Differential Display Technique Research Articles

The nifk gene is widely expressed in mouse tissues and is up-regulated in denervated hind limb muscle.

Denervation of skeletal muscle alters the expression of many genes, which may be important for establishing optimal conditions for reinnervation. Using the differential display technique we have attempted to discover neurally regulated genes in skeletal muscle. An mRNA that is up-regulated in denervated hind limb muscle was identified and cloned. The cDNA encodes an RNA-binding protein, which was discovered during the course of this work to be a nucleolar protein interacting with the fork-head associated domain of the proliferation marker protein Ki-67, and named NIFK. We show that the nifk gene is widely expressed in adult mouse tissues and that the expression is up-regulated in denervated hind limb muscle. No difference between expression in perisynaptic and extrasynaptic portions of muscle was observed. The widespread expression in adult tissues suggests that the NIFK protein has other functions in addition to its interaction with Ki-67, which is only expressed in proliferating cells.

Isolation of cDNAs differentially expressed in response to drought stress and characterization of the Ca-LEAL1 gene encoding a new family of atypical LEA-like protein homologue in hot pepper ( Capsicum annuum L. cv. Pukang)

Among various abiotic stresses, water deficit is one of the most severe environmental factors responsible for the reduction of crop yield in many parts of the world. By means of the mRNA differential display technique, seven cDNAs (pCa-DIs for Capsicum annuum drought induced) have been isolated that are rapidly induced when hot pepper plants are subjected to water stress (5–20% loss of fresh weight). For all of the isolated Ca-DIs, database search provided significant sequence similarity to previously described genes from different plant species. The predicted proteins encoded by the Ca-DI genes are putatively involved in processes as diverse as primary metabolism, protein degradation, cell wall modification and stress response, suggesting the complexity of cellular responses to drought stress in hot pepper plants. Particularly, we analyzed the detailed structural property and expression pattern of the Ca-DI4 ( Ca-LEAL1) gene. Sequence homology studies indicate that Ca-LEAL1 ( M r=19.3 kDa) belongs to a new family of atypical hydrophobic late embryogenesis-abundant-like (LEA-like) proteins. Expression analysis showed that Ca-LEAL1 was strongly activated by drought and salt stresses, and also in response to mechanical wounding in both local and systemic leaves. Moreover, the level of Ca-LEAL1 transcript was rapidly enhanced by exogenous application of ABA and ethylene. These results are consistent with the notion that an atypical hydrophobic Ca-LEAL1 protein is subject to control by diverse environmental factors and that ethylene, in conjunction with ABA, plays an important role in the regulation of the stress gene in hot pepper plants. The possible physiological functions of Ca-LEAL1 as well as other Ca-DI proteins in the adaptive process against drought stress are discussed

In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis.

Several clinical trials have revealed that individuals who were given beta-carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo; rather, vitamin A could actually have caused an adverse effect in the lungs of smokers [Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S. & Hammar, S. N. Engl. J. Med (1996) 334, 1150-1155; Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner, B., Cook, N.R., Belanger, C., LaMotte, F., Gaziano, J.M., Ridker, P.M., Willet, W. & Peto, R. (1996) N. Engl. J. Med. 334, 1145-1149]. Using differential display techniques, an initial survey using rats showed that liver RNA expression of c-H-Ras was decreased and p53 increased in rats with chronic vitamin A deficiency. These findings prompted us to evaluate the expression of c-Jun, p53 and p21WAF1/CIF1 (by RT-PCR) in liver and lung of rats. This study showed that c-Jun levels were lower and that p53 and p21WAF1/CIF1 levels were higher in chronic vitamin A deficiency. Vitamin A supplementation increased expression of c-Jun, while decreasing the expression of p53 and p21WAF1/CIF1. Western-blot analysis demonstrated that c-Jun and p53 showed a similar pattern to that found in the RT-PCR analyses. Binding of retinoic acid receptors (RAR) to the c-Jun promoter was decreased in chronic vitamin A deficiency when compared to control hepatocytes, but contrasting results were found with acute vitamin A supplementated cells. DNA fragmentation and cytochrome c release from mitochondria were analyzed and no changes were found. In lung, an increase in the expression of c-Jun produced a significant increase in cyclin D1 expression. These results may explain, at least in part, the conflicting results found in patients supplemented with vitamin A and illustrate that the changes are not restricted to lung. Furthermore, these results suggest that pharmacological vitamin A supplementation may increase the risk of adverse effects including the risk of oncogenesis.

A Novel Mitochondrial Ca2+-dependent Solute Carrier in the Liver Identified by mRNA Differential Display

Pancreatic AR42J cells have the feature of pluripotency of the precursor cells of the gut endoderm. Dexamethasone converts them to exocrine cells or liver cells. Using mRNA differential display techniques, we have identified a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily, which is expressed during the course of differentiation, and have designated it MCSC. The corresponding cDNA comprises an open reading frame of 1407 base pairs encoding a polypeptide of 469 amino acids. The carboxyl-terminal-half of MCSC has high similarity with other mitochondrial carriers, and the amino-terminal-half has three canonical elongation factor-hand motifs and has calcium binding capacity. The deduced amino acid sequence revealed 79.1% homology to the rabbit peroxisomal Ca2+-dependent member of the mitochondrial superfamily, but the subcellular localization of the protein was exclusively mitochondrial, not peroxisomal. Northern blot and Western blot analyses revealed its predominant expression in the liver and the skeletal muscle. In the liver, the expression level of MCSC was higher in the adult stage than in the fetal stage, and MCSC was highly up-regulated in dexamethasone-treated AR42J cells before the expression of albumin. Taken together, MCSC may play an important role in regulating the function of hepatocytes rather than in differentiation in vivo.

Human T-cell leukemia virus type 1 Tax protein stimulates the interferon-responsive enhancer element via NF-κB activity

Gene expression in Rat-1 fibroblast cells transformed by Tax from human T-cell leukemia virus type 1 was studied using the reverse transcriptase polymerase chain reaction differential display technique. The analysis revealed eight genes that were upregulated and one gene that was suppressed in Tax-transformed cells. Interestingly, at least four of the upregulated genes were interferon-stimulated genes. Promoter analysis of the 2′,5′-oligoadenylate synthetase gene, which was activated in both Tax-transformed Rat-1 cells and primary adult T-cell leukemia cells, demonstrated that Tax indirectly activates its interferon-responsive enhancer element in a nuclear factor-κB pathway-dependent manner, indicating a close association of interferon signaling with the transformation by Tax.

Identification of Multiple Differentially Expressed Messenger RNAs in Normal and Pathological Trophoblast

In an attempt to assess the molecular basis of phenotypic alterations present in the gestational trophoblastic diseases (GTDs) and to identify genes whose expression is specifically associated to these placental proliferative disorders we performed differential display techniques. Initially 19 candidate gene fragments were identified and differential expression was confirmed in eight of these fragments by Northern blot analysis. At the mRNA level ribosomal L26 (rL26), ribosomal L27 (rL27), a new Krüppel type zinc finger protein and TIS11d were preferentially expressed in normal early placenta (NEP) relative to complete hydatidiform mole (CHM), persistent gestational trophoblastic disease (PGTD) and choriocarcinoma JEG-3 cell line. In contrast, heterogeneous ribonucleoprotein A1 (hnRNPA1), the ferritin light chain mRNA, and the uncharacterized protein KIAA0992 were predominantly expressed in JEG-3 cell line. Finally, decorin, a prototype member of an expanding family of small leucine-rich proteoglycans, showed high expression in CHM. In addition we demonstrated by immunohistochemistry analysis that increased decorin mRNA in CHM reflected a genuine augmentation in average steady state mRNA levels within cells. Taken together, these findings provide several interesting candidates for regulation of tumorigenic expression as well as early placentation development, including those involved in protein synthesis (rL26 and rL27), metabolism (ferritin light chain), intercellular communication (decorin) and regulation of gene expression (Krüppel-like zinc finger, TIS11d and hnRNPA1). Information about such alterations in gene expression could be useful for elucidating the genetic events associated to gestational trophoblastic pathogenesis, developing new diagnostic markers, or determining novel therapeutic targets.

Can we identify novel angiogenesis inhibitors from cultured embryonic cells?

The first vascular structures in embryos are formed by vasculogensis, the de novo formation of blood vessels from angioblasts. However, angiogenesis, the sprouting of capillaries from pre-existing blood vessels, are also involved in the development of embryonic vascular system in certain organs, such as the kidney or the brain. These facts led us to speculate that embryonic tissues could produce potent angiogenesis inhibitors as well to refine the primitive vascular development. In the present study, we found that two different types of cultured human embryonic cells, 293T and WI-38, produced soluble factors that completely inhibited angiogenic effects of vascular endothelial growth factor on microvascular endothelial cells. Since angiogenesis is related to various pathological states, including tumor growth and metastasis and diabetic retinopathy, to identify novel potent angiogenesis inhibitors from cultured embryonic cells by differential display techniques or DNA microarray technology might be a valuable strategy to develop a novel therapeutic approach.

Differential display techniques to identify tumor suppressor gene pathways.

Differential display techniques to identify tumor suppressor gene pathways.

Identification of cellular mRNA targets for RNA-binding protein Sam68.

Sam68 (Src-associated in mitosis, 68 kDa), a nuclear RNA-binding protein, has been postulated to play a role in cell-growth control as a modulator of signal transduction and activation of RNA metabolism. Although Sam68 was demonstrated to bind to the UAAA sequences in synthetic oligoribonucleotides and poly(U) homopolymers in vitro, the legitimate cellular mRNA target remained unclear. By using the differential display and cDNA-representational difference analysis techniques, followed by reverse transcription polymerase chain reaction of RNAs co-immunoprecipitated with Sam68 from a HeLa cell lysate, we identified 10 mRNA species that bind in vivo to Sam68 in an RNA-binding domain-dependent manner. Among them, the mRNA species for hnRNP A2/B1 and beta-actin were found to bind prominently in vivo as well as in vitro, suggesting the possible involvement of Sam68 in the post- transcriptional regulation of these genes. Mapping of the Sam68-binding sequence revealed that Sam68 associates with these mRNAs through different nucleotide motifs, UAAA for hnRNP A2/B1 mRNA and UUUUUU for beta-actin mRNA, and that both binding sequences must reside in a loop structure for recognition by Sam68. The results indicated that Sam68 recognizes both the UAAA motif and poly(U) sequences in vivo for binding to cellular target mRNAs.

Signaling pathways mediate the neuroprotective effects of GDNF.

We show that the glial cell line-derived neurotrophic factor (GDNF) activates the PI3K/Akt-signaling pathway in human neuroblastoma cells that express functional Ret-receptor complexes. Consistent with this finding we show PI3K-dependent Bad-inactivation by binding to 14-3-3 proteins in response to GDNF. Using differential display techniques we detected several cDNA clones differentially expressed after treatment with GDNF or 6-OHDA.

Cloning of a grapevine Botrytis-responsive gene that has homology to the tobacco hypersensitivity-related hsr203J.

A cDNA encoding a putative HSR203J-like protein (BIG8.1) was obtained from total RNA isolated from Botrytis cinerea-infected grapevine leaves using differential display and RACE techniques. Real time RT-PCR analysis confirmed that the level of mRNA corresponding to BIG8.1 increased in grapevine leaves during the infection progress by B. cinerea. No significant change in mRNA level was observed in leaves after UV exposure. This expression pattern suggests that BIG8.1 could be a HR-specific marker in grapevine like hsr203J in tobacco.

Interleukin-13 Receptor in Psoriatic Keratinocytes: Overexpression of the mRNA and Underexpression of the Protein

Although several cytokines and their receptors have been involved in the development of psoriasis, the etiology is still unknown. In this study we looked for genes possibly involved in the disease by the reverse transcription-polymerase chain reaction differential display technique in lesional and nonlesional skin biopsies from psoriatic patients. We found the mRNA of the alpha1 chain of the interleukin-13 receptor expressed differentially in psoriatic biopsies. By reverse transcription-polymerase chain reaction, we confirmed an overexpression of the alpha1 chain of the IL-13 receptor and alpha chain of the interleukin-4 receptor mRNA in lesional skin psoriatic biopsies, when compared with skin biopsies from healthy subjects (p<0.01). The nonlesional skin obtained from a region close to a lesional zone in psoriatic patients presented also an overexpression of these mRNA in 50% of the samples. Interleukin-13 and interleukin-4 were not detected either as mRNA or as the proteins in any of the biopsies from psoriatic patients or healthy subjects. A monoclonal antibody to the alpha1 chain of the interleukin-13 receptor detected the receptor in the epidermal keratinocytes of psoriatic patients and of healthy subjects; however, the positive antibody reaction was stronger in skin tissue from healthy subjects than in psoriatic lesional skin tissue (p<0.01), although the mRNA was overexpressed. As interleukin-13 is a pleiotropic immunoregulatory cytokine with a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes, we suggest, based on our results, that the interleukin-13 receptor possibly plays an important part in the early inflammatory process of psoriasis; however, its function is lost in the psoriatic keratinocytes.

CHANGES IN GROWTH AND GENE EXPRESSION INDUCED BY SULFUR DEFICIENCY IN GARLIC

ABSTRACT Sulfur deficiency in garlic Allium sativum L. caused a reduction in growth together with chlorosis and necrosis of leaves. Large differences in shoot sulfur and sulphate concentrations between deficient and high sulfur treatments were only observed after 54 days growth. Using the mRNA differential display technique, a novel cDNA was isolated from shoots grown in sulfur depleted nutrient solution for 24 days. This novel cDNA was constitutively expressed in the shoots during further growth in sulfur depleted solution, but it was undetectable following 30 days recovery with sulfur supplementation. The cDNA sequence demonstrated a high degree of identity with a coat protein gene of a garlic latent carlavirus. The results suggest a possible relationship between low plant sulfur status and the induction of a latent carlavirus in garlic.

CigA, a light-inducible gene involved in vegetative growth in Mucor circinelloides is regulated by the carotenogenic repressor crgA

A strategy based on the differential-display technique has been applied to identify and to isolate potential target genes controlled by crgA, a negative regulator of the light-inducible carotenogenesis in the fungus Mucor circinelloides. This approach resulted in the identification and cloning of the gene cigA, whose expression is repressed by the crgA gene. After a light pulse, there is an immediate though transient accumulation of transcripts of cigA gene. A similar result was obtained when mycelia were exposed to continuous illumination, suggesting the existence of a photoadaptation mechanism in M. circinelloides. The protein sequence deduced from the nucleotide sequence of cigA shows homology to the mouse and human GDP-fucose protein O-fucosyltransferases. Null cigA mutants were generated by gene replacement. Lack of cigA function does not affect carotenogenesis but provokes a slight reduction in vegetative growth that is independent of light. Introduction of wild-type cigA alleles into a cigA − strain restored the wild-type phenotype for vegetative growth. These results suggest that cigA is involved in vegetative growth and, moreover, that crgA controls cellular processes other than carotenogenesis.

Overexpression of FKBP51 in idiopathic myelofibrosis regulates the growth factor independence of megakaryocyte progenitors

Idiopathic myelofibrosis (IMF) is a chronic myeloproliferative disorder characterized by megakaryocyte hyperplasia and bone marrow fibrosis. Biologically, an autonomous megakaryocyte growth and differentiation is noticed, which contributes to the megakaryocyte accumulation. To better understand the molecular mechanisms involved in this spontaneous growth, we searched for genes differentially expressed between normal megakaryocytes requiring cytokines to grow and IMF spontaneously proliferating megakaryocytes. Using a differential display technique, we found that the immunophilin FKBP51 was 2 to 8 times overexpressed in megakaryocytes derived from patients' CD34(+) cells in comparison to normal megakaryocytes. Overexpression was moderate and confirmed in 8 of 10 patients, both at the mRNA and protein levels. Overexpression of FKBP51 in a UT-7/Mpl cell line and in normal CD34(+) cells induced a resistance to apoptosis mediated by cytokine deprivation with no effect on proliferation. FKBP51 interacts with both calcineurin and heat shock protein (HSP)70/HSP90. However, a mutant FKBP51 deleted in the HSP70/HSP90 binding site kept the antiapoptotic effect, suggesting that the calcineurin pathway was responsible for the FKBP51 effect. Overexpression of FKBP51 in UT-7/Mpl cells induced a marked inhibition of calcineurin activity. Pharmacologic inhibition of calcineurin by cyclosporin A mimicked the effect of FKBP51. The data support the conclusion that FKBP51 inhibits apoptosis through a calcineurin-dependent pathway. In conclusion, FKBP51 is overexpressed in IMF megakaryocytes and this overexpression could be, in part, responsible for the megakaryocytic accumulation observed in this disorder by regulating their apoptotic program.

Identification of cadmium-sensitive genes in the Antarctic fish Chionodraco hamatus by messenger RNA differential display

To investigate the ability of cadmium to affect gene transcription in fish, the messenger RNA (mRNA) differential display technique was used to analyze gene expression in the Antarctic icefish Chionodraco hamatus exposed to sublethal doses of cadmium salt. Seven DNA complementary to RNA (cDNA) bands whose steady-state levels of expression significantly changed in response to cadmium exposure were identified. The results obtained show that two groups of genes are affected by cadmium in icefish liver. The first group comprises genes that are up-regulated by the metal: in particular, a gene encoding the heat-shock protein HSP70 and another encoding a protein homologous to GP49 of Sparus aurata egg envelope. The other group comprises genes down-regulated by cadmium. These are the transferrin gene and a gene encoding a protein presenting homology to mouse T2K, a kinase having a role in the prevention of apoptosis. Three cDNAs had no homology to known gene sequences, thus suggesting that may either encode not yet identified proteins, or correspond to untranslated regions of mRNA molecules.

Gene expression changes in Schistosoma mansoni sporocysts induced by Biomphalaria glabrata embryonic cells.

Biomphalaria glabrataembryonic (Bge) cells have been shown to provide favourable environmental conditions for the development of Schistosoma mansoni sporocysts. We investigated the effect of Bge excretory-secretory products on metabolic activity and gene transcription in S. mansoni mother sporocysts. Using the differential-display technique, we identified several sporocyst transcripts regulated by exposure to Bge soluble components. Research in databases indicated that six of the eight differential products analysed were homologous to sequences already present in databases. Two transcripts appeared of interest for schistosome development since they could be associated with cell division and protein synthesis in developing sporocysts. Their up-regulation following contact with cell products was confirmed by semi-quantitative RT-PCR. The first fragment coded for a part of the chaperonin containing T-complex protein gamma subunit-like protein of S. mansoni (SmTCP 1-C). The second one represented a new S. mansoni expressed sequence tag encoding a protein homologous to various glutaminyl-tRNA synthetases (GlnRS). The full-length sequence of SmGlnRS was cloned from adult schistosomes and its primary sequence was compared to other GlnRS. The overexpression of SmTCP-1 and SmGlnRS could be correlated with the metabolic changes observed in Bge-exposed sporocysts.

Isolation of differentially expressed genes from white spot virus (WSV) infected Pacific blue shrimp (Penaeus stylirostris).

To isolate novel cellular factors that are activated or repressed upon WSV infection, the RNA fingerprints of healthy and WSV infected blue shrimp ( Penaeus stylirostris) were compared using the mRNA differential display technique. Thirty-two unique differentially expressed, and one constitutively expressed, cDNA sequences were retrieved. Six of 32 cDNAs showed similarities with the database entries: cDNA 10G32-142 to a shrimp arginine kinase, 22C48-201 to shrimp mitochondrial ATPase gene; 22C47-197, 21G49-203 and 20A55-268 to shrimp ESTs and 20G50-206 to a WSV gene, ORF 116. The constitutively expressed gene showed significant similarity to a yeast elongation factor 1-alpha gene. The expression of a subset of differentially expressed genes (13 of 32) was further evaluated by real-time RT-PCR. Ten of 13 genes showed statistically significant changes in expression between healthy and WSV infected animals suggesting that these genes may play an important role in WSV pathogenesis.

Mammaglobin A: A Promising Marker for Breast Cancer

Most tumor markers in routine clinical practice lack organ specificity (1). For example, CA 15-3, BR 27.29, and carcinoembryonic antigen, which are widely used serum markers for breast cancer, can be increased in patients with most types of adenocarcinoma, especially if distant metastases are present [for a review, see Ref. (2)]. Using a modified differential display technique, Watson and Fleming (3)(4) identified a novel gene that appeared to be expressed only in breast tissue. This gene, which was designated mammaglobin ( MG ), was found to encode a 93-amino acid protein with a predicted molecular mass of 10.5 kDa (4). MG mRNA expression was reported to be increased at least 10-fold relative to nonmalignant breast tissue in 8 of 35 (23%) breast carcinomas (4), and MG protein was detected by immunohistochemistry in 81 of 100 breast cancers (5). MG was detected by quantitative reverse transcription-PCR in 31 of 42 (74%) primary breast cancers (6) and was either undetectable or present at low concentrations in small numbers of nonbreast tissues (4)(5)(6). In 1998, Becker et al. (7) identified a new gene with marked sequence identities to MG . This new gene was named mammaglobin B ( MGB ) (7), and the original MG was redesignated mammaglobin A ( MGA ). Both MGA and MGB and several related genes [e.g., Clara cell 10-kDa protein, lipophilin A, and lipophilin B ( LPB )] are localized in a dense cluster on chromosome 11q12.2 …

Increased expression of a COOH-truncated nucleophosmin resulting from alternative splicing is associated with cellular resistance to ionizing radiation in HeLa cells.

We previously demonstrated that transfecting HeLa cells with the 24 kDa basic fibroblast growth factor-2 (FGF-2) isoform dramatically increased cell survival after irradiation. To investigate genes implicated in this radioresistance acquisition, we compared mRNA expression between radioresistant 24 kDa FGF-2-expressing cells (HeLa 3A) and radiosensitive control HeLa PINA cells using the differential display technique. Of the 32 differentially expressed mRNAs, 1 presented a significant homology with a known gene. This 378 bp fragment presented 100% identity with exon 11 and 12 of human nucleophosmin (NPM) but differed by including a part of intron 9 in its 5' end. The differential expression of this fragment was confirmed using an RNase protection assay. We then cloned the entire corresponding mRNA and showed that it contained all the exons of NPM plus intron 9, suggesting that it was a splicing product of the NPM gene. This variant encoded for a 35-amino acid truncated NPM (NPM2). NPM2 expression was increased in HeLa 3A. To investigate NPM2's role in radioresistance acquisition, we transfected HeLa cells with NPM2 cDNA and analyzed survival after irradiation of the clones obtained. After transfection with NPM2, radiosensitive HeLa cells exhibited a dramatic increase in cell survival after irradiation. Taken together, our results demonstrate that expression of a COOH-truncated NPM form resulting from the alternative splicing of NPM mRNA is able to increase cell survival after irradiation and suggests that it might be involved in cellular response to ionizing radiation.