time RT-PCR analysis. Exposure of RINm5F cells to IL-1beta or to a cytokine mixture (IL-1beta, TNF-alpha, IFN-gamma) for 6 h resulted in the differential expression of a functional gene cluster. Apart from the well-known up-regulation of the cytokine-responsive genes iNOS, NF-kappaB, MnSOD and Hsp70, several genes that belong to the functional cluster of the endocytotic pathway were identified. These endocytotic genes comprised: clathrin, megalin, synaptotagmin and calcineurin, which were up-regulated by IL-1beta or the cytokine mixture. In contrast, the expression of the calcineurin inhibitor CAIN and of the GDP/GTP exchange protein Rab3 was down-regulated by cytokines. Other up-regulated cytokine-responsive genes were: agrin, murine adherent macrophage protein mRNA ( MAMA) and transport-associated protein ( TAP1/MTP), whereas the plasma membrane calcium ATPase ( PMCA) 2 and PMCA 3 genes were down-regulated by cytokines. Our results indicate that genes of the endocytotic pathway are regulated by pro-inflammatory cytokines. This might affect the density of cytokine receptors at the beta cell surface and concomitantly the sensitivity of the cells to cytokine toxicity. A better understanding of the functional cross-talk between endocytotic and cytokine signalling pathways could further the development of novel strategies to protect pancreatic beta cells against toxic effects of pro-inflammatory cytokines.

Gene expression profile of human bone marrow stromal cells determined by restriction fragment differential display analysis.

Using an in vitro osteogenic culture system, we carried out a restriction fragment differential display (RFDD-PCR) to identify genes expressed by these cells in their undifferentiated stage and not expressed, or expressed at a lower level, in a closely related but distinct cell type: bone marrow stromal cells (BMSC)-derived osteoblasts (BDO). Forty-seven candidate regulated genes, selected by RFDD, were analyzed by RT-PCR analysis in three cell clones and in primary cultures from seven different donors. A subset of three genes were confirmed as upregulated in BMSC relative to BDO in every primary culture and cloned population examined: betaIG-h3, IGFbp3, and LOXL2. Their differential expression was confirmed by Northern analysis and the corresponding proteins were detected by immunolocalization in BMSC.

Differential expression of the enolase gene under in vivo versus in vitro growth conditions of Aeromonas hydrophila

Aeromonas hydrophila is an emerging human pathogen that leads to gastroenteritis and other invasive diseases. By using a murine peritoneal culture (MPC) model, we identified via restriction fragment differential display PCR (RFDDPCR) five genes of A. hydrophila that were differentially expressed under in vivo versus in vitro growth conditions. The gene encoding enolase was among those five genes that were differentially up regulated. Enolase is a glycolytic enzyme and its surface expression was recently shown to be important in the pathogenesis of a gram-positive bacterium Streptococcus pyogenes. By Western blot analysis and Immunogold staining, we demonstrated secretion and surface expression of enolase in A. hydrophila. We also showed that the whole cells of A. hydrophila had strong enolase activity. Using an enzyme-linked immunosorbant assay and sandwich Western blot analysis, we demonstrated binding of enolase to human plasminogen, which is involved in the fibrinolytic system of the host. We cloned the A. hydrophila enolase gene, which exhibited 62% homology at the DNA level and 57% homology at the amino acid level when compared to S. pyogenes enolase. This is a first report describing the increased expression of enolase gene in vivo that could potentially contribute to the pathogenesis of A. hydrophila infections.

Restriction fragment differential display using BA 46 from subjects with schizophrenia: A role for cell adhesion and signal transduction in the pathology of the disorder

The elucidation of the genetic changes of hepatocellular carcinoma (HCC) is very important for understanding the molecular mechanism of liver carcinogenesis. In order to identify the gains or losses in DNA sequence copy number in HCC, we used comparative genomic hybridisation to study 40 cases (44 tumours) of HCC. Tumour DNA and DNA from non-neoplastic liver tissue were labelled with different fluorochromes and then simultaneously hybridised to normal metaphase spread chromosomes. An image acquisition system was used to quantitate signal intensities contributed by tumour and reference DNA along the entire length of each chromosome. Regions of amplification and deletion were demonstrated as quantitative alterations. Losses were prevalent on chromosome regions 16q (43%), 17p (20%), 13q (20%), 4q (15%) and 8p (15%). Gains frequently occurred on 8q (30%), 1q (20%), 6p (20%) and 17q (18%). Hepatitis B virus carriers had a significantly higher frequency of losses on chromosome 16q. Furthermore, the minimal region of losses was narrowed down to 16q11-q22. This study confirms the presence of previously known chromosomal aberrations in HCC and highlights a new significant correlation between losses on chromosome 16q and hepatitis B virus carriers.

Cellular responses to environmental salinity in the halophilic black yeast Hortaea werneckii.

The development of crop plants with increased salt tolerance necessitates the study of naturally salt-tolerant eukaryotic species. We studied the bio-synthesis of glycerol as a compatible solute in the halophilic eukaryotic microorganism, black yeast Hortaea werneckii. A restriction fragment-differential display technique was used to investigate the transcriptome of the organism. Eight differentially expressed genes were identified in response to growth at different salinities. Although the putative functions of their products, P-type ATPase, ubiquinone reductase, aconitase, RNA helicase, Asn-tRNA ligase, isoamyl alcohol oxidase, and phosphatidylinositol-3-kinase, are not intimately related within the cellular machinery, the results presented here are sufficient to propose a model which describes how H. werneckii adapts to extremely high salinities. Some of these mechanisms of adaptation to raised environmental salinity are similar to those in other salt-sensitive species, e.g. glycerol accumulation, there also appear to be novel mechanisms present such as the use of different energy production mechanisms and post-transcriptional regulation of gene expression. Our results have also provided new data on two genes from two other fungal species, the Neurospora crassa B1D1.130 gene and the Aspergillus ustus amdS-A gene.

Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii.

We have used restriction fragment differential display for isolating genes of the unicellular green alga Chlamydomonas reinhardtii that exhibit elevated expression on exposure of cells to high light. Some of the high light-activated genes were also controlled by CO2 concentration. Genes requiring both elevated light and low CO2 levels for activation encoded both novel polypeptides and those that function in concentrating inorganic carbon (extracellular carbonic anhydrase, low CO2-induced protein, ABC transporter of the MRP subfamily). All the genes in this category were shown to be under the control of Cia5, a protein that regulates the responses of C. reinhardtii to low-CO2 conditions. Genes specifically activated by high light, even under high-CO2 conditions, encoded a 30 kDa chloroplast membrane protein, a serine hydroxymethyltransferase, a nuclease, and two proteins of unknown function. Experiments using DCMU, an inhibitor of photosynthetic electron transport, and mutants devoid of either photosystem I or photosystem II activity, showed aberrant expression of all the genes regulated by both CO2 and high light, suggesting that redox plays a role in controlling their expression. In contrast, there was little effect of DCMU or lesions that block photosynthetic electron transport on the activity of genes that were specifically controlled by high light.

Mammalian Resistance to Oxidative Stress: A Comparative Analysis

Changes in gene expression represent a major protective mechanism, and enforced overexpression of individual genes has been shown to protect cells. However, no large-scale comparison of genes involved in mammalian oxidative stress protection has yet been described. Using filter microarray and restriction fragment differential display technology, hydrogen peroxide (H2O2)-resistant variants of hamster HA-1 fibroblasts and human HL-60 promyelocytes were found to possess a surprising lack of commonality in specific modulated genes with the single exception of catalase, supporting the hypothesis that catalase overexpression is critical for resistance to H2O2. Comparison of two cell lines from the same species (hamster) selected with an exogenous oxidative stressing agent (H2O2) and an endogenous metabolic oxidative stressing agent (95% O2) also revealed little commonality in modulation of specific mRNAs with the exception of glutathione S-transferase enzymes and catalase. Acute oxidative stress in HL-60 led to the modulation of a limited subset of the genes associated with chronic oxidative stress resistance. Overall, these results suggest that mammalian resistance to oxidative and perhaps other stress does not require a significant number of common genes but rather only a limited number of key genes (e.g., catalase in our model systems) in combination with others that are cell type and stress agent specific.

Analysis of gene expression following oxidative stress.

Abstract The modulation of gene expression by cellular stress is a universal phenomena that has been described in organisms ranging from bacteria to animals to plants, and in response to a wide range of stress agents including heat shock, glucose deprivation, oxidants, radiation, and heavy metals (1). For oxidative stress alone, many genes have been identified whose steady-state mRNA product levels are modulated by a multitude of oxidant stress agents (1). These genes were identified using various techniques that are still popular, such as two-dimensional gel electrophoresis, subtractive hybridization, differential display (DD), restriction fragment differential display (RFDD), serial analysis of gene expression (SAGE), and gene arrays; as well as other techniques that have lost their popularity, including one-dimensional protein gel electrophoresis and differential hybridization (1). Differential display (DD) is a polymerase chain reaction (PCR)-based technique that requires only small amounts of sample, is relatively sensitive, and able to identify both induced and reduced species (2–3). Its main drawbacks are a high number of false-positives and products that are strongly biased for the ’3’ untranslated region of the mRNA. These drawbacks have been addessed by a more recent technique called restriction fragment differential display (RFDD), which uses restriction enzyme-generated cDNA fragments as PCR templates (1). This modification allows for the use of much higher temperature and more specific annealing conditions as well as the ability to generate products that include the coding region and ’5’ untranslated region.KeywordsDifferential DisplayPolymerase Chain Reaction TemplateReverse Northern BlotOxidant Stress AgentPolymerase Chain Reaction ThermocyclerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.