Differential Display Technique Research Articles

Differential expression of cytochrome oxidase and ALY-family genes in resistant and susceptible tomato cultivars (Solanum lycopersicum) inoculated with Tomato bushy stunt virus

Tomato cultivars (TY20, TY70/84 and TY70/70) were mechanically inoculated with purified virus preparations of Tomato bushy stunt virus (TBSV). Inoculated plant tissues were collected after one day, one week and two weeks post-inoculation, respectively. The P19 gene was amplified using specific RT-PCR from the three examined tomato cultivars. Our main result is that the expression of the P19 gene was high in both TY70/84 and TY70/70, but it was very low in case of TY20 tomato cultivars. Messenger RNA of the inoculated and healthy plants was scanned using the differential display technique to discover the up- and down-regulated genes induced or suppressed in the infected plants. Three down-regulated and four up-regulated genes were observed. Sequence analysis revealed that the 400 bp up-regulated genes amplified from cultivars TY70/84 and TY70/70 were cytochrome oxidase genes. Expression of these genes was highest in cultivar TY70/84, followed by cultivar TY70/70; it was considerably low in cultivar TY20 and the control plants. The other four up-regulated genes belonged to the ALY gene family. We conclude that the expression of P19, cytochrome oxidase, and ALY family genes were highly expressed in inoculated plants with TBSV compared to the control plants, which suggest that these genes are members of the plant innate immune system. Keywords Differential expression; Tomato (Solanum lycopersicum); Tomato bushy stunt virus (TBSV); P19 gene; Cytochrome oxidase; Aly-family genes

Screening of differentially expressed genes of gonads from the pond-reared black tiger shrimp (<I>Penaeus monodon</I>)

In order to clone and analyze the differentially expressed genes of gonads of domesticated broodstock Penaeus monodon Fabricius,total RNA was extracted from the ovary and testis,and differential display technique was performed to screen the differentially expressed genes by using a set of 3 anchored primers for cDNA synthesis and 8 arbitrary primers in the PCR. The differential display DNA bands were separated by denaturing urea-polyacrylamide gel electrophoresis (PAGE) and displayed by silver staining. Finally 173 cDNA fragments were isolated,and 44 cDNA fragments were excised. After cloning and sequencing,11 differences in expression of the screened cDNA fragments were confirmed by real-time RT-PCR,and 10 cDNA fragments proved to be real difference. Results show that these 10 fragments were expressed differently between ovary and testis,7 fragments were expressed significantly higher level in ovary,two fragments were expressed significantly higher level in testis,one fragment was particularly expressed in testis. The nucleotide sequence analysis revealed that two fragments were not homologous to of the known genes or ESTs,may be novel genes. TC1 was only expressed in testis,and there was no any difference between female and male DNA level. TG6-3 revealed a preferential expression level in testes than ovaries,and it was only expressed in male brain but no expression in female brain. This research provided a foundation for further studies of the molecular mechanism of sexual differentiation and gonadal development of Penaeus monodon.

Transient expression of βC1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions.

BackgroundGeminiviruses are emerging plant pathogens that infect a wide variety of crops including cotton, cassava, vegetables, ornamental plants and cereals. The geminivirus disease complex consists of monopartite begomoviruses that require betasatellites for the expression of disease symptoms. These complexes are widespread throughout the Old World and cause economically important diseases on several crops. A single protein encoded by betasatellites, termed βC1, is a suppressor of gene silencing, inducer of disease symptoms and is possibly involved in virus movement. Studies of the interaction of βC1 with hosts can provide useful insight into virus-host interactions and aid in the development of novel control strategies. We have used the differential display technique to isolate host genes which are differentially regulated upon transient expression of the βC1 protein of chili leaf curl betasatellite (ChLCB) in Nicotiana tabacum.ResultsThrough differential display analysis, eight genes were isolated from Nicotiana tabacum, at two and four days after infitration with βC1 of ChLCB, expressed under the control of the Cauliflower mosaic virus 35S promoter. Cloning and sequence analysis of differentially amplified products suggested that these genes were involved in ATP synthesis, and acted as electron carriers for respiration and photosynthesis processes. These differentially expressed genes (DEGs) play an important role in plant growth and development, cell protection, defence processes, replication mechanisms and detoxification responses. Kegg orthology based annotation system analysis of these DEGs demonstrated that one of the genes, coding for polynucleotide nucleotidyl transferase, is involved in purine and pyrimidine metabolic pathways and is an RNA binding protein which is involved in RNA degradation.ConclusionβC1 differentially regulated genes are mostly involved in chloroplast and mitochondrial functions. βC1 also increases the expression of those genes which are involved in purine and pyrimidine metabolism. This information gives a new insight into the interaction of βC1 with the host and can be used to understand host-virus interactions in follow-up studies.

Gene expression associated with the onset of hearing detected by differential display in rat organ of Corti

The exquisite performance of the mammalian hearing organ results from a finely orchestrated array of cell types, and their highly specialized functions are determined by their gene expression profile. In rodents, this profile is established mainly during the first 2 weeks of postnatal maturation. In this paper, we used the differential display technique on the rat organ of Corti to uncover transcripts upregulated in expression between postnatal stages P0 and P14. A total of 176 different genes were identified, the mRNA amount of which increased during early postnatal development. The transcripts code for proteins serving a broad spectrum of cellular functions including intracellular signaling, control of growth/differentiation, regulation of protein synthesis/degradation/modification, metabolism and synaptic function. In addition, the set of upregulated transcripts contained several proteins of yet unknown function, as well as hypothetical proteins and so far unknown mRNA sequences. Thus, this study unravels the broad and specific transcription program that operates the maturation of the mammalian hearing organ. Further, as 49 of the genes found here map to at least one unspecified deafness locus, our study provides candidate genes for these and novel deafness loci.

EINE SEGREGATE FORM VON SALIX CA-PREA, DIE DURCH STECKLINGE VER-MEHRT WERDEN KANN

Differential display technique is an important method to isolate differentially expressed gene. Comparing to other methods like representational difference analysis, suppression subtractive hybridization and serial analysis of gene expression, differential display technique is used in higher frequency. Since it was established in 1992, it has overcome many disadvantages and widened its practical fields through improvements and enhancements by global researchers. In this paper the principle and the main advantages and disadvantages of differential display technique were briefly introduced. Meanwhile, the four progressed aspects in designing primer, reducing false positives, identifying differentially expressed gene and techniques derived from DD were introduced in detail.

Fluorescent differential display analysis of Lactobacillus sakei strains under stress conditions

Lactobacillus ( Lb.) sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors such as temperature, pH and salt concentration. We studied the behaviour of Lb. sakei strains under various growth conditions relative to acid, osmotic and heat stress treatments by a novel fluorescent differential display (FDD) technique. This study obtained the development and the optimization of a technique that allows the identification of genome expression changes, associated with differential microbial behaviour under different stress conditions with a better stress response definition and a better discrimination of starter cultures. DNA sequence information from the FDD products provided an important tool to assess and observe the response to a variety of environmental stimuli and the adaptation to bacterial stress. Our work provided an innovative FDD method, with a high level of reproducibility and quality for studying and probing the knowledge of the relation between differential genome expression and different stresses tolerance.

Identification of genes associated with growth cessation and bud dormancy entrance using a dormancy-incapable tree mutant.

BackgroundIn many tree species the perception of short days (SD) can trigger growth cessation, dormancy entrance, and the establishment of a chilling requirement for bud break. The molecular mechanisms connecting photoperiod perception, growth cessation and dormancy entrance in perennials are not clearly understood. The peach [Prunus persica (L.) Batsch] evergrowing (evg) mutant fails to cease growth and therefore cannot enter dormancy under SD. We used the evg mutant to filter gene expression associated with growth cessation after exposure to SD. Wild-type and evg plants were grown under controlled conditions of long days (16 h/8 h) followed by transfer to SD (8 h/16 h) for eight weeks. Apical tissues were sampled at zero, one, two, four, and eight weeks of SD and suppression subtractive hybridization was performed between genotypes at the same time points.ResultsWe identified 23 up-regulated genes in the wild-type with respect to the mutant during SD exposure. We used quantitative real-time PCR to verify the expression of the differentially expressed genes in wild-type tissues following the transition to SD treatment. Three general expression patterns were evident: one group of genes decreased at the time of growth cessation (after 2 weeks in SD), another that increased immediately after the SD exposure and then remained steady, and another that increased throughout SD exposure.ConclusionsThe use of the dormancy-incapable mutant evg has allowed us to reduce the number of genes typically detected by differential display techniques for SD experiments. These genes are candidates for involvement in the signalling pathway leading from photoperiod perception to growth cessation and dormancy entrance and will be the target of future investigations.

Gene expression profiling of phytoplasma-infected Madagascar periwinkle leaves using differential display

Phytoplasmas are small (0.2-0.8 μm), wall-less, pleiomorphic prokaryotes responsible of numerous economically important plant diseases. They are characterized by a very small genome and are obligate parasites of phloem tissues and some insects that act as vectors of infection. To investigate molecular mechanisms involved in pathogenesis, the differential display technique was here applied to identify plant genes whose transcription was significantly altered in leaves of Madagascar periwinkle (Catharanthus roseus (L.) G.Don) infected by 'Candidatus Phytoplasma pyri'. We detected, reamplified, cloned, and sequenced 16 putative differentially expressed cDNA fragments. Northern blot analysis revealed that seven of the 16 genes identified were up-regulated following phytoplasma infection, while three genes were down-regulated. The remaining six genes did not show significant changes in the level of expression. Identified genes are mainly involved in plant defence/stress responses, protein metabolism and transport, transcriptional regulation, vesicle trafficking, and carbohydrate metabolism. The possible role played by these genes in the phytoplasma infection is discussed.

A novel porcine gene, USP7, differentially expressed in the musculus longissimus from Wujin and Large White pigs

The mRNA differential display technique was performed to investigate differences in the gene expression in the <I>musculus longissimus</I> from Wujin and Large White pigs. A fragment of one differentially expressed gene was isolated and sequenced. A complete cDNA sequence was obtained using the rapid amplification of cDNA ends (RACE) method. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 1 103 amino acids, which is homologous with the ubiquitin specific peptidase 7 (<I>USP7</I>) of four species, rat (identity 98%), human (98%), mouse (98%) and chicken (95%), so it can be defined as the porcine <I>USP7</I> gene. The differences in the USP7 gene expression in muscles from Wujin and Large White pigs were confirmed using semi-quantitative RT-PCR. The gene expression analysis in eight tissues of a Wujin × Large White cross showed that <I>USP7</I> was expressed in all the tissues, except for fat.

Identification of biomarker for determining genotypic potential of nitrogen-use-efficiency and optimization of the nitrogen inputs in crop plants

Worldwide, the nitrogen use efficiency (NUE) for crop plants is of great concern. The burgeoning world population needs crop genotypes that respond to higher nitrogen and show a direct relationship to yield with use of nitrogen inputs, i.e. high nitrogen-responsive genotypes. However, for fulfilling the high global demand of organic produce, it requires the low nitrogen responsive genotypes with greater NUE and grain yields. The lack of knowledge about precise regulatory mechanisms to explain NUE in crop plants hampers the goal of agricultural productivity. Understanding the molecular basis of NUE will enable to provide handle for crop improvement through biotechnological means. With the advent of modern genomics and proteomics approaches such as subtractive hybridization, differential display, and microarray techniques are revolutionizing to identify the candidate genes which play a pivotal role in the regulation of NUE. Beside it, quantitative real-time polymerase chain reaction technology is also being used to establish marker-trait association for NUE. The identification of potential candidate genes/proteins in the regulation of NUE will serve as biomarker(s) for screening genotypes for their nitrogen responsiveness for optimization of nitrogen input in agriculture. This paper describes the molecular basis of NUE with respect to nitrogen metabolism and its intimate relationship with carbon metabolism, use of molecular-physiological-genetics approaches for understanding the role of various genes/proteins, and their validation to use as biomarker(s) for determining genotypic potential for NUE. Since NUE in plants is a complex trait which not only involves the primary process of nitrogen uptake and assimilatory pathways but also a series of events, including metabolite partitioning, secondary remobilization, C-N interactions, as well as molecular signalling pathways and regulatory control outside the metabolic cascades. Therefore, identification of novel nitrogen responsive genes and their cis- and trans-acting gene elements is essential. Thus, fishing out a single gene, biomarker or a master regulator controlling complex trait of NUE could serve as an appropriate strategy for nitrogen management in agriculture.

Development and Optimization of a Fluorescent Differential Display PCR System for Analyzing the Stress Response in Lactobacillus sakei Strains

Lactobacillus sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors. We studied the differential expression of genome in response to different stresses by the fluorescent differential display (FDD) technique. This study resulted in the development and optimization of an innovative technique, with a high level of reproducibility and quality, which allows the identification of gene expression changes associated with different microbial behaviours under different growth conditions.

A novel porcine gene, MAPKAPK3, is differentially expressed in the pituitary gland from mini-type Diannan small-ear pigs and large-type Diannan small-ear pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the pituitary gland from mini-type Diannan small-ear pigs and large-type Diannan small-ear pigs. One novel gene differentially expressed was identified through semi-quantitative RT-PCR and its cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. Nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence analysis revealed that the open reading frame of this gene encodes a protein of 384 amino acids has high homology with the mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) of eight species--cattle (96%), horse (96%), rhesus monkey (93%), rabbit (77%), human (98%), chimpanzee (98%), mouse (94%) and rat (91%)--so that it can be defined as swine MAPKAPK3 gene. This gene was finally assigned GENE ID: 100233192. Computer assisted analysis revealed that the genomic DNA of open reading frame of the swine MAPKAPK3 gene consists of ten exons and nine introns. The phylogenetic tree analysis revealed that the swine MAPKAPK3 gene has a closer genetic relationship with the MAPKAPK3 of cattle. The tissue expression analysis indicated that the MAPKAPK3 mRNA expression levels of large-type Diannan small-ear pigs are ubiquitously increased in various tissues compared to the mini-type Diannan small-ear pigs. Our experiment suggested that the swine MAPKAPK3 gene might play an important role in the growth and development differences between mini-type Diannan small-ear pigs and large-type Diannan small-ear pigs.

Nitric oxide as a signaling molecule in the fission yeast Schizosaccharomyces pombe

Nitric oxide synthases (NOS) catalyze the synthesis of ubiquitous signaling molecule nitric oxide (NO) which controls numerous biological processes. Using a spectrofluorometric NOS assay, we have measured the rate of total NO production in the crude cell extracts of Schizosaccharomyces pombe. NO production was reduced in the absence of NOS cofactors calmodulin and tetrahydrobiopterin, and a competitive NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was able to cause a statistically significant inhibition on the rate of total NO production. These results, for the first time, provide evidence that an enzyme with a NOS-like activity may be present in the fission yeast. In order to assess the possible regulatory roles of NO as a signaling molecule in this yeast, using the differential display technique, we screened for NO-responsive genes whose expression decreased upon exposure to L-NAME and increased in response to an NO donor, sodium nitroprusside treatment. Differential expression patterns of byr1, pek1, sid1, and wis1 genes were confirmed by quantitative real-time PCR. The physiological experiments performed based on the functions and molecular interactions of these genes have pointed to the possibility that NO production might be required for sporulation in S. pombe. Taken together, these findings suggest that NO may function as a signaling molecule which can induce both transcriptional and physiological changes in the fission yeast. Hence, these data also imply that S. pombe can be used as a model system for investigating the mechanisms underlying NO-related complex signaling pathways.

DIFFERENTIAL GENE EXPRESSION IN CHESTNUT BUDS FOLLOWING INFESTATION BY GALL-WASP (DRYOCOSMUS KURIPHILUS YASUMATSU, HYMENOPTERA: CYNIPIDAE)

The chestnut gall-wasp Dryocosmus kuriphilus Yasumatsu, an insect native to China, was found in 2002 for the first time in Europe, and specifically in North western Italy (Cuneo province). The cynipid lays eggs into dormant buds and, in the following year, causes the development of galls on leaves, sprouts and inflorescences, due to the feeding activity of larvae. The wasp has an effect both on growth and yield of the tree. Complete resistance to D. kuriphilus was recently found in the hybrid Bouche de Betizac (C. sativa × C. crenata) and the genetic bases regulating this resistance are being investigated. Accessions of the cultivars Marrone (susceptible) and Bouche de Betizac (resistant) were subjected to an intensive attack of D. kuriphilus, with the purpose of getting infested buds to be examined at different stages of development of larvae. At budburst and just afterwards (March-April) buds were collected for the extraction and isolation of mRNA. cDNA was obtained by reverse transcription and analysed using the differential display technique. Two protocols, differing for the primer design, were applied and the results showed that 13 mer primers in combination with one-base anchored oligo-dT primers produced the best scorable profiles. Differentially expressed bands were cloned, sequenced and blasted with sequences deposited in NCBI and TIGR databases; putative genes were identified. The results showed that early activity of larvae in susceptible genotypes particularly influences the expression of genes involved in differentiation and nourishing; other genes found differentially expressed in infested tissues are involved in the plant response to external stimuli.

Differential display reveals a novel pig gene, PRPF3, which is differentially expressed in Large White versus Wujin skeletal muscle tissues

The mRNA differential display technique was performed to investigate gene expression differences in the longissimus dorsi muscle from Wujin and Large White pigs. A fragment of one differentially expressed gene was isolated and sequenced. A complete cDNA sequence of the gene was obtained using the rapid amplification of cDNA ends method. The open reading frame of this gene encodes a protein of 683 amino acids, which is homologous with the PRP3 pre-mRNA processing factor 3 (PRPF3) of five species: bovine (99%), human (99%), sumatran orangutan (99%), mouse (99%) and chicken (94%). This newly identified gene was respectively defined as the swine PRPF3 gene. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic tree analysis revealed that the swine PRPF3 gene has a closer genetic relationship with the PRPF3 gene of sumatran orangutan.

Differential gene expression profiles in embryos of the lizard Podarcis sicula under in ovo exposure to cadmium

Screening for differentially expressed genes is a straightforward approach to study the molecular basis of contaminant toxicity. In this paper, the mRNA differential display technique was applied to analyze transcriptional regulation in response to cadmium exposure in the lizard embryos. Lizard eggs may be particularly susceptible to soil contamination and in ovo exposure may interfere or disrupt normal physiological function in the developing embryo, including regulation of gene expression. Fertilized eggs of the lizard Podarcis sicula were incubated in cadmium-contaminated soil at 25 °C for 20 days. Gene expression profiling showed 5 down- and 9 up-regulated genes. Four 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. Four fragments exhibited significant sequence similarity with genes encoding novel proteins or ESTs derived from other vertebrates. The remaining genes are mainly involved in molecular pathways associated with processes such as membrane trafficking, signal transduction, cytoskeletal organization, cell proliferation and differentiation. Cadmium also affected the expression of factors actively involved in the regulation of the transcription machinery. Down-regulated genes are mainly associated with cellular metabolism and cell-cycle regulation and apoptosis. All of these differentially expressed genes may represent candidates that function in cadmium responses. The present study leads to an increased understanding of genes and/or the biochemical pathways involved in perturbation of embryo development following cadmium exposure.

A Novel Porcine Gene-erlin2, Differentially Expressed in the Liver Tissues from Meishan and Large White Pigs

The messenger RNA differential display technique was performed to investigate the differences of gene expression in the liver tissues from Meishan and Large White pigs. A fragment of one differentially expressed gene was isolated and sequenced. A complete complementary DNA (cDNA) sequence was obtained using the rapid amplification of cDNA end method. Nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 339 amino acids which have high homology with those of the ER lipid-raft-associated 2 isoform 2 (ERLIN2) of eight species--human (97%), rhesus monkey (97%), rat (96%), horse (97%), cattle (97%), mouse (97%), dog (95%), and red jungle fowl (90%)--so that it can be defined as the swine erlin2 gene. The phylogenetic tree analysis revealed that the swine erlin2 gene has a closer genetic relationship with the erlin2 genes of human and rhesus monkey. The tissue expression profile analysis indicated that the swine erlin2 gene is differentially expressed in detected tissues from Meishan and Large White pigs. Our experiment suggested that the swine erlin2 gene might play an important role in the superabundant fat deposition of Chinese pigs.

Identification of genes expressed as a result of lindane exposure in Oreochromis niloticus using differential display

In order to assess the effect of lindane exposure on gene expression in tilapia ( Oreochromis niloticus), twenty male fish were individually weighted and injected intraperitoneally with a single dose of lindane (19.09 mg/kg bw) using corn oil as a carrier vehicle, while a second group of twenty male fish (controls) was only injected with the carrier vehicle. Groups of four fish each were then sacrificed at 3, 6, 12, 18 and 24 h after treatment application and total RNA was extracted from liver tissue. The differential display (DD) technique was then used to identify differentially expressed cDNA fragments between treatment and control fish. A total of fifty cDNA fragments were isolated and sequenced, from which only four showed homology with genes previously described in other fish species, namely the immunoglobulin heavy chain (IgH), coagulation factor V (FV), casein kinase 2 alpha (CK2a), and the receptor protein-tyrosine-like phosphatase (RPT-LP). The expression of such genes was confirmed using quantitative real time-polymerase chain reaction (QRT-PCR). Results showed that lindane exposure triggered the differential expression of these genes during the first 6, 18 and 24 h subsequent to treatment application, suggesting that lindane exposure can trigger a rapid immune system response in tilapias.

Smoke-water-induced changes of expression pattern in Grand Rapids lettuce achenes

AbstractAerosol smoke and smoke-water can break dormancy and promote seed germination of many plant species. In this study we investigated changes in gene expression after imbibition of light-sensitive Lactuca sativa L. cv. ‘Grand Rapids’ achenes with dilute smoke-water compared to water control samples kept in the dark or continuous light, using the fluorescent differential display technique. Although no difference was detected in the smoke-water versus water control samples germinated in light, smoke-water treatment resulted in the differential display of several expressed sequence tags (ESTs) when compared to water control samples kept in the dark. The most pronounced fragments isolated correspond to known genes related to germination, with functions in cell wall expansion, regulation of translation, the cell division cycle, carbohydrate metabolism and abscisic acid (ABA) regulation. Real-time polymerase chain reaction (PCR) validation revealed that the transcript abundance of the genes, HVA22, short-chain dehydrogenase/reductase and late embryogenesis abundant protein, are upregulated after smoke treatment when compared to control achenes kept in the light. The results indicate that smoke has a dual effect. On the one hand, the smoke can induce genes that may be linked to ABA action, whereas, on the other hand, it elicits a faster germination rate by inducing a similar pattern in gene expression as light treatment. Smoke effects could be manifested mainly through the induction of the cell division cycle, cell wall extension and storage mobilization.

Pituitary tumours: all silent on the epigenetics front

Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to 'uncover' or 'unmask' silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.