DD RT-PCR Research Articles

Identification of estrogen-regulated genes in the mouse uterus using a delayed-implantation model.

Gonadal steroid hormones are known to modulate the implantation of the blastocyst, but how the controlling genetics are regulated remains largely unknown. Using a delayed-implantation model, we examined estrogen-regulated genes (ERGs) in the mouse uterus using the differential-display reverse transcription-polymerase chain reaction (DD RT-PCR). Pregnant mice were ovariectomized and injected daily with progesterone (P, 1 mg/mouse), followed by a single injection of estrogen (E, 200 ng/mouse); 24 or 48 hr later, total RNA was extracted from the uterus. Reverse Northern analysis verified the expression patterns of 36 clones out of thousands of RNA species. Only five clones had mRNA levels that were modified, whereas other mRNAs were unchanged or not detectable. Sequence analysis of these, using the Basic Local Alignment Search Tool (BLAST) service, revealed that four of these clones were novel; one clone, designated ERG10, was found to be the mouse homologue of that deleted in oral cancer DOC-1. DOC-1 mRNA was detected all tissues examined, but only in the uterus and cervix was markedly increased 12 hr after E administration, it returned to basal level by 48 hr. One of the novel genes, designated ERG8, had three different forms of mRNAs and was expressed ubiquitously in all examined tissues. In the uterus, the mRNA level of ERG8 also increased 12 hr after E administration. These results suggest that during the implantation process, E differentially regulates several genes depending on cell type. Uterine-specific induction of newly found genes, such as ERG8 and 10, by E appears to be important for the early implantation process.

Changes in T-plastin expression with human trophoblast differentation

During the first trimester of pregnancy, the human placenta is an actively dividing and highly invasive tumour-like tissue, while near term, it represents a fully developed, non-invasive unit. In order to understand the molecular basis of this marked difference in the placental phenotypes, an approach based on a differential display–reverse transcription–polymerase chain reaction (DD–RT–PCR) was adopted to analyse changes in gene expression, using total RNA isolated from first-trimester and term placental villi. Using this approach, T-plastin was initially identified as being differentially expressed in the human first-trimester placenta. T-plastin is an actin-bundling protein and is known to be highly expressed in actively dividing cells and up-regulated in several carcinomas. Using a homogenous population of cytotrophoblasts and syncytiotrophoblasts isolated from human placentae, the present authors demonstrate the differential expression of T-plastin in cytotrophoblasts compared with the terminally differentiated syncytiotrophoblasts. The down-regulation of T-plastin expression is further demonstrated in human trophoblastic BeWo cells induced to differentiate using transforming growth factor (TGF)β1, a growth factor known for its anti-proliferative and anti-invasive response in placental cells. These studies suggest that expression of T-plastin in the placental context may indeed be associated with the enhanced replicative potential of placental trophoblasts.

Interferon β Secreted from Human Hair Dermal Papilla Cells Inhibits the Growth of Outer Root Sheath Cells Cultured in Vitro

Hair growth is tightly regulated by the epithelial–mesenchymal interaction of hair follicle cells. To investigate the molecular interaction between epithelial and mesenchymal cells of the hair follicle, the outer root sheath (ORS) cells were cultured with the dermal papilla (DP)-conditioned medium. Using the techniques of differential display reverse transcription–polymerase chain reaction (DD RT–PCR) and Northern blot analysis, we identified that an interferon-inducible gene 6–16 is regulated in the ORS cells when cultured with the DP-conditioned medium. We further studied the effect of the DP-conditioned medium and found that interferon β, a predominant interferon secreted by the fibroblasts, is expressed by the DP cells. Moreover, the anti-interferon β neutralized DP-conditioned medium dramatically inhibited the 6–16 mRNA expression. Interferon β or DP-conditioned medium suppressed the growth of the in vitro cultured ORS cells, suggesting that interferon β secreted from the DP cells negatively regulates the growth of ORS cells. Together these results reveal the potential importance of the interferon β as a negative regulator of hair growth.

Fumonisin B1 Induces Apoptosis in LLC-PK1 Renal Epithelial Cells via a Sphinganine- and Calmodulin-Dependent Pathway

Fumonisins are a family of mycotoxins produced by Fusarium moniliforme, which is the most common mold found on corn throughout the world. These compounds are both toxic and carcinogenic for animals, and perhaps humans, with the kidney being the most sensitive organ to fumonisin toxicity. The molecular mechanism of fumonisin toxicity appears to involve disruption of de novo biosynthesis of sphingolipids and accumulation of sphinganine. The goals of this study were to determine whether fumonisin B1 kills LLC-PK1 renal kidney epithelial cells by inducing apoptosis and to identify genes affected by sphinganine that mediate fumonisin B1-induced cell death. Fumonisin B1 produced morphological changes (i.e., cell shrinkage, membrane blebbing) and time-dependent increases in DNA fragmentation demonstrating that the toxin induces apoptosis. Simultaneously, fumonisin B1 blocked sphingolipid biosynthesis and caused accumulation of sphinganine. To further investigate the role of sphinganine in fumonisin B1-induced apoptosis, β-fluoroalanine (βFA) was used to inhibit serine palmitoyltransferase, which catalyzes an earlier step in the sphingolipid biosynthetic pathway. βFA blocked sphinganine accumulation and prevented fumonisin B1-induced DNA fragmentation, confirming that apoptosis induced by fumonisin B1 is dependent upon accumulation of sphinganine. To examine gene expression, differential display reverse transcriptase polymerase chain reaction (DDRT–PCR) was applied to RNA isolated after 16 h of exposure to fumonisin B1. Differential expression in response to fumonisin B1 of a gene identified as calmodulin has been verified by Northern analysis. Sphinganine appears to mediate the effect because βFA reduces induction of calmodulin mRNA by fumonisin B1. Fumonisin B1 also increases calmodulin protein in a concentration-dependent manner and the calmodulin antagonist W7 blocks fumonisin B1-induced DNA fragmentation, supporting a role for calmodulin in fumonisin B1-induced apoptosis. In contrast, fumonisin B1 had no effect on expression of bcl-2 family genes (bax, bcl-2, and bcl-x). These findings demonstrate that fumonisin B1 kills LLC-PK1 kidney cells by inducing apoptosis. Further, the results establish a sequence of events for fumonisin B1-induced apoptosis involving initial disruption of sphingolipid metabolism and accumulation of sphinganine (or a metabolite), which, in turn, induces expression of calmodulin.

Identification of drug resistant related cDNA in lung adenocarcinoma cell lines

Objective: To clone multidrug resistance (MDR) related genes in lung adenocarcinoma cell lines. Methods: The differentially expressed cDNA fragments between A549 and A549 DDP cells were analyzed by mRNA differential display PCR(DD RT-PCR). The fragments thus obtained were further analyzed by DNA sequencing and Northern blotting. Results: Three differentially expressed cDNA fragments were obtained and confirmed by Northern blot. Sequence analysis revealed that two of them were novel and one was 100% identical with ICE gene. Conclusion: Analyzing differentially expressed fragment between A549 and A549 DDP cells may be helpful for finding new MDR related genes. The drug resistance of A549 DDP cells may be related to the inhibition or down-regulation of ICE gene.

RNAs That Interact with the Fragile X Syndrome RNA Binding Protein FMRP

The Fragile X protein FMRP is an RNA binding protein whose targets are not well known; yet, these RNAs may play an integral role in the disease's etiology. Using a biotinylated-FMRP affinity resin, we isolated RNAs from the parietal cortex of a normal adult that bound FMRP. These RNAs were amplified by differential display (DDRT–PCR) and cloned and their identities determined. Nine candidate RNAs were isolated; five RNAs, including FMR1 mRNA, encoded known proteins. Four others were novel. The specificity of binding was demonstrated for each candidate RNA. The domains required for binding a subset of the RNAs were delineated using FMRP truncation mutant proteins and it was shown that only the KH2 domain was required for binding. Binding occurred independently of homoribopolymer binding to the C-terminal arginine–glycine-rich region (RGG box), suggesting that FMRP may bind multiple RNAs simultaneously.

Mkp-1 encoding mitogen-activated protein kinase phosphatase 1, a verotoxin 1 responsive gene, detected by differential display reverse transcription-PCR in Caco-2 cells.

The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing Escherichia coli is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release. Four differentially expressed genes, including mkp-1 (encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells. Northern blot analysis showed the induction of mkp-1 mRNA 6 h after VT1 stimulation. Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced mkp-1 mRNA, but mkp-1 mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor. Therefore, we concluded that the A subunit of VT1 was essential for mkp-1 induction. Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin. Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1. This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins.

Refinements of the Differential Display Reverse Transcription–Polymerase Chain Reaction Technique: Use of Oligo(dT)-Based Anchored Primers with Escherichia coli Messenger RNA Identifies a Salt-Induced Promoter in the dcw Gene Cluster

Differential display reverse transcription PCR (DDRT–PCR)2 is a popular technique used to identify differences in messenger RNA species expressed under different conditions (1–4). The use of this technique in prokaryotic systems has been greatly hampered by the lack of a poly(A) tail at the 39 end of these messages, resulting in the use of random primers to define both ends of the PCR products, in turn leading to lower probability of identifying any differentially expressed gene.

HBRAG, a novel B cell lineage cDNA encoding a type II transmembrane glycoprotein potentially involved in the regulation of recombination activating gene 1 (RAG1).

The different display reverse transcription-PCR (DD RT-PCR) technique was used to identify novel cDNA detecting mRNA transcripts co-expressed with human recombination activating gene-1 (RAG1). A 5.0-kb transcript detected by the differential display amplicon 3G1 was found to correlate strongly with RAG1 mRNA expression in various human cell lines. Subsequent screenings of a pre-B cDNA library with 3G1 led to the identification of a complete cDNA we have termed hBRAG (human B-cell RAG-Associated Gene). The hBRAG cDNA encodes a 503-amino acid (aa) protein with no known homology to any nucleotide or protein sequence. The predicted molecular mass of 55 kDa was confirmed by in vitro translation. Based on sequence analysis, the predicted open reading frame encodes for a type II transmembrane spanning glycoprotein with the N-terminal 81 -aa in the cytoplasm, a 17-aa transmembrane domain, and a C-terminal 405-aa extracellular domain with four potential N-glycosylation sites. Northern blot analysis indicated a close association of the 5.0-kb hBRAG mRNA transcript with RAG1 in numerous human pro-B, pre-B and mature B cell lines assessed, but not in human T cell lines. In human tissues, hBRAG is expressed at highest levels in B cell-enriched tissues, but is not expressed in fetal or adult thymus. Southern blotting analysis revealed that this gene is conserved across eukaryotes, is expressed as a single copy in the human genome, and is likely not a multigene family member. The hBRAG gene was localized to the long arm of chromosome 10 (10q26). Transfection of the full-length hBRAG cDNA increased levels of human RAG1 transcripts in the B cell line OCI LY8-C3P, but not in the non-lymphoid line K562, suggesting a B cell-specific role for the hBRAG product in regulating RAG expression.

Molecular Dissection of Dimethylnitrosamine (DMN)-Induced Hepatotoxicity by mRNA Differential Display

Differential display reverse transcription–polymerase chain reaction (DDRT–PCR) was used to catalogue altered hepatic transcript expression during dimethylnitrosamine (DMN) exposurein vivo.Mice were administered DMN (1.5 or 5 mg/kg) or vehicle (phosphate-buffered saline) ip once daily for up to 7 days, and livers were collected 6 h post-injection. Total RNA was reverse transcribed and cDNA subsets were selectively amplified by PCR. DDRT–PCR products were fractionated on denaturing polyacrylamide gels, and differentially expressed bands were excised, reamplified, and subsequently cloned into a plasmid vector. This study identified 23 cDNAs that were induced and 25 cDNAs that were suppressed during DMN exposure. Altered expression during DMN exposure for cDNA clones was confirmed by Northern blotting, RNase protection, orin situhybridization analyses. DNA sequence information indicated that four cDNAs suppressed during DMN exposure encode cytochrome P450 isoenzyme-cholesterol 7α-hydroxylase (CYP7), a monokine, a myeloid cell differentiation protein, and mouse major urinary protein (MUP). We further observed a DMN-induced increase in transcripts for complement factor 3 (C3) and serum amyloid A (SAA). In contrast, the remaining differentially expressed transcripts detected by DDRT–PCR during DMN exposure demonstrated no similarity to sequences present in Genbank, suggesting that they may encode previously unreported gene products.In situhybridization showed MUP transcripts to be expressed by hepatic centrilobular areas that undergo necrosis during subchronic DMN exposure. Thus, the utilization of DDRT–PCR has identified several differentially expressed hepatic mRNAs associated with various doses and stages of DMN exposure.

Immunohistorical localization of insulin-like growth factor-I (IGF-I), IGF-I receptor and IGF-binding protein-1 (IGFBP-1) in the human anchoring villi

During the first trimester of pregnancy, the human placenta is an actively dividing and highly invasive tumour-like tissue, while near term, it represents a fully developed, non-invasive unit. In order to understand the molecular basis of this marked difference in the placental phenotypes, an approach based on a differential display–reverse transcription–polymerase chain reaction (DD–RT–PCR) was adopted to analyse changes in gene expression, using total RNA isolated from first-trimester and term placental villi. Using this approach, T-plastin was initially identified as being differentially expressed in the human first-trimester placenta. T-plastin is an actin-bundling protein and is known to be highly expressed in actively dividing cells and up-regulated in several carcinomas. Using a homogenous population of cytotrophoblasts and syncytiotrophoblasts isolated from human placentae, the present authors demonstrate the differential expression of T-plastin in cytotrophoblasts compared with the terminally differentiated syncytiotrophoblasts. The down-regulation of T-plastin expression is further demonstrated in human trophoblastic BeWo cells induced to differentiate using transforming growth factor (TGF)β1, a growth factor known for its anti-proliferative and anti-invasive response in placental cells. These studies suggest that expression of T-plastin in the placental context may indeed be associated with the enhanced replicative potential of placental trophoblasts.

Regulation of macrophage gene expression following invasion by Mycobacterium tuberculosis

Introduction: Mycobacteria are intracellular pathogens which survive and grow in host macrophages. M. tuberculosis bacilli enter the macrophage via binding to several distinct cell surface molecules. Following phagocytosis, sustained intracellular bacterial growth depends on the ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. We used differential display reverse transcription polymerase chain reaction (DD RT-PCR) to identify host genes which are regulated during infection and hence which might be involved in the host-parasite cross talk. Results: Live M. tuberculosis (strain H 37Rv) was used to infect Balb/c peritoneal murine macrophages. mRNA from infected and uninfected macrophages was isolated at different time intervals after phagocytosis and subjected to DD RT-PCR. Oligo dT 12NV and random 10mer primers were used for PCR amplification of cDNA. Macrophage genes which appeared to be differently regulated during infection were subjected to further reamplification by PCR in order to clone and sequence them. The differential expression of the selected bands was further analysed by an RNA protection assay and a Northern blot. Results: Several differentially regulated bands were identified. One band, of 158 bp, was down regulated after infection. Sequencing of this band revealed a high level of homology (95% identity) to mouse cytochrome c oxidase subunit VIIc. The downregulation was specific for live virulent Mtb, while live BCG, heat killed Mtb and latex beads-mediated phagocytosis did not affect the transcriptional level of this enzyme. Conclusions: The cytochrome oxidase enzyme complex of the inner mytochondrial membrane catalyzes the reaction between ferrocytochrome c and oxygen. The reaction is the terminal event in the electron transport scheme. Downregulation of cytochrome c oxidase subunit VIIc could interfere with: (1) the host apoptotic programme; or (2) the host respiratory burst.

Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression

Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences that appear to be upregulated in the presence of testosterone show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression.

Identification of Dioxin-Responsive Genes in Hep G2 Cells Using Differential mRNA Display RT–PCR

Differential mRNA display RT–PCR (DD RT–PCR) offers a tool to identify genes which are regulated or responsive to certain receptors or chemicals such as dioxin (TCDD). Treatment of Hep G2 cells with TCDD followed by DD analysis of a gel with series of different primers revealed a significantly different pattern from the control for a number of mRNAs. The differentially displayed mRNAs were isolated and reamplified. A GenBank search of four mRNAs revealed two known and two unknown sequences. Northern blot analysis revealed that two known sequences, fibrinogen γ chain and plastin mRNAs were down regulated by TCDD in a time-dependent manner, whereas two unknown mRNAs were induced by TCDD treatment. The function of these genes in TCDD toxicity is not known; however, the application of DD RT–PCR in the studies of TCDD-induced responses could be very useful in the discovery of other unknown genes important for TCDD toxicity.