Display Reverse Transcriptase Polymerase Chain Research Articles

New gene functions are involved in the thermotolerance of the wild wheat relative Aegilops umbellulata

Wheat is one of the most important food crops in the world for human consumption, like all plants it is exposed to environmental stresses including high temperatures. The deleterious effect of high temperatures negatively affects plant growth and development, leading to reduced viability and yield. These effects can be reduced by improvement of thermotolerance through innovative breeding strategies, based on the expansion of the genetic pool available, by exploring important genetic functions from wheat wild progenitors. Improving the genetic thermotolerance characteristics of wheat requires greater understanding of genetic bases of thermotolerance, through identification of high temperature stress related genes. A good source of new useful alleles is given by Aegilops species characterized by thermotolerant habits.In this study we have classified as thermotolerant or thermosensitive, on the basis of physiologic tests, some accessions of wheat wild relative species belonging to Aegilops and Triticum genera. A thermotolerant accession of Aegilops umbellulata (AUM5) was selected, subjected to different thermal treatments and analyzed at transcriptional level. By differential display reverse transcriptase polymerase chain reaction (DDRT-PCR), we investigated modulation of gene expression elicited by heat treatments. This approach allowed the identification of various transcript-derived fragments (TDFs) produced by AUM5 in response to different thermal treatments. The functions of the inducible unique genes in the molecular determination of thermotolerance process are discussed.

Photoperiodic mediation of differential gene expression in Scytosiphon lomentaria (Phaeophyceae)

SUMMARYScytosiphon lomentaria (Lyngbye) Link is a species of brown algae common in the eulittoral and sublittoral zones of temperate oceans. Some populations exhibit an asexual heteromorphic life cycle that is regulated by photoperiod. While this response is mediated by blue light, virtually nothing is known regarding downstream genes that are activated or repressed during photo‐morphogenesis in this organism. This study examined photoperiodic mediation of differential gene expression in S. lomentaria using isolates obtained from the eastern coast of Virginia, USA. Putative differentially expressed transcripts were produced by differential display reverse transcriptase polymerase chain reaction (PCR), cloned and sequenced. The sequences where identified using bioinformatics tools, and the expression patterns analyzed and validated by quantitative Real‐Time PCR. Of the 133 putative differentially expressed bands selected, 92 were successfully sequenced and analyzed. Of these, 27 could not be matched to known sequences. Twenty‐eight transcripts were found to have high similarity to sequences in the databases and transcript levels were quantified. Seventeen were upregulated under a short day, one was downregulated, five showed no expression differences, four did not amplify and one was the reference gene. These analyses provide new insights into the genetic response to photoperiod‐mediated morphogenesis in S. lomentaria.

Differential display analysis of cDNA fragments potentially involved in Nostoc flagelliforme response to osmotic stress

Nostoc flagelliforme is a terrestrial, highly drought-tolerant cyanobacterium. However, the molecular mechanism of its drought adaptation still remains little known. In this paper, we attempted to isolate those osmotic stress-regulated cDNA fragments by traditional differential display reverse transcriptase–polymerase chain reaction (DDRT-PCR). The PCR primers were chosen and evaluated from the reported HIP1D sequence-based primers and Ea/Es primers. The physiologically recovered samples were treated with 0.7 M sorbitol for 3 h. A total of 21 cDNA fragments were obtained by DDRT-PCR, and among them, 16 are up-regulated by osmotic stress. BlastX analyses showed that these up-regulated cDNA fragments are most related to ATPases, methyltransferases, transporters, glutathione synthase, signal transduction-associated components, and other unknown proteins. They are potentially important genes for N. flagelliforme to cope with drought stress since they are distinctly up-regulated by osmotic stress. In addition, five cDNA fragments are down-regulated by osmotic stress, such as the cDNA fragment of PSII protein. They may represent those genes involved in normal physiological processes and thus were inhibited by the osmotic stress. Taken together, the interpretation of the functional clues of these osmotic stress-responsive cDNA fragments has provided us an insight into the molecular mechanism of N. flagelliforme coping with the early phase of drought stress.

Identification of salt stress-induced transcripts in barley leaves by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR)

Identification of salt stress-induced transcripts in barley leaves by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR)

Differential Display RT-PCR Reveals Genes Associated with Lithium-Induced Neuritogenesis in SK-N-MC Cells

Lithium is shown to be neurotrophic and protective against variety of environmental stresses both in vitro as well as in vivo. In view of the wider clinical applications, it is necessary to examine alterations in levels of expression of genes affected by lithium. Lithium induces neuritogenesis in human neuroblastoma cell line SK-N-MC. Our aim was to elucidate genes involved in lithium-induced neuritogenesis using SK-N-MC cells. The differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) technique was used to study gene expression profiles in SK-N-MC cells undergoing lithium-induced neuritogenesis. Differential expression of genes in control and lithium (2.5 mM, 24 h)-treated cells was compared by display of cDNAs generated by reverse transcription of mRNA followed by PCR using arbitrary primers. Expression of four genes was altered in lithium-treated cells. Real-time PCR was done to confirm the levels of expression of each of these genes using specific primers. Lithium significantly up-regulated NCAM, a molecule known to stimulate neuritogenesis, occludin, a molecule participating in tight junctions and PKD2, a molecule known to modulate calcium transport. ANP 32c, a gene whose function is not fully known yet, was found to be down-regulated by lithium. This is the first report demonstrating altered levels of expression of these genes in lithium-induced neuritogenesis and contributes four hitherto unreported candidates possibly involved in the process.

Gene Expression Profiling of Metabolism-Related Genes between Top Round and Loin Muscle of Korean Cattle (Hanwoo)

Using differential display reverse transcriptase polymerase chain reaction, we detected 11 differentially expressed genes between top round and loin muscle in Korean cattle (Hanwoo). In the loin muscle, the lightness (L*) value (P<0.01) and marbling fat content (P<0.01), which are important factors in determining meat quality, were higher than in top round muscle. Three of the 11 genes were validated as significant genes between two types of muscle by real-time polymerase chain reaction (P<0.05). To determine whether the three genes were associated with meat quality traits, a regression analysis was preformed. The result demonstrated that two genes (NADH dehydrogenase 2 and cytochrome oxidase III), which are involved in oxidative phosphorylation in mitochondria, were significantly correlated with marbling fat content in the loin muscle (P<0.01), while two genes were not significant with marbling fat content in top round muscle. No significant effects for two genes on other meat quality traits such as meat color (redness and yellowness value), Warner-Bratzler shear force, and water-holding capacity were detected in this study.

Exploration of senescence-associated genes by differential display reverse transcription polymerase chain reaction: Prosaposin as a novel senescence-associated gene

To understand the process and molecular mechanism of cellular aging, we explored novel senescence-associated genes using rat tissues with different age. Using total RNAs from 6 and 24-month old rat tissues, differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) were performed and 12 differentially expressed genes (DEGs) which were down- or up-regulated in aged rat tissues were found. Among these DEGs, the level of prosaposin mRNA was elevated of in senescent human dermal fibroblast (HDF) and human umbilical vein endothelial cells (HUVECs). It was further confirmed that expression of prosaposin was up-regulated in prematurely senescent HUVECs induced by hydrogen peroxide or interferon-gamma treatment. Taken together, we report the up-regulation of prosaposin in the senescent HDF and HUVECs as well as in hydrogen peroxide or interferon-gamma induced prematurely senescent HUVECs, suggesting that prosaposin might be a novel senescence-associated gene.

Molecular Identification and Characterization of a Gene Associated with the Onset of Tapping Panel Dryness (TPD) Syndrome in Rubber Tree (Hevea brasiliensis Muell.) by mRNA Differential Display

In rubber tree (Hevea brasiliensis), tapping panel dryness (TPD) syndrome is considered as a complex physiological disorder which affects latex biosynthesis. To identify differentially expressed genes between healthy and TPD-affected trees, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) analysis was performed. We isolated 10 differentially expressed cDNA fragments of which one cDNA encoding a putative TOM20 like protein was identified. The cDNA (1,024 bp), corresponding to the HbTOM20 gene (H evea b rasiliensis Translocase of the Outer Mitochondrial Membrane), contained an open reading frame to code for 202 amino acid protein with a theoretical pI value of 9.5 and the calculated protein M (W) was 23.5 kDa. The predicted amino acid sequence contained conserved domains of TOM20 like proteins in the N-terminal. The protein HbTOM20 has 32% and 27% similarity to Populus TOM20 and Solanum TOM20, respectively. Both semi-quantitative RT-PCR and Northern blot results revealed that the HbTOM20 expression was significantly down-regulated in TPD-affected trees compared to healthy one. Accumulation of HbTOM20 mRNA transcripts was significantly higher in the bark tissues collected from healthy region than that of partially affected by TPD (partially dried) while barely detectable in completely TPD-affected area. Differential expression pattern was noticed in three rubber clones representing various degrees of TPD tolerance. These results suggest that down-regulation of HbTOM20 in TPD-affected trees may play an important role in alteration of mitochondrial metabolism resulting in impaired latex biosynthesis.

Changes in gene expression following androgen receptor blockade is not equivalent to androgen ablation by castration in the rat ventral prostate

Involution of the rat ventral prostate and concomitant modulation of gene expression post-castration is a well- documented phenomenon. While the rat castration model has been extensively used to study androgen regulation of gene expression in the ventral prostate,it is not clear whether all the gene expression changes post-castration are due to androgen depletion alone. To obtain insights into this, we performed differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) which resulted in the identification of castration and/or flutamide-regulated genes in the rat ventral prostate. These include clusterin, methionine adenosyl transferase II alpha, and prostate-specific transcripts such as PBPC1BS, S100RVP and A7. While clusterin, PBPC1BS and methionine adenosyl transferase II alpha are regulated by both castration and flutamide, S100 RVP and A7 are regulated by castration alone. Interestingly, we show that flutamide, unlike castration, does not induce apoptosis in the rat ventral prostate epithelium, which could be an underlying cause for the differential effects of castration and flutamide treatment. We propose that castration leads to enrichment and depletion of stromal and epithelial cell types, respectively, resulting in erroneous conclusions on some of the cell type-specific transcripts as being androgen regulated.

Comparison of gene expression patterns between porcine cumulus-oocyte complexes and naked oocytes

Several layers of cumulus cells surround the oocyte in the antral ovarian follicle throughout the follicular development until ovulation. The cumulus cells play an important supportive and regulative role in oocyte development and maturation via intercellular communications between oocytes and cumulus cells. Using the differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) and semi-quantitative RT-PCR methods, we compared the mRNA expression patterns in porcine oocytes from two sources: cumulus-oocyte complexes (COCs) and naked oocytes (NOs). After a small scale screen, 14 differentially expressed mRNAs were cloned, sequenced, and their expression patterns were verified by semi-quantitative RT-PCR. It was confirmed that four mRNAs including three genes (PELP1, Myo5b and CAST) and a new EST (Expressed Sequence Tag) are expressed preferentially in the oocytes of COCs rather than in NOs. Previous studies of the three genes suggested that they are mainly involved in oestrogen receptor regulation, membrane trafficking, organelle transport, cellular signalling and some other cellular processes. These results suggest that the aberrant of gene expression patterns detected in the oocytes of NOs compared with COCs explains their reduced quality in terms of development and maturation. In conclusion, these differentially expressed mRNAs may be involved in cellular interactions between oocytes and cumulus cells and thus could be considered as essential genes for the competence of oocytes. Keywords: Differential gene expression, DD-RT-PCR, porcine oocytes, cumulus South African Journal of Animal Science Vol. 37 (1) 2007: pp. 57-63

Novel flutamide regulated genes in the rat ventral prostate: differential modulation of their expression by castration and flutamide treatments

To identify flutamide regulated genes in the rat ventral prostate. Total RNA from ventral prostates of control and flutamide treated rats were isolated. Differentially expressed transcripts were identified using differential display reverse transcriptase polymerase chain reaction. The effect of castration on the expression of flutamide-regulated transcripts was studied. We have identified beta2-microglobulin, cytoplasmic FMR1 interacting protein 2 and pumilio 1 as flutamide induced and spermine binding protein and ribophorin II as flutamide repressed targets in the rat ventral prostate. Although flutamide treatment caused an induction of pumilio 1 mRNA, castration had no effect. Castration and flutamide treatments exert differential effects on gene expression. Flutamide might also have direct AR independent effects, which might have implications in the emergence of androgen independent prostate cancer and the failure of flutamide therapy.

Differential gene expression during terminal erythroid differentiation

Terminal erythroid differentiation in mammals is the process whereby nucleated precursor cells accumulate erythroid-specific proteins such as hemoglobin, undergo extensive cellular and nuclear remodeling, and ultimately shed their nuclei to form reticulocytes, which then become mature erythrocytes in the circulation. Little is known about the mechanisms that enable erythroblasts to undergo such a transformation. We hypothesized that genes involved in these mechanisms were likely expressed at restricted times during the differentiation process and used differential display reverse transcriptase polymerase chain reaction as a first step in identifying such genes. We identified three differentially expressed cDNAs that we termed late erythroblast (LEB) 1–3. None of these cDNAs were previously identified as being expressed in erythroblasts and their patterns of expression indicated they are likely to be involved in the differentiation process. LEB-1 cDNA was derived from the gene A330102K04Rik (approved gene symbol Apoll1), and shares homology with members of the apolipoprotein L family in humans. LEB-3 cDNA was derived from the novel gene D930015E06Rik , that has no known function. LEB-2 cDNA was derived from the gene ranBP16 (approved gene symbol Xpo7), a nuclear exportin. D930015E06Rik mRNA is also strongly expressed in the testis and was localized to a region of the seminiferous tubule where secondary spermatocytes and early spermatids are found, suggesting a role for D930015E06Rik in spermatogenesis as well as terminal erythroid differentiation. We have thus identified three genes not previously described as being expressed in erythroblasts that could be relevant in elucidating mechanisms involved in terminal erythroid differentiation.

420 POSTER Measuring alpha-folate receptor expression levels on ascites tumour cells may help to identify patients that are more likely to respond to alpha-FR targeted therapy

The action of retinoic acid (RA) in normal development and differentiation is mediated by changes in the expression of RA-responsive target genes. We used differential display reverse transcriptase polymerase chain reaction (RT-PCR) to identify RA-responsive genes expressed in embryonic stem (ES) cells and found that murine folate receptor-α (FR-α) expression is rapidly induced by RA treatment. The observed increase in FR-α expression occurs within 3 h, is independent of protein synthesis and does not occur when ES cells are differentiated by removal of leukaemia inhibitory factor (LIF), evidence that the response to RA is both direct and specific. Two messenger RNA (mRNA) isoforms of FR-α featuring novel sequence in the 5′ untranslated region (UTR) were cloned, and both were found to be upregulated by RA. Other splice variants in both the 5′ UTR and 3′ UTR of FR-α mRNA were also identified. There is a striking similarity between these splicing patterns and those reported for human FR-α, which also generates multiple isoforms by alternative splicing in the 5′ and 3′ UTR. The conservation of these splicing patterns in the non-coding regions of the FR-α gene between mouse and human suggests that these regions, and in particular the 5′ UTR, play an important role in regulating expression of this gene.

Differential expression and antibacterial activity of WFDC10A in the monkey epididymis

The ability of the epididymis to perform its diverse functions stems from its regionalized gene and protein expression patterns. The differences in the gene expression patterns of the caput and cauda regions of the bonnet monkey epididymis were compared using the technique of differential display reverse transcriptase polymerase chain reaction. A transcript showing homology to human whey acidic protein 10 (hWFDC10A) was highly expressed in the monkey caput region. A peptide P2 was designed spanning a region of the monkey WFDC10A (mWFDC10A), which could inhibit the growth of gram-negative bacterial strains of Escherichia coli. P2 could permeabilize the bacterial cell membrane but was unable to permeabilize mammalian cells as evidenced by the lack of hemolysis upon incubation with the peptide. Expression of genes such as mWFDC10A may be essential in providing the first line of defense against microbial infections to the epididymal tract and thus rendering protection to the male gametes sheltered within the epididymis.

Early activation of cell wall strengthening-related gene transcription in cotton by a Verticillium dahliae elicitor

Differential expression of genes in cultured cotton cells and leaf disks that have been challenged with a purified elicitor from Verticillium dahliae, was investigated in order to identify genes involved in the early defense response of cotton. The mRNA differential display reverse transcriptase polymerase chain reaction was used to identify differentially expressed genes 5 h after application of 50 μ gm L �1 Verticillium dahliae elicitor. Sequence analysis of selected amplicons revealed homologies with genes involved in metabolism of carbohydrate precursors for cell wall synthesis. cDNAs identified that were up-regulated after elicitor treatment coded for homologs to a UDP-N-acetylglucosamine pyrophosphorylase-like protein, a glucosyltransferase-like protein, a beta-1,4-N-acetylglucosaminyltransferase, a cellulose synthase-like protein, a 3-deoxy-D-manno-octulosonic acid transferase-like protein and a hydroxyproline-rich glycoprotein. In addition, one cell wall-related cDNA that was down-regulated after elicitor treatment, coded for a proline-rich protein family member homolog. The differential expression of the cDNAs up-regulated after the Verticillium dahliae elicitor treatment was confirmed with reverse Northern dot blots. These results indicate that metabolic reprogramming through the enhanced synthesis of carbohydrate precursors accompanies the activation of de novo cell wall synthesis. As such it is important for the understanding of early defense related responses in cotton and for their biotechnological manipulation. © 2006 SAAB. Published by Elsevier B.V. All rights reserved.

Differentially expressed genes in the prostate cancer cell line LNCaP after exposure to androgen and anti-androgen

Androgens play an important role in growth and maintenance of prostate cells. The actions of androgens are mediated by the androgen receptor (AR), a transcription factor member of the super-family of nuclear hormone receptors. Androgen regulated genes (ARGs) are potential markers for early diagnosis and treatment of prostate cancer patients. In the present study, we used DDRT-PCR (differential display reverse transcriptase polymerase chain reaction) technique in order to investigate differentially expressed genes in the prostate cancer cell line LNCaP after treatment with dihydrotestosterone and bicalutamide for 6, 24, and 48 hours. Fifty-five differentially expressed fragments were isolated, cloned, and sequenced. Sequencing analysis of these fragments revealed 56 different transcripts that showed homology to transcription factors, cell cycle regulators, metabolic enzymes, and hypothetical proteins. Among the differentially expressed genes, SPA17 and DDEF2 were further validated using quantitative real time RT-PCR (qPCR) in a series of 25 prostate tumor samples. The DDEF2 gene is involved in adhesion and cell migration of monocytes, and the SPA17 gene might be involved in cellular signal transduction. The transcripts of both, SPA17 and DDEF2 genes, showed altered pattern of expression in the group of prostate tumors analyzed by qPCR. The differentially expressed genes identified in this study might provide new insights into the androgen signaling pathways in prostate cells.

Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display

Citrus unshiu is freeze tolerant to − 10 °C when fully acclimated after exposure to cold, nonfreezing temperatures. To gain an understanding of its cold tolerance mechanism, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) and quantitative relative RT-PCR were used to study gene expression under a gradual cold-acclimation temperature regime. Six up-regulated and two down regulated genes were identified based on their amino acid sequences. The identified proteins encoded by the up-regulated genes were: 14-3-3 protein, 40S ribosomal protein S23, putative 60S ribosomal protein L15, nucleoside diphosphate kinase III protein, regulator of chromosome condensation-like protein, and amino acid permease 6. The proteins encoded by the two down-regulated genes were: miraculin-like protein and beta-galactosidase. Their individual function has been briefly reviewed based on published information. In addition to the findings in this study, we compared the function of cold responsive genes of Poncirus trifoliata, a very cold hardy relative of Citrus species that is freeze tolerant to − 30 °C when fully acclimated, to the function of genes in the current study.

Identification of putative rat ribonuclease III by differential display: a novel rat mRNA expressed in a circadian manner in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus constitutes the principal site responsible for the generation and entrainment of circadian rhythms in mammals. The mechanisms of the circadian clock involve periodic gene expression. Here we report the use of differential display reverse transcriptase polymerase chain reaction to identify a novel rat mRNA sequence which is highly homologous to human ribonuclease III. Analysis of its expression in the rat brain by in situ hybridization histochemistry showed this transcript to be expressed at differing intensities at various sites. Temporal variation in expression was observed in the SCN, with a peak at circadian time (CT) 2 and a nadir at CT14. No significant changes in its expression were detected across the cycle within the supraoptic nucleus, cingulate cortex or caudate putamen.

Oxidized low-density lipoproteins may induce expression of monocyte chemotactic protein-3 in atherosclerotic plaques

Genes induced or suppressed by oxidized low-density lipoproteins (oxLDL) in human monocytic THP-1 cells were searched using the differential display reverse transcriptase polymerase chain reaction. One of the differentially expressed (up-regulated) cDNA fragments was found to contain sequences corresponding to monocyte chemotactic protein-3 (MCP-3). The stimulatory effect of the oxLDL on the expression of MCP-3 mRNA was both time- and dose-dependent. Treatment with GF109203X and genistein, inhibitors of protein kinase C and tyrosine kinase, respectively, had no effect on the induction of MCP-3 mRNA by oxLDL, while treatment with cycloheximide inhibited the induction. The induction was reproduced by the lipid components in oxLDL such as 9-HODE and 13-HODE, which are known to activate the peroxisome proliferator-activated receptor γ (PPARγ). Introduction of an endogenous PPARγ ligand, 15d-PGJ2, in the culture of THP-1 cells resulted in the induction of MCP-3 gene expression. Furthermore, analyses of human atherosclerotic plaques revealed that the expressional pattern of MCP-3 in the regions of neointimal and necrotic core overlapped with that of PPARγ. These results suggest that oxLDL delivers its signal for MCP-3 expression via PPARγ, which may be further related to the atherogenesis.

Up-regulation of Bfl-1/A1 via NF-κB activation in cisplatin-resistant human bladder cancer cell line

The potent anti-cancer agent cis-diamminedichloroplatinum (II) (cisplatin) is currently used for treating bladder cancer. However, clinical use of this drug for long periods is often limited because of the appearance of cisplatin-resistant bladder tumor cells. We employed the method of a differential display reverse transcriptase polymerase chain reaction to identify the differentially expressed genes in the parental human bladder cancer cell line, T24 and three cisplatin-resistant cell lines. We report here that cisplatin-resistant cell lines overexpress Bcl-2 family protein Bcl-2-related gene expressed in fetal liver (Bfl-1)/A1 as compared with their parental cell. Cisplatin and γ-irradiation induced expression of Bfl-1/A1 in T24R2 cells but not in T24 cells. Among Bcl-2 family members, Bfl-1/A1 showed the most significant alteration of the expression level in resistant cells. The nuclear translocation of nuclear factor-kappaB (NF-κB) by cisplatin and γ-irradiation selectively occurred in T24R2 cells. Mitochondrial depolarization and cell death by cisplatin were also prevented in T24R2 cells. Moreover, Bfl-1/A1 inhibited cisplatin- and TNF-α-induced apoptosis in BOSC23 cells. Our findings suggest that the induction of Bfl-1/A1 by NF-κB may be important in controlling resistance to cisplatin responses in bladder tumor cells.