Unlabeled Probe Research Articles

Expression of Fetal Hemoglobin among Adult Human Erythroblasts Is Associated with an Altered Expression Pattern for the Transcription Factor c-Myb.

The c-Myb transcription factor plays a central role in regulating growth and differentiation during definitive erythropoiesis. In addition, c-Myb expression has been previously linked to GATA-1 expression and hereditary persistence of fetal hemoglobin. Here we examined a potential role for c-Myb in signaled expression of fetal hemoglobin (HbF) among primary adult human erythroblasts. Initial experiments were performed to determine the expression pattern of c-Myb under conditions of low fetal hemoglobin production (CD34 cells cultured over 14 days in media supplemented with erythropoietin (EPO) alone). Quantitative PCR performed on cells sampled on days 2, 5, 8, 11, and 14 demonstrated highly regulated c-Myb expression. A dramatic increase (>30 fold) in c-Myb mRNA was measured between days 5 and 8 as the committed progenitor cells differentiated to the proerythroblast stage. That increase was followed by a 3-fold reduction in c-Myb during terminal differentiation. Western blotting of nuclear extracts obtained on days 7 and 14 in EPO cultures corroborated with the quantitative PCR analyses. Electrophoretic mobility shift assays performed using day 7 and day 14 nuclear extracts showed a shifted major band with a c-Myb consensus probe. That major band was competed by unlabeled c-Myb probe on both days. Moreover, day 14 extracts repeatedly revealed two additional bands that were not competed suggesting the possibility of other complexes binding to the Myb motif or changes in c-Myb conformation during terminal differentiation. Chromatin immunoprecipitation assays also revealed preliminary evidence for Myb binding directly to beta globin locus. When compared to low HbF culture conditions (EPO; HbF <1%), cells grown in high HbF conditions (erythropoietin + stem cell factor + TGF-beta (EST); HbF>35%) demonstrated a distinct pattern of c-Myb expression. Relative to the low HbF conditions, high HbF expressing cells had reduced c-Myb levels on day 7 followed by increased levels on day 14. These results suggest that c-Myb possesses highly regulated patterns of expression and DNA binding during erythroid maturation. In addition, the association of changes in c-Myb expression patterns with HbF production suggests that this growth-related transcription factor may help regulate fetal hemoglobin synthesis in patients with beta-hemoglobinopathies.

Unlabeled Oligonucleotide Probes Modified with Locked Nucleic Acids for Improved Mismatch Discrimination in Genotyping by Melting Analysis

With a frequency of 1 in 1000 bp, single nucleotide polymorphisms (SNPs) are used to study complex inherited diseases (1,2). A universal concern in genotyping techniques is that rare variants may interfere. Melting analysis addresses this concern and is a homogeneous and simple method for genotyping (3,4). For example, the 2-probe/2-fluorophore system HybProbe

High-Resolution DNA Melting Analysis for Simultaneous Mutation Scanning and Genotyping in Solution

High-resolution DNA melting analysis with saturation dyes for either mutation scanning of PCR products or genotyping with unlabeled probes has been reported. However, simultaneous PCR product scanning and probe genotyping in the same reaction has not been described. Asymmetric PCR was performed in the presence of unlabeled oligonucleotide probes and a saturating fluorescent DNA dye. High-resolution melting curves for samples in either capillaries (0.3 degrees C/s) or microtiter format (0.1 degrees C/s) were generated in the same containers used for amplification. Melting curves of the factor V Leiden single-nucleotide polymorphism (SNP) and several mutations in exons 10 and 11 of the cystic fibrosis transconductance regulator gene were analyzed for both PCR product and probe melting transitions. Independent verification of genotype for simple SNPs was achieved by either PCR product or probe melting transitions. Two unlabeled probes in one reaction could genotype many sequence variants with simultaneous scanning of the entire PCR product. For example, analysis of both product and probe melting transitions genotyped DeltaF508, DeltaI507, Q493X, I506V, and F508C variants in exon 10 and G551D, G542X, and R553X variants in exon 11. Unbiased hierarchal clustering of the melting transitions identified the specific sequence variants. When DNA melting is performed rapidly and observed at high resolution with saturating DNA dyes, it is possible to scan for mutations and genotype at the same time within a few minutes after amplification. The method is no more complex than PCR and may reduce the need for resequencing.

Two for Typing: Homogeneous Combined Single-Nucleotide Polymorphism Scanning and Genotyping

More than 20 years after the first in vitro amplification of DNA by PCR, this technique is firmly established for diagnostic purposes in many clinical laboratories. PCR technology is an exciting field that has continually advanced. Evolutionary steps have been, for example, the transition from conventional cycling to rapid-cycling (1), from post-PCR to real-time product analysis (2), and from gel-based to probe-based PCR product identification (3) and genotyping with energy transfer probes (4). One of the many possible applications of PCR in the clinical laboratory is genotyping. In this issue of Clinical Chemistry , Zhou et al., from Carl Wittwer’s group (5), contribute another innovation to this field. Their study reports a homogeneous technique based on simultaneous high-resolution melting of a whole PCR product and an unlabeled probe in the presence of saturating fluorescent DNA dye. It appears as the logical continuation of underlying previous work. The use of fluorescence resonance energy transfer probe pairs for genotyping by analysis of the melting curve of a probe covering the mutation site was followed by hundreds of published applications since its first description (4). Single-nucleotide polymorphisms (SNPs) under a probe affect probe melting. The degree of destabilization depends on the type of SNP itself and on the bases that lie next to the SNP. By using a nearest-neighbor model, the melting temperatures of matched and mismatched probes can be predicted …

Control of ACAT2 liver expression by HNF1

ACAT catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. There are two known genes encoding the two ACAT enzymes, ACAT1 and ACAT2 (also known as Soat1 and Soat2). In adult humans, ACAT1 is present in most tissues, whereas ACAT2 is localized to enterocytes and hepatocytes. In this report, we elucidate the mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified hepatic nuclear factor 1 (HNF1) as an important liver-specific trans-acting element for the human ACAT2 gene using the human hepatocellular carcinoma cell lines HuH7 and HepG2. Targeted deletion of the HNF1 binding site in the DNA sequence abolished not only the basal promoter function in HepG2 and HuH7 cells but also the induction of the ACAT2 promoter by HNF1. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the transcription factors HNF1alpha and HNF1beta interact with this region in the human ACAT2 gene in vitro and in vivo. These data indicate that a) the identified HNF1 binding site serves as a positive regulator sequence, b) the binding site is functionally active both in vivo and in vitro, and c) the transcription factors HNF1alpha and HNF1beta, which bind to this site, play an important part in the regulation of the human ACAT2 promoter.

Histone Deacetylase Inhibitors Suppress the Induction of c-Jun and Its Target Genes Including COX-2

Cyclooxygenase-2 (COX-2) is considered to be a target for anticancer therapy. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity, but the mechanisms of action are incompletely understood. We investigated whether HDAC inhibitors blocked AP-1-mediated activation of COX-2 transcription. Trichostatin A and suberoylanilide hydroxamic acid, two structurally related inhibitors of HDAC activity, blocked AP-1-mediated induction of COX-2 expression and prostaglandin E2 biosynthesis. Chromatin immunoprecipitation assays indicated that HDAC inhibitors suppressed c-Jun binding to the COX-2 promoter and thereby blocked transcription. The observed reduction in binding reflected reduced levels of c-Jun. HDAC inhibitors suppressed the induction of c-jun transcription by blocking the recruitment of the preinitiation complex (RNA polymerase II and TFIIB) to the c-jun promoter. HDAC3 but not HDAC1 or HDAC2 was required for AP-1-mediated stimulation of c-jun expression. Because HDAC inhibitors suppressed the induction of c-jun gene expression, resulting in reduced COX-2 transcription, it was important to determine whether other known AP-1 target genes were also modulated. Cyclin D1 and collagenase-1 are AP-1-dependent genes that have been implicated in carcinogenesis. HDAC inhibitors suppressed the induction of both cyclin D1 and collagenase-1 transcription by inhibiting the binding of c-Jun to the respective promoters. Taken together, these results suggest that HDAC inhibitors block the induction of c-jun transcription by inhibiting the recruitment of the preinitiation complex to the c-jun promoter. This led, in turn, to reduced expression of several activator protein-1-dependent genes (COX-2, cyclin D1, collagenase-1). These findings provide new insights into the mechanisms underlying the antitumor activity of HDAC inhibitors.

The RNA-binding Protein HuR Binds to Acetylcholinesterase Transcripts andRegulates Their Expression in Differentiating Skeletal MuscleCells

During myogenic differentiation, acetylcholinesterase (AChE) transcript levels are known to increase dramatically. Although this increase can be attributed in part to increased transcriptional activity, posttranscriptional mechanisms have also been implicated in the high levels of AChE mRNA in myotubes. In this study, we observed that transfection of a luciferase reporter construct containing the full-length AChE 3'-untranslated region (UTR) resulted in significantly higher (5-fold) luciferase activity in differentiated myotubes versus myoblasts. RNA-electrophoretic mobility shift assays (REMSAs) performed with a full-length AChE 3'-UTR probe and the AU-rich element revealed that the intensity of RNA-binding protein complexes increased as myogenic differentiation proceeded. Using several complementary approaches including supershift REMSA, mRNA-binding protein pull-down assays, and immunoprecipitation followed by reverse transcription-PCR, we found that the mRNA-stabilizing protein HuR interacts directly with AChE transcripts. Stable overexpression of HuR in C2C12 cells increased the expression of endogenous AChE transcripts as well as that of the luciferase reporter construct containing the AChE 3'-UTR. In vitro stability assays performed with protein extracts from these cells versus controls resulted in a slower rate of AChE mRNA decay. The down-regulation of HuR expression mediated through small interfering RNA further confirmed the role of HuR in the regulation of AChE mRNA levels. Taken together, these studies demonstrate that HuR interacts with the AChE 3'-UTR to regulate posttranscriptionally the expression of AChE mRNA during myogenic differentiation.

Regulation of the Expression of Caspase-9 by the Transcription Factor Activator Protein-4 in Glucocorticoid-induced Apoptosis

Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-xL, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5'-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at -1.55 and -1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases -1140 and -944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus

SummaryCell‐free extracts from Streptomyces clavuligerus, purified by elution from heparin‐agarose with an ARE‐containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (AREccaR). This sequence is located upstream of the ccaR gene, encoding the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty‐one nucleotides upstream of brp another ARE sequence (AREbrp) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp‐deleted mutant, S. clavuligerus Δbrp::neo1, produced 150–300% clavulanic acid and 120–220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Δbrp::neo1 lacked a high‐mobility band‐shift due to Brp but still showed a slow‐mobility band‐shift observed in the wild‐type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C biosynthesis by its action on ccaR gene expression.

Mint Represses Transactivation of the Type II Collagen Gene Enhancer through Interaction with αA-crystallin-binding Protein 1

Collagen type II is an extracellular matrix protein important for cartilage and bone formation, and its expression is controlled by multiple cis- and trans-acting elements, including the zinc finger transcription factor alpha A-crystallin-binding protein 1 (CRYBP1). Here we show that MSX2-interacting nuclear target protein (MINT), a conserved transcriptional repressor, associates with CRYBP1 and negatively regulates the transactivation of the collagen type II gene (Col2a1) enhancer. We identified CRYBP1 as a binding partner of MINT by screening a mouse embryonic cDNA library using the yeast two-hybrid system. We demonstrated that the C terminus of MINT interacts with the C terminus of CRYBP1 using the mammalian cell two-hybrid assay, glutathione S-transferase pull-down, and co-immunoprecipitation analyses. Furthermore, MINT and CRYBP1 form a complex on the Col2a1 enhancer, as shown by chromatin immunoprecipitation and gel shift assays. In the presence of CRYBP1, overexpression of MINT or its C-terminal fragment in cells repressed a reporter construct driven by the Col2a1 enhancer elements. This transcription repression is dependent on histone deacetylase, the main co-repressor recruited by MINT. The present study shows that MINT is involved in CRYBP1-mediated Col2a1 gene repression and may play a role in regulation of cartilage development.

Up-regulation of Human myo-Inositol Oxygenase by Hyperosmotic Stress in Renal Proximal Tubular Epithelial Cells

myo-Inositol oxygenase (MIOX) catalyzes the oxidative cleavage of myo-inositol (MI) to give d-glucuronic acid, a committed step in MI catabolism. d-Glucuronic acid is further metabolized to xylitol via the glucuronate-xylulose pathway. Although accumulation of polyols such as xylitol and sorbitol is associated with MI depletion in diabetic complications, no causal relationship has been established. Therefore we are examining the role of MIOX in diabetic nephropathy. Here we present evidence that the basis for the depletion of MI in diabetes is likely to be mediated by the increased expression of MIOX, which is induced by sorbitol, mannitol, and xylitol in a porcine renal proximal tubular epithelial cell line, LLC-PK1. To understand the molecular mechanism of regulation of MIOX expression by polyols, we have cloned the human MIOX gene locus of 10 kb containing 5.6 kb of the 5' upstream sequence. Analysis of the 5' upstream sequence led to the identification of an osmotic response element (ORE) in the promoter region, which is present approximately 2 kb upstream of the translation start site. Based on luciferase reporter and electrophoretic mobility shift assays, polyols increased the ORE-dependent expression of MIOX. In addition, we demonstrate that the activity of the promoter is dependent on the binding of the transcription factor, tonicity element-binding protein, or osmotic response element-binding protein, to the ORE site. These results suggest that the expression of MIOX is up-regulated by a positive feedback mechanism where xylitol, one of the products of MI catabolism via the glucuronate-xylulose pathway, induces an overexpression of MIOX.

Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres

The aim was to introduce a new strategy based on peptide nucleic acid (PNA) probes and competitive displacement for using fluorescence in-situ hybridization (FISH) analysis on human blastomeres. Sequential FISH analysis with PNA probes and competitive displacement was performed using three different probe sets. The first set consisted of labelled probe only. The second and third sets included labelled as well as unlabelled probe, corresponding to the labelled probes in the previous cycles. The probes for enumeration were for chromosome 1, 13, 16, 17, 18, 21, X and Y. The performance of PNA probes was similar to the established DNA probes. The strategy of competitive displacement resulted in a destabilization of already bound probe before the next FISH cycle at only 50 degrees C, which allowed for up to five sequential FISH cycles without loss of signal. PNA probes are a good alternative to DNA probes in the present set-up, since the low temperature required both for binding and destabilization of PNA probes minimizes the loss of signal, and several FISH cycles can therefore be carried out before FISH errors occur.

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus

Cell-free extracts from Streptomyces clavuligerus, purified by elution from heparin-agarose with an ARE-containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE(ccaR)). This sequence is [corrected] located upstream of the ccaR gene, encoding [corrected] the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty-one nucleotides upstream of brp another ARE sequence (ARE(brp)) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp-deleted mutant, S. clavuligerus Deltabrp::neo1, produced 150-300% clavulanic acid and 120-220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Deltabrp::neo1 lacked a high-mobility band-shift due to Brp but still showed a [corrected] slow-mobility band-shift observed in the wild-type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C [corrected] biosynthesis by its action on ccaR gene expression.

Carbon Monoxide Differentially Modulates STAT1 and STAT3 and Inhibits Apoptosis via a Phosphatidylinositol 3-Kinase/Akt and p38 Kinase-dependent STAT3 Pathway during Anoxia-Reoxygenation Injury

Carbon monoxide (CO), previously considered a toxic waste product of heme catabolism, is emerging as an important gaseous molecule. In addition to its important role in neurotransmission, exogenous CO protects against vascular injury, transplant rejection, and acute lung injury. However, little is known regarding the precise signaling mechanisms of CO. We have recently shown that CO attenuates endothelial cell apoptosis during anoxia-reoxygenation injury by activating MKK3/p38alpha mitogen-activated protein kinase (MAPK) pathways. Our current study is the first to demonstrate that CO can differentially modulate STAT1 and STAT3 activation and, specifically, that STAT3 activation by CO is responsible for the anti-apoptotic effect in endothelial cells. In addition, we show that the anti-apoptotic effects of CO depend upon both phosphatidylinositol 3-kinase/Akt and p38 MAPK signaling pathways in endothelial cells, whereas previous reports have implicated only the MKK3/p38 MAPK pathway. Using chemical inhibitors and dominant negative constructs, we show that CO enhances STAT3 activation via phosphatidylinositol 3-kinase/Akt and p38 MAPK pathways with subsequent attenuation of Fas expression and caspase 3 activity. These data highlight the anti-apoptotic signaling mechanisms of CO and, importantly, delineate potential therapeutic strategies to prevent ischemia-reperfusion or anoxia-reoxygenation injury in the vasculature.

Transcriptional Activity of the Paired-like Homeodomain Proteins CHX10 and VSX1

CHX10 and VSX1 are homeodomain (HD) proteins essential for normal retinal development. CHX10 is required first for retinal progenitor cell proliferation and later for bipolar cell differentiation, whereas VSX1 is important in the terminal differentiation of a subset of bipolar cells. Elucidating the transcriptional activity of CHX10 and VSX1 is required to understand how these factors control retinal development. We show that CHX10 and Vsx1 can function as transcriptional repressors. When tethered to a promoter by a heterologous LexA DNA-binding domain or its HD, CHX10 repressed multiple classes of activators in different immortalized cell lines. CHX10 blocked TATA-containing and TATA-less promoters, repressed at a distance, and inhibited a complex enhancer positioned upstream or downstream of the reporter gene, whereas retinoblastoma protein (RB) inhibited the downstream enhancer only. Interestingly, CHX10 mildly potentiated a subset of activators in chick neuronal cultures. Thus, CHX10 is both a versatile repressor and a context-specific weak activator. The CHX10 HD and CVC domains were sufficient for DNA binding and repression. VSX1 contains closely related homeo and CVC domains and, like CHX10, also repressed transcription. A VSX1 HD mutation, R166W, that impairs DNA binding and causes keratoconus in humans, hindered repressor function. Therefore, CHX10 and VSX1 may control retinal bipolar cell specification or differentiation by repressing genes required for the development of other cell types.

Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter

This study was designed to explore the possible existence and location of estrogen response elements (EREs) in the human insulin receptor (hIR) gene promoter. Transfections of U-937 cells with the reported plasmids phIR(−1819)-GL2, phIR(−1473)-GL2, and phIR(−876)-GL2, that contain the −1819 to −271 bp fragment of the hIR promoter (wild-type promoter) and progressive 5′ deletions of this promoter, revealed that while the activity of the wild-type promoter, was repressed 36% by treatment with 17β-estradiol (E 2), the activities of progressive 5′ deletions of this promoter were reduced by 26% and by 0%, by this hormone. This suggests that E 2 needs the wild-type promoter for full transcriptional repression of this gene and it also suggests the presence of putative EREs in the region between −1819/−877 bp of this promoter. To identify these EREs we performed a computer search, using the SEQFIND programme developed in our laboratory, by homology with the consensus vit-ERE (5′ GGTCAnnn TGACC3′) of the Xenopus vitellogenin A 2 gene promoter. The results of our search indicated no sequence identical to this consensus ERE, and neither was any sequence found to show 9 or 8 of the 10 bases of this consensus in this promoter. Nevertheless, a putative hIR ERE1 (5′ AGTGAaac TGGCC3′) showing 7 bases of the consensus vit-ERE, and 10 bases of the optimal binding sequence ERE (5′CA/G GGTCAnnn TGACCT/CG3′), was identified between −1430/−1418 bp of the hIR promoter. An AP-1-like site was covering the 3′ half-element of this ERE; another AP-1-like site was overlapping the first AP-1-like site, and finally a third AP-1-like site was located beside to the 5′ half-element. In addition, another putative hIR ERE2 (5′ GCTCCtag CAAAC3′) showing 5 bases of the consensus vit-ERE, and 9 bases of the optimal binding sequence ERE, was located upstream of the hIR promoter, between −1567/−1555 bp. An AP-1-like site was located downstream of the 3′ half-element of this ERE, and another AP-1-like site was beside the 5′ half-element. EMSA analysis using nuclear extracts of E 2-treated cells and natural sequences, including these putative EREs, indicated that ERβ – the only isoform expressed in U-937 cells – specifically recognized both EREs because ERβ–DNA complexes were efficiently competed by the corresponding unlabelled probe and supershifted by the anti-human ERβ (L-20) antibody. These data provide the first identification of EREs complexed with AP-1-like sites in the hIR promoter, which account for the transcriptional repression of the hIR gene mediated by ERβ in U-937 cells.

Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye.

Homogeneous PCR methods for genotyping usually require fluorescently labeled oligonucleotide probes. Amplicon melting with the DNA dye LCGreen I was recently introduced as a closed-tube method of genotyping that does not require probes or real-time PCR. However, some single-nucleotide polymorphisms (SNPs) could not be completely genotyped without addition of a known genotype, and high-resolution melting techniques were necessary. A 3'-blocked, unlabeled oligonucleotide probe and the saturating dye, LCGreen I, were added to standard PCR reagents before amplification. After PCR, the samples were melted at 0.1-0.3 degrees C/s in high-resolution (HR-1), high-throughput (LightTyper), and rapid-cycle, real-time (LightCycler) instruments, and fluorescence melting curves were recorded. Derivative melting curves of the probe-target duplexes were characteristic of the genotype under the probe. With synthetic plasmid templates, all SNP base combinations could be genotyped. For human genomic DNA, the technique was demonstrated with mutations associated with cystic fibrosis, including SNPs (G542X, I506V, and F508C) and 3-bp deletions (F508del and I507del). Genotyping of SNPs and small deletions by melting analysis of an unlabeled probe in the presence of LCGreen I is simple and rapid. Only three unlabeled oligonucleotides (two primers and one probe), a saturating DNA dye, PCR, and a melting instrument are required. The method is closed-tube, does not require fluorescently labeled probes or real-time PCR, and can be completed in <10 min on any instrument capable of monitoring melting curves by fluorescence.

Identification of putative transcription factor binding sites in rodent selenoprotein W promoter

To understand transcriptional regulation of the selenoprotein W (SeW) gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter and antibodies, we observed that specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as to the metal response element (MRE). Although competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore, the SeW gene may be a target for Sp1 whose binding to various regulatory sequences of the SeW promoter may activate or repress the transcription of SeW. The MRE, GRE, AP-1 and LF-A1 sites were also tested but no evidence was obtained for specific binding as indicated by lack of competition with unlabeled probes.

Neural-restrictive silencers in the regulatory mechanism of pituitary adenylate cyclase-activating polypeptide gene expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a pleiotropic neuropeptide and is present abundantly in central nervous system. During a detailed analysis of the 5′-flanking region of the mouse PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element, and are termed neural-restrictive silencer-like elements 1 and 2 (NRSLE1 and NRSLE2). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. In the electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells and individual oligonucleotide probes for NRSLE1 and NRSLE2, a specific complex was observed to have the same migration as compared with the NRSE probe of rat type II sodium channel gene (NaII). Furthermore, these complexes were efficiently competed by the unlabeled NaII probe. In the luciferase reporter assay, the reporter gene constructs containing NRSLEs, driven by heterologous SV40 promoter, exhibited repression of luciferase activity almost equal to basal level in Swiss-3T3 cells. In contrast, the repression was not observed in differentiated PC12 cells with NGF. These results suggested that the neural-restrictive silencer system might be involved in the regulatory mechanism of neuron-specific PACAP gene expression.

Characterization of DNA end-binding activities in higher plants

DNA double-strand-breaks (DSB) are the most severe lesion in cells exposing to ionizing radiation and many other stress environments. Repair of DNA DSB is therefore critical to cellular survival. In this work, we observed the double-stranded DNA end-binding (DEB) like activities in rice ( Oryza sativa L. cv. TN5) suspension cells and hypocotyls from etiolated mung bean ( Vigna radiata L. TN5) seedlings. Higher plant DEB-like protein binds primarily to linearized double-stranded DNA ends. Competition of unlabeled probe was examined in double-stranded DEB assay of cell extracts from rice and mung bean. DEB-like activities of higher plants did not depend on sequence and types of double-stranded DNA ends. Distinct electrophoretic mobility shift patterns and binding features further indicate that DEB-like factors from various sources might not share identical structure and function, and probably belong to different types of DEB proteins from higher plants. Our evidence suggests that DEB proteins are certainly ubiquitous in all organisms probably for repairing and processing double-stranded DNA breaks from formidable lethal lesion.