Differential Display Approach Research Articles

Identification of Differentially Expressed Genes between “Honeycrisp” and “Golden Delicious” Apple Fruit Tissues Reveal Candidates for Crop Improvement

Cultivars of the same species exhibit a large degree of variation in fruit quality traits, which can be directly influenced by differences in gene expression due to allelic variations and interactions with the environment. For Malus × domestica Borkh. (apple), fruit quality traits, including color, texture, aroma, flavor profile, and shelf life, are of utmost economic importance. In order to identify genes potentially influencing these traits, a direct comparative transcriptome profiling approach, based on the differential display technique, was performed using “Golden Delicious” and “Honeycrisp” apple endocarp and peel tissues. A total of 45 differentially expressed sequence tags were identified between the two apple varieties. Reanalysis of a previously published fruit developmental microarray expression experiment revealed that only one of the 45 sequence tags was represented on the array. Differential expression of 31 sequence tags from the peel tissue was validated using quantitative reverse transcription PCR, confirming the robustness of the differential display approach to quickly identify differentially expressed sequence tags. Among these were genes annotated to be involved in ripening, phytohormone signaling, transcription factors, and fruit texture. This work demonstrates yet again the utility of the differential display technique to rapidly identify genes related to desirable traits.

IDENTIFICATION OF CDNA FRAGMENT TIGHTLY LINKED TO NV AND TM-2 LOCI IN TOMATO

Tm-2 is a resistance gene in tomato to Tomato Mosaic Virus (ToMV), located in heterochromatic region of chromosome nine. Since map based cloning difficult to perform for identify the gene on that region, we apply differential display approach by using two near-isogenic tomato lines (NILs), one without Tm-2 and the other with Tm-2 to identify cDNAs of the transcripts from the region surrounding the Tm-2 locus. Among the 150 combinations of three anchor primers and fifty arbitrary primers, 10 combinations generated cDNA polymorphic bands. Out of them, only one combination of CA6, exhibited polymorphic band under southern blot analysis, subsequently a genetic experiment showed that the CA6 locus tightly linked to the Tm-2 locus. The CA6 fragment also hybridized to genomic DNA fragments from a tomato line carrying Tm-2a, a line of L. peruvianum from which Tm-2a originated, and a tomato line carrying another Tm-2-like gene. A northern hybridization blotting result suggested that the gene corresponding to CA6 fragment was constitutively transcribed.

PnTgs1-like expression during reproductive development supports a role for RNA methyltransferases in the aposporous pathway.

BackgroundIn flowering plants, apomixis (asexual reproduction via seeds) is widely believed to result from failure of key regulators of the sexual female reproductive pathway. In the past few years, both differential display and RNA-seq comparative approaches involving reproductive organs of sexual plants and their apomictic counterparts have yielded extensive lists of candidate genes. Nevertheless, only a limited number of these genes have been functionally characterized, with few clues consequently available for understanding the molecular control of apomixis. We have previously identified several cDNA fragments with high similarity to genes involved in RNA biology and with differential amplification between sexual and apomictic Paspalum notatum plants. Here, we report the characterization of one of these candidates, namely, N69 encoding a protein of the S-adenosyl-L-methionine-dependent methyltransferases superfamily. The purpose of this work was to extend the N69 cDNA sequence and to characterize its expression at different developmental stages in both sexual and apomictic individuals.ResultsMolecular characterization of the N69 cDNA revealed homology with genes encoding proteins similar to yeast and mammalian trimethylguanosine synthase/PRIP-interacting proteins. These proteins play a dual role as ERK2-controlled transcriptional coactivators and mediators of sn(o)RNA and telomerase RNA cap trimethylation, and participate in mammals and yeast development. The N69-extended sequence was consequently renamed PnTgs1-like. Expression of PnTgs1-like during reproductive development was significantly higher in floral organs of sexual genotypes compared with apomicts. This difference was not detected in vegetative tissues. In addition, expression levels in reproductive tissues of several genotypes were negatively correlated with facultative apomixis rates. Moreover, in situ hybridization observations revealed that PnTgs1-like expression is relatively higher in ovules of sexual plants throughout development, from premeiosis to maturity. Tissues where differential expression is detected include nucellar cells, the site of aposporous initials differentiation in apomictic genotypes.ConclusionsOur results indicate that PnTgs1-like (formerly N69) encodes a trimethylguanosine synthase-like protein whose function in mammals and yeast is critical for development, including reproduction. Our findings also suggest a pivotal role for this candidate gene in nucellar cell fate, as its diminished expression is correlated with initiation of the apomictic pathway in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0297-0) contains supplementary material, which is available to authorized users.

Characterization of rainbow trout gonad, brain and gill deep cDNA repertoires using a Roche 454-Titanium sequencing approach

Rainbow trout, Oncorhynchus mykiss, is an important aquaculture species worldwide and, in addition to being of commercial interest, it is also a research model organism of considerable scientific importance. Because of the lack of a whole genome sequence in that species, transcriptomic analyses of this species have often been hindered. Using next-generation sequencing (NGS) technologies, we sought to fill these informational gaps. Here, using Roche 454-Titanium technology, we provide new tissue-specific cDNA repertoires from several rainbow trout tissues. Non-normalized cDNA libraries were constructed from testis, ovary, brain and gill rainbow trout tissue samples, and these different libraries were sequenced in 10 separate half-runs of 454-Titanium. Overall, we produced a total of 3million quality sequences with an average size of 328bp, representing more than 1Gb of expressed sequence information. These sequences have been combined with all publicly available rainbow trout sequences, resulting in a total of 242,187 clusters of putative transcript groups and 22,373 singletons. To identify the predominantly expressed genes in different tissues of interest, we developed a Digital Differential Display (DDD) approach. This approach allowed us to characterize the genes that are predominantly expressed within each tissue of interest. Of these genes, some were already known to be tissue-specific, thereby validating our approach. Many others, however, were novel candidates, demonstrating the usefulness of our strategy and of such tissue-specific resources. This new sequence information, acquired using NGS 454-Titanium technology, deeply enriched our current knowledge of the expressed genes in rainbow trout through the identification of an increased number of tissue-specific sequences. This identification allowed a precise cDNA tissue repertoire to be characterized in several important rainbow trout tissues. The rainbow trout contig browser can be accessed at the following publicly available web site (http://www.sigenae.org/).

Identification of differentially expressed genes in ovaries of chicken attaining sexual maturity at different ages

In poultry as well as in other birds, sexual maturity is one of the important factors influencing female reproduction and egg production. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) differential display approach was used to identify genes related to sexual maturity. Using 54 EcoR I/Mse I selective primer combinations, totally 403 differentially expressed transcript-derived fragments (TDFs) were isolated, 27 of which belong to 25 unigenes. By real-time quantitative PCR (qPCR), the expression pattern of 13 genes was confirmed; among them, four genes including ZNF183 (P < 0.01), KIAA0700, CCT6A, and 23e 15 (P < 0.05) are significantly up-regulated and one gene (Loc418883) is significantly down-regulated (P < 0.01) in sexually mature ovaries compared to immature ones. The mRNA expression dynamics of ZNF183, CCT6A, 23e 15 and Loc418883 were further investigated in ovaries of 70-, 300- and 500-day-old commercial egg-laying hens: the expression level of CCT6A was the highest in 300-day-old hens (P < 0.05), while that of Loc418883 in 500-day-old hens was significantly higher than the other two stages (P < 0.01). The expression levels of ZNF183 and 23e 15 in ovary increase significantly from 70-day-old hens (P < 0.01) and 300-day-old (P < 0.05) to 500-day-old hens, respectively. The consistence of CCT6A expression and egg-laying performance suggests that CCT6A likely plays important role in sexual maturity in hens.

Characterization of a F-box gene up-regulated by phytohormones and upon biotic and abiotic stresses in grapevine

F-box proteins are key components of the ubiquitin (Ub)/26S proteasome pathway that mediates selective degradation of regulatory proteins involved in a wide variety of cellular processes affecting eukaryotic cells. In plants, F-box genes form one of the largest multigene superfamilies and control many important biological functions. Among the F-box genes characterized to date only few have been involved in the regulation of plant defense responses. Moreover, no F-box genes have been studied and characterized in grapevine. Using a differential display approach we isolated a F-box gene (BIG-24.1), which is up-regulated during Botrytis cinerea infection of grapevine leaves. BIG-24.1 encodes a polypeptide of 386 amino acids with a conserved F-box domain in the N-terminus region and a kelch domain. By investigating expression profiles of BIG-24.1, we show that the gene expression is strongly stimulated in B. cinerea infected berries and in grapevine cells challenged by MAMP rhamnolipids, a non-host bacterium and an endophytic rhizobacterium. The gene is also strongly induced by abiotic stresses including UV-C and wounding or by salicylic acid, methyl-jasmonate, ethylene and abscisic acid that are known to be involved in defense signalling pathways. In addition, sequence analysis of the BIG-24.1 promoter revealed the presence of several regulatory elements involved in the activation of plant defense responses.

Isolation, characterization and expression of an elongation factor 1α gene in potato (Solanum tuberosum) cell cultures

Using a differential display (DDRT‐PCR) approach to screen genes involved in response to osmotic stress, we isolated six cDNA clones from potato cell cultures. One of these, clone #54, is a member of the gene family encoding the α‐subunit of translation elongation factor 1 (EF‐1α). After sequencing, this clone showed a deduced amino acid sequence of 447 residues. Southern blot analysis revealed that this EF‐1α is a member of a family consisting of at least nine genes. The expression of EF‐1α increased upon osmotic stress mediated by polyethylene glycol (PEG) but was unaffected by abscisic acid (ABA) treatment. Salt also increased EF‐1α expression but with a different kinetics. These results indicate that we cloned an EF‐1α in potato which is ABA responsive, independent of osmotic stress. Abbreviations: DDRT‐PCR, differential display reverse transcriptase‐polymerase chain reaction; PEG, polyethylene glycol; RACE, rapid amplification of cDNA ends; RT‐PCR, reverse transcriptase‐polymerase chain reaction

Negative regulation of TIMP1 is mediated by transcription factor TWIST1

TWIST1 is involved in tumor invasion and metastasis by promoting epithelial-to-mesenchymal transition. However, the molecular target of TWIST1 involving this mechanism is poorly understood. To identify the TWIST1-regulated genes, we established the Saos-2 cells stably expressing TWIST1 gene by transfecting TWIST1 cDNA into the cells and performed a differential display approach by using annealing control primers. Here, we report that one of the genes that were down-regulated in TWIST1 expressing cells is predicted to be TIMP1. TIMP1 has been reported as the naturally occurring specific inhibitor of matrix metalloproteinases (MMPs). Overexpression of TWIST1 gene suppressed the expression of TIMP1 mRNA but had no effect on TIMP2 and MMP-2 expression, as determined by semi-quantitative RT-PCR. We showed that TWIST1 was up-regulated in SCCBHY cells, which have a strong capacity of invasion into mandibular bone compared with SCCHN cells. Our present results suggest that TWIST1 is involved in tumor invasion by regulating the expression of TIMP1.

THE OIL PALM "MANTLED" SOMACLONAL VARIATION: A MODEL FOR EPIGENETIC STUDIES IN HIGHER PLANTS

Epigenetics is the study of heritable changes in gene function that occur without a change in the DNA sequence. In plants, although epigenetic mechanisms help to protect cells from parasitic elements, this defense can complicate the genetic engineering process through transcriptional gene silencing. These phenomena have economic relevance in the case of somaclonal variation: a genetic and phenotypic variation among clonally propagated plants from a single donor genotype. In oil palm (Elaeis guineensis Jacq), approximately 5% of somatic embryo-derived palms show abnormalities in their floral development. The mantled variant phenotype offers a very interesting model, because somaclonal variation alters the expression of genes coding, directly or indirectly for the flower structure and DNA hypomethylation has already been clearly demonstrated in somaclonal variants. Methylation sensitive DNA markers have been investigated using MSAP and MS-RFLP techniques and the three different classes of DNA methyltransferases have been studied comparatively in normal and variant material. Differential display and microarray approaches are currently developed in order to provide a range of putative candidate genes displaying expression which might be regulated through DNA methylation and/or chromatin remodeling. We have identified floral homeotic genes of the MADS box transcription factor family which are affected by the chain of events resulting in the mantled abnormality. Our aim is to describe specific molecular events which could be used for the development of markers of epigenetic instability in plants. These markers will be integrated in a strategy aimed at the identification of in vitro treatments which are prone to generate epigenetic variability in somatic embryogenesis-based micropropagation processes. (Resume d'auteur)

Induction of Dlk1 by PTTG1 Inhibits Adipocyte Differentiation and Correlates with Malignant Transformation

Pituitary tumor-transforming gene-1 (PTTG1) is an oncogene highly expressed in a variety of endocrine, as well as nonendocrine-related cancers. Several tumorigenic mechanisms for PTTG1 have been proposed, one of the best characterized being its capacity to act as a transcriptional activator. To identify novel downstream target genes, we have established cell lines with inducible expression of PTTG1 and a differential display approach to analyze gene expression changes after PTTG1 induction. We identified dlk1 (also known as pref-1) as one of the most abundantly expressed PTTG1 targets. Dlk1 is known to participate in several differentiation processes, including adipogenesis, adrenal gland development, and wound healing. Dlk1 is also highly expressed in neuroendocrine tumors. Here, we show that PTTG1 overexpression inhibits adipogenesis in 3T3-L1 cells and that this effect is accomplished by promoting the stability and accumulation of Dlk1 mRNA, supporting a role for PTTG1 in posttranscriptional regulation. Moreover, both pttg1 and dlk1 genes show concomitant expression in fetal liver and placenta, as well as in pituitary adenomas, breast adenocarcinomas, and neuroblastomas, suggesting that PTTG1 and DLK1 are involved in cell differentiation and transformation.

Differential Display Analysis of cDNA Involved in Microbial Mats Response after Heavy Fuel Oil Contamination

Pristine microbial mats from Camargue salterns (France) maintained in microcosm were contaminated by Erika fuel oil in order to identify gene sequences induced in response to heavy fuel oil contamination. The differential display approach was adapted to detect differentially expressed mRNA in complex bacterial communities. Among the six differentially expressed (DD) cDNA fragments isolated, one was identified and associated with an ABC-type efflux pump. A second DD-fragment was related to a conserved hypothetical protein found in many different bacterial species. Despite differentially expressed fragments could not be clearly identified, this study reveals new perspectives for the improvement of our knowledge on the response of microbial community after petroleum contamination.

Panicum repensとAcacia mangiumの酸性ストレス応答遺伝子のディファレンシャルディスプレイ法による網羅的解析

Panicum repensとAcacia mangiumの酸性ストレス応答遺伝子のディファレンシャルディスプレイ法による網羅的解析

Identification and characterization of novel senescence‐associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.

A New Prokaryotic Farnesyldiphosphate Synthase from the Octocoral Eunicea Fusca: Differential Display, Inverse PCR, Cloning, and Characterization

We recently reported that the biosynthesis of fuscol, a diterpene from the octocoral Eunicea fusca, is inducible by the application of plant signaling factors such as salicylic acid to the coral's algal symbiont. In this study, an mRNA differential display approach has been employed with the dinoflagellate symbiont of this octocoral which has led to the isolation of a farnesyldiphosphate synthase (FPPS) that was transcriptionally activated under conditions that led to an induction of fuscol biosynthesis. Using a degenerate primer based on the aspartate-rich motifs found in prenylsynthases and a cassette ligation strategy, we report the cloning of the complete FPPS associated with the E. fusca dinoflagellate symbiont Symbiodinium sp. The protein exhibited the enzymatic properties associated with FPPS, namely, the synthesis of farnesyl diphosphate from geranyldiphosphate and isopentenyl diphosphate. The amino acid sequence of this FPPS has a high sequence similarity (82%) to known archaeal isoprenyl diphosphate synthases. This is the first description of a prokaryotic FPPS derived from a marine source.

SEPS1 Gene is Activated during Astrocyte Ischemia and Shows Prominent Antiapoptotic Effects

Contrarily to neurons, astrocytes can survive short periods of ischemia. We have searched for genes implicated in astrocyte resistance to ischemia using oxygen and glucose deprivation (OGD) as a stroke model. A RNA differential display approach uncovered the OGD induction of selenoprotein-S-encoding gene SEPS1. This endoplasmic reticulum (ER) resident protein is known to promote cell survival regulating the ER stress as well as inflammation. We found that suppression of SEPS1 by small interfering RNA severely increases astrocyte injure caused by OGD, suggesting that selenoprotein S protects astrocytes against ischemia. Our data also support that modulation of ER stress is implicated in this effect.

Identification, isolation and characterization of a CC-NBS-LRR candidate disease resistance gene family in grapevine

Plasmopara viticola causes downy mildew of grapevine, one of the most important diseases in viticulture. Resistance to this oomycete is present in American and Asian Vitis species, while traditional European Vitis vinifera cvs. for wine and table grape production are susceptible. Breeding aims to achieve resistance through introgression, but the molecular mechanisms are still unknown. Therefore, the differential display approach was used to detect grapevine genes involved in defense. P. viticola sporangia were applied to the lower leaf surface of in vitro plants of the resistant Vitis riparia selection ‘Gloire de Montpellier’ and susceptible cv. ‘Riesling’. Controls were treated with sterile water. Messenger RNAs extracted 12 h post infection were subjected to differential display. Seven transcripts appeared specifically induced during the incompatible interaction. Sequencing showed that they build three classes. One of them, named VRP1, represented by three transcripts of almost identical sequence but differing lengths, showed clear homology to resistance genes of the NBS-LRR type from other plants. Northern hybridizations confirmed its elevated expression in the resistant ‘Gloire de Montpellier’. Redundancy of VRP1 PCR products from V. riparia prevented PCR-walking, so the VRP1 genes were isolated from a BAC-library of the resistant cv. ‘Regent’. Three genes matching the original VRP1 sequences were found within a BAC clone carrying a 134,392 bp insertion. These were referred to as VRP1-1, 1-2 and 1-3. They encode proteins of 798, 811 resp. 813 amino acids and exhibit the structure of CC-NBS-LRR resistance genes. They were genetically mapped to linkage group 10.

VERTICILLIUM DAHLIAE TOXINS INDUCE CHANGES IN EXPRESSION OF A PUTATIVE HISTONE H2B AND A PUMILIO/PUF RNA-BINDING PROTEIN IN ARABIDOPSIS THALIANA

To gain insight into the molecular mechanisms of plant defence response to Verticillium dahliae, we identified the genes whose expression patterns were altered during early response to V. dahliae toxins (VD-toxins) in Arabidopsis seedlings using the mRNA differential display approach. Four true positive bands (DD1, DD2, DD3 and DD4) were confirmed from the 30 differentially displayed bands. The deduced nucleotide sequence of the DD1 and DD2 clones showed strong homology with genes encoding a putative histone H2B and a pumilio /Puf RNA-binding protein, respectively. Quantitative real-time RT-PCR assays suggested that the expression pattern of genes of DD1 and DD2 in two ecotypes of Arabidopsis were clearly different. The results indicate that the putative histone H2B and pumilio /Puf RNA-binding protein encoded by DD1 and DD2 genes may be involved in regulation of the defence responses against VD-toxins in Arabidopsis and the pumilio/Puf RNA-binding protein plays a more important role in the defence responses.

Calcipotriol Affects Keratinocyte Proliferation by Decreasing Expression of Early Growth Response-1 and Polo-like Kinase-2

Calcipotriol is a potent drug for topical treatment of psoriasis because it manages to inhibit keratinocyte proliferation. In the present study we investigated the effects of calcipotriol on gene expression in human keratinocytes in terms of mechanism of how calcipotriol decreases proliferation. Cell proliferation was analyzed by MTT assay. The differential display approach together with qPCR was used to assess the gene expression after treatment. In addition, Western immunoblotting revealed differences on the protein level. Finally, transfection of the KCs with specific small interfering RNA determined the genes necessary to inhibit proliferation. KCs proliferation was decreased in a concentration-dependent manner. Moreover, calcipotriol dowregulated the expression of two proliferation factors: early growth response-1 (EGR1) and polo-like kinase-2 (PLK2). The protein levels of EGR1 and PLK2 were also decreased. Specific siRNA against EGR1 and PLK2 in KCs resulted in marked reduction of EGR1 and PLK2 expression. In both cases, the reduction resolved in the decreased proliferation of KCs. This study provides a new insight into how calcipotriol affects proliferation of keratinocytes by decreasing the expression of EGR1 and PLK2. Furthermore, the results offer groundwork for developing novel compounds for the treatment of hyperproliferative skin disorders like psoriasis.

The WD40-repeat protein Pwp1p associates in vivo with 25S ribosomal chromatin in a histone H4 tail-dependent manner

The tails of core histones (H2A, H2B, H3 and H4) are critical for the regulation of chromatin dynamics. Each core histone tail is specifically recognized by various tail binding proteins. Here we screened for budding yeast histone H4-tail binding proteins in a protein differential display approach by two-dimensional gel electrophoresis (2DGE). To obtain highly enriched chromatin proteins, we used a Mg2+-dependent chromatin oligomerization technique. The Mg2+-dependent oligomerized chromatin from H4-tail deleted cells was compared with that from wild-type cells. We used mass spectrometry to identify 22 candidate proteins whose amounts were reduced in the oligomerized chromatin from the H4-tail deleted cells. A Saccharomyces Genome Database search revealed 10 protein complexes, each of which contained more than two candidate proteins. Interestingly, 7 out of the 10 complexes have the potential to associate with the H4-tail. We obtained in vivo evidence, by a chromatin immunoprecipitation assay, that one of the candidate proteins, Pwp1p, associates with the 25S ribosomal DNA (rDNA) chromatin in an H4-tail-dependent manner. We propose that the complex containing Pwp1p regulates the transcription of rDNA. Our results demonstrate that the protein differential display approach by 2DGE, using a histone-tail mutant, is a powerful method to identify histone-tail binding proteins.

Induction of the adenoma-carcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells

TFF1 is overexpressed in inflammatory diseases and human cancers of the digestive and urogenital systems. To examine the transforming potential of TFF1 in human colon epithelial cells, premalignant PC/AA/C1 adenoma cells (PC) derived from a patient with familial adenomatous polyposis (FAP) were transformed by the TFF1 cDNA and used as a model of the adenoma-carcinoma transition. Constitutive expression of TFF1 increased anchorage-independent cell growth in soft agar, and induced or potentiated the growth of colon PC-TFF1 and kidney MDCKts.src-TFF1 tumor xenografts in athymic mice. This resulted in reduction of thapsigargin-induced apoptosis and promotion of collagen type I invasion through several oncogenic pathways. Using the differential display approach to identify TFF1 target genes, we found that the dual specific phosphatases Cdc25A and B implicated in cell cycle transitions are strongly upregulated under active forms in both PC-TFF1 and HCT8/S11-TFF1 colon cancer cells. Accordingly, TFF1 expression is absent in normal human colon crypts but is induced in correlation with Cdc25a and b transcript levels and tumor grade in familial and sporadic colon adenomas and carcinomas. We propose that TFF1 and Cdc25A-B cooperate with other dominant oncogenic pathways to induce the adenoma and adenocarcinoma transitions. Agents that target TFF1/Cdc25 signaling pathways may be useful for treating patients with TFF1-positive solid tumors.