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Target Sequences (target + sequence)
Selected AbstractsORIGINAL ARTICLE: Suppression of Mamu-AG by RNA InterferenceAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2009Jessica G. Drenzek Problem, The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu-AG, the rhesus monkey homology of human leukocyte antigen (HLA)-G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function. Method of study, A series of potential target short hairpin RNA (shRNA) sequences to suppress Mamu-AG expression was screened, which identified an optimal sequence to use in transfection experiments. Knockdown in two different Mamu-AG-expressing cell lines was measured by flow cytometry. Cytotoxicity assays were performed to correlate Mamu-AG expression with NK cell cytotoxicity. Results, Decreased expression of Mamu-AG by short interfering RNA (siRNA) (70,80%) in cell types tested was associated with increased lysis of Mamu-AG target cells. Conclusion, Target sequences have been identified that knocked down Mamu-AG expression by RNAi and increased lysis by NK cells. This supports the concept that NK cell receptors recognize this placental non-classical MHC class I molecule. [source] Human natural Treg microRNA signature: Role of microRNA-31 and microRNA-21 in FOXP3 expressionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2009Redouane Rouas Abstract Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3, untranslated region (3, UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3, UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells. [source] ,-MSH and cAMP signalling in normal human melanocytesEXPERIMENTAL DERMATOLOGY, Issue 9 2004R. Buscà Melanocytes are neural crest-derived skin cells specialized in the synthesis of melanin pigments responsible, in human, for skin and hair colour. The pro-opiomelanocortin peptide, ,-MSH is a strong melanogenic agent secreted by keratinocytes following UV radiation. ,-MSH through the binding to the MC1R and activation of the cyclic AMP pathway plays a pivotal role in melanocyte differentiation and in the regulation of skin pigmentation. During the last few years, we have elucidated the molecular events linking the cAMP pathway to melanogenesis upregulation. This cascade involves the activation of protein kinase A and CREB transcription factor, leading to the upregulation of the expression of microphthalmia-associated transcription factor (MITF). MITF binds and activates the melanogenic gene promoters thereby increasing their expression, which results in an increased melanin synthesis. Beyond this simplified scheme, other intracellular signalling pathways are regulated by cAMP and participate to the regulation of melanocyte differentiation. Indeed, cAMP inhibits the phosphatidyl inositol 3-kinase pathway, leading to the inhibition of AKT and to the activation of GSK3,. This kinase phosphorylates MITF and allows its binding to the target sequence. Such pathways are involved in the upregulation of melanogenesis. ,-MSH and cAMP signalling also regulate melanocyte dendricity, and melanosome transport through the inhibition of the Rho GTPase cascade that function downstream the PI3 kinase. It should be also mentioned that cAMP activates the ERK pathway through a melanocyte-specific pathway involving Ras and B-Raf. The activation of ERK and RSK1 leads to the phosphorylation of MITF and target MITF to the proteasome degradation pathway. Interestingly, several proteins involved in melanocyte differentiation by ,-MSH (MC1R, PI3K, B-Raf and MITF) have also been implicated in the development of melanoma, suggesting that the cAMP pathway could influence melanocyte transformation. [source] Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response elementFEBS JOURNAL, Issue 13 2001Iwona Grad The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. Our previous study [Niedziela-Majka, A., Kochman, M., O,yhar, A. (2000) Eur. J. Biochem.267, 507,519] on EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5, half-site of the pseudo-palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. Here, we analyzed in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition , helix and the roles of the base pairs of the UspDBD target sequence have been probed by site-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5, half-site of the element may be used for the fine-tuning of the 20E-response element specificity and consequently the physiological response. [source] Quantitative analysis of total mitochondrial DNA: Competitive polymerase chain reaction versus real-time polymerase chain reactionJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2004Hari K. Bhat Abstract An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 ± 1.01 × 104 molecules/ng total genomic DNA using competitive PCR vs 4.90 ± 0.84 × 104 molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:180,186, 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20024 [source] Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplificationJOURNAL OF FISH DISEASES, Issue 4 2006P J Millard Abstract A new method for the molecular detection of the fish pathogens, infectious haematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV), is described. By employing molecular padlock probe (MPP) technology combined with rolling circle amplification (RCA) and hyperbranching (Hbr), it is possible to detect RNA target sequence from these viruses at levels comparable with those detected by the polymerase chain reaction (PCR), but without prior reverse transcription. The use of MPP technology combined with RCA and Hbr for the detection of IHNV and ISAV in fish exhibited selectivity comparable with that of PCR while potentially reducing the time and cost required for analysis. The method described was used to detect as few as 104 DNA oligonucleotide targets and was sequence-specific at the single base level. Viral RNA could be detected directly, either alone or in the presence of non-viral RNA from fish tissue. This technology is applicable for detecting a variety of microbes, in addition to IHNV and ISAV, and is ideal for further integration into a biosensor platform for on-site diagnosis of pathogen infection in fish. [source] Efficient recognition of protein fold at low sequence identity by conservative application of Psi-BLAST: application,JOURNAL OF MOLECULAR RECOGNITION, Issue 2 2005F. J. Stevens Abstract Based on a study involving structural comparisons of proteins sharing 25% or less sequence identity, three rounds of Psi-BLAST appear capable of identifying remote evolutionary homologs with greater than 95% confidence provided that more than 50% of the query sequence can be aligned with the target sequence. Since it seems that more than 80% of all homologous protein pairs may be characterized by a lack of significant sequence similarity, the experimental biologist is often confronted with a lack of guidance from conventional homology searches involving pair-wise sequence comparisons. The ability to disregard levels of sequence identity and expect value in Psi-BLAST if at least 50% of the query sequence has been aligned allows for generation of new hypotheses by consideration of matches that are conventionally disregarded. In one example, we suggest a possible evolutionary linkage between the cupredoxin and immunoglobulin fold families. A thermostable hypothetical protein of unknown function may be a circularly permuted homolog to phosphotriesterase, an enzyme capable of detoxifying organophosphate nerve agents. In a third example, the amino acid sequence of another hypothetical protein of unknown function reveals the ATP binding-site, metal binding site, and catalytic sidechain consistent with kinase activity of unknown specificity. This approach significantly expands the utility of existing sequence data to define the primary structure degeneracy of binding sites for substrates, cofactors and other proteins. Copyright © 2004 John Wiley & Sons, Ltd. [source] The effect of target lithology on the products of impact meltingMETEORITICS & PLANETARY SCIENCE, Issue 12 2008G. R. OSINSKI Impact events generate pressures and temperatures that can melt a substantial volume of the target; however, there remains considerable discussion as to the effect of target lithology on the generation of impact melts. Early studies showed that for impacts into crystalline targets, coherent impact melt rocks or "sheets" are formed with these rocks often displaying classic igneous structures (e.g., columnar jointing) and textures. For impact structures containing some amount of sedimentary rocks in the target sequence, a wide range of impact-generated lithologies have been described, although it has generally been suggested that impact melt is either lacking or is volumetrically minor. This is surprising given theoretical constraints, which show that as much melt should be produced during impacts into sedimentary targets. The question then arises: where has all the melt gone? The goal of this synthesis is to explore the effect of target lithology on the products of impact melting. A comparative study of the similarly sized Haughton, Mistastin, and Ries impact structures, suggests that the fundamental processes of impact melting are basically the same in sedimentary and crystalline targets, regardless of target properties. Furthermore, using advanced microbeam analytical techniques, it is apparent that, for the structures under consideration here, a large proportion of the melt is retained within the crater (as crater-fill impactites) for impacts into sedimentary-bearing target rocks. Thus, it is suggested that the basic products are genetically equivalent but they just appear different. That is, it is the textural, chemical and physical properties of the products that vary. [source] OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in ricePLANT BIOLOGY, Issue 5 2009C. Fu Abstract Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP (http://rgp.dna.affrc.go.jp). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3, which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation. [source] Using a gel-free PCR-ELISA for the molecular identification of wheat genotypes carrying wheat,rye translocationsPLANT BREEDING, Issue 1 2008J. Zuñiga Abstract Current techniques to identify wheat lines possessing the 1RS chromosome are generally unsuitable in relation to the speed and cost needs in modern wheat breeding programmes. A gel-free, direct amplicon capture PCR-ELISA assay was developed and evaluated, aiming at speeding the identification of wheat genotypes possessing the 1RS chromosome arm in breeding programmes. The chosen target sequence was the repetitive, interspersed rye-specific element RIS-1. Primers were end-labelled with digoxigenin and biotin, and amplicons captured on to straptavidine-coated microplates. Subsequent immunodetection of the digoxigenin moiety readily distinguished 1RS from non-1RS control genotypes tested. When a nursery consisting of 120 winter and spring wheat lines was screened by PCR-gel electrophoresis and the PCR-ELISA, a perfect agreement between both techniques was observed. Test robustness, as measured by the tolerance to variations in DNA input, was better for PCR-ELISA than PCR-gel electrophoresis. In conclusion, a simple, robust, fast and scalable technique for the detection of 1RS chromosome carriers in wheat breeding programmes is now available. [source] Heritable targeted mutagenesis in maize using a designed endonucleaseTHE PLANT JOURNAL, Issue 1 2010Huirong Gao Summary The liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I- CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I- CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium -mediated transformation of immature embryos, and transgenic T0 plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T0 plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant. [source] Trex-1 deficiency in rheumatoid arthritis synovial fibroblastsARTHRITIS & RHEUMATISM, Issue 9 2010Michel Neidhart Objective To explore whether the increased expression of long interspersed nuclear element 1 (LINE-1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex-1, an exonuclease involved in the metabolization of L1 DNA:RNA hybrids. Methods Chromatin immunoprecipitation was used to detect L1-related p40 protein (L1-ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1-ORF1p and pGL3-TS3 carrying the target sequence for L1-ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex-1 measured by flow cytometry. The expression of Trex-1 mRNA and protein was also compared using real-time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex-1 in the L1-ORF1p,mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex-1 expression vector. Results Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3-TS3. L1-ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex-1. Levels of Trex-1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex-1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex-1, however, resulted in an enhancement of L1-ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex-1 inhibited 5-azacytidine,induced expression of p38, MAPK, a gene carrying the TS3 sequence. Conclusion The deficiency of Trex-1 in RASFs allows a longer half-life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a "spontaneous" aggressive behavior. [source] Regulation of targeted gene repair by intrinsic cellular processesBIOESSAYS, Issue 2 2009Julia U. Engstrom Targeted gene alteration (TGA) is a strategy for correcting single base mutations in the DNA of human cells that cause inherited disorders. TGA aims to reverse a phenotype by repairing the mutant base within the chromosome itself, avoiding the introduction of exogenous genes. The process of how to accurately repair a genetic mutation is elucidated through the use of single-stranded DNA oligonucleotides (ODNs) that can enter the cell and migrate to the nucleus. These specifically designed ODNs hybridize to the target sequence and act as a beacon for nucleotide exchange. The key to this reaction is the frequency with which the base is corrected; this will determine whether the approach becomes clinically relevant or not. Over the course of the last five years, workers have been uncovering the role played by the cells in regulating the gene repair process. In this essay, we discuss how the impact of the cell on TGA has evolved through the years and illustrate ways that inherent cellular pathways could be used to enhance TGA activity. We also describe the cost to cell metabolism and survival when certain processes are altered to achieve a higher frequency of repair. [source] Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system,BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Jung-Won Keum Abstract Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components. Biotechnol. Bioeng. 2009;102: 577,582. © 2008 Wiley Periodicals, Inc. [source] Crystallization of a ZRANB2,RNA complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2008Fionna E. Loughlin ZRANB2 is a zinc-finger protein that has been shown to influence alternative splice-site selection. The protein comprises a C-terminal arginine/serine-rich domain that interacts with spliceosomal proteins and two N-terminal RanBP2-type zinc fingers that have been implicated in RNA recognition. The second zinc finger bound to a six-nucleotide single-stranded RNA target sequence crystallized in the hexagonal space group P6522 or P6122, with unit-cell parameters a = 54.52, b = 54.52, c = 48.07,Å; the crystal contains one monomeric complex per asymmetric unit. This crystal form has a solvent content of 39% and diffracted to 1.4,Å resolution using synchrotron radiation. [source] A Molecular Probe for the Detection of Homopurine SequencesCHEMBIOCHEM, Issue 1 2007Ivan Trkulja The highly selective detection of homopurine target strands with a triplex-forming molecular probe is described. The binding of a clamp-type oligonucleotide containing two terminally attached pyrene molecules to the target sequence is easily monitored through excimer formation. The oligonucleotide probe allows the efficient discrimination of single nucleotide mismatches because of the high mismatch sensitivity of the triplex formation. [source] Stabilisation of RNA Bulges by Oligonucleotide Complements Containing an Adenosine AnalogueCHEMBIOCHEM, Issue 11 2003Annemieke Madder Abstract Incorporation of 2,-deoxy-2,- , -(1-naphthylmethyl)tubercidin into an oligodeoxyribonucleotide mostly has little or a slightly negative effect on the Tmvalues of complexes with DNA complements. With the same naphthylmethyl-substituted nucleoside at the 3,-end of a 2,-O-methyloligoribonucleotide, however, a stabilisation of 1,2,°C in the corresponding complexes with both DNA and RNA is observed. When the target sequence is an RNA fragment forming a two- or three-nucleotide bulge, complexes with (naphthylmethyl)tubercidin-modified oligodeoxyribonucleotides, as well as with the corresponding 2,-O-methyloligoribonucleotides, give stabilisations of 1,2,°C for the three-nucleotide bulge and of almost 4,°C for the two-nucleotide bulge. This stabilisation is specific to RNA, since the corresponding complexes with the DNA fragments do not display this effect. Thus, the (naphthylmethyl)tubercidin-containing oligonucleotides are the first reported oligonucleotide modifications that specifically stabilise bulged RNA. [source] Label-Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste ElectrodesELECTROANALYSIS, Issue 24 2002Pinar Kara Abstract A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label-free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects. [source] Simultaneous detection of genetically modified organisms by multiplex ligation-dependent genome amplification and capillary gel electrophoresis with laser-induced fluorescenceELECTROPHORESIS, Issue 13 2010Virginia García-Cañas Abstract In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis. [source] Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probesENVIRONMENTAL MICROBIOLOGY, Issue 5 2009Christina M. Preston Summary A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10,1.98 and 4.43, 12.54 fmole ml,1 homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton. [source] Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone librariesENVIRONMENTAL MICROBIOLOGY, Issue 11 2002Andreas Schramm Summary A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated Td values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. [source] LPXN, a member of the paxillin superfamily, is fused to RUNX1 in an acute myeloid leukemia patient with a t(11;21)(q12;q22) translocationGENES, CHROMOSOMES AND CANCER, Issue 12 2009Hai-Ping Dai RUNX1 (previously AML1) is involved in multiple recurrent chromosomal rearrangements in hematological malignances. Recently, we identified a novel fusion between RUNX1 and LPXN from an acute myeloid leukemia (AML) patient with t(11;21)(q12;q22). This translocation generated four RUNX1/LPXN and one LPXN/RUNX1 chimeric transcripts. Two representative RUNX1/LPXN fusion proteins, RL and RLs, were both found to localize in the nucleus and could bring the CBFB protein into the nucleus like the wild-type RUNX1. Both fusion proteins inhibit the ability of RUNX1 to transactivate the CSF1R promoter, probably through competition for its target sequences. Unlike RL and RLs, the LPXN/RUNX1 fusion protein LR was found to localize in the cytoplasm. Thus, we believe it has little impact on the transcriptional activity of RUNX1. We also found that fusion proteins RL, RLs, LR, and wild-type LPXN could confer NIH3T3 cells with malignant transformation characteristics such as more rapid growth, the ability to form colonies in soft agar, and the ability to form solid tumors in the subcutaneous tissue of the BALB/c nude mice. Taken together, our data indicated that the RUNX1/LPXN and LPXN/RUNX1 fusion proteins may play important roles in leukemogenesis and that deregulation of cell adhesion pathways may be pathogenetically important in AML. Our study also suggests that LPXN may play an important role in carcinogenesis. © 2009 Wiley-Liss, Inc. [source] Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients,HUMAN MUTATION, Issue 1 2008Fanyi Zeng Abstract Multiplex ligation-dependent probe amplification (MLPA) is widely used to screen genes of interest for deletions and duplications. Since MLPA is usually based on size-separation of the amplification products, the maximum number of target sequences that can be screened in parallel is usually limited to ,40. We report the design of a robust array-based MLPA format that uses amplification products of essentially uniform size (100,120,bp) and distinguishes between them by virtue of incorporated tag sequences. We were thus able to increase probe complexity to 124, with very uniform product yields and signals that have a low coefficient of variance. The assay designed was used to screen the largest set studied so far (249 patients) of unrelated Duchenne muscular dystrophy (DMD) cases from the Chinese population. In a blind study we correctly assigned 98% of the genotypes and detected rearrangements in 181 cases (73%); i.e., 163 deletions (65%), 13 duplications (5%), and five complex rearrangements (2%). Although this value is significantly higher for Chinese patients than previously reported, it is similar to that found for other populations. The location of the rearrangements (76% in the major deletion hotspot) is also in agreement with other findings. The 96-well flow-through microarray system used in this research provides high-throughput and speed; hybridization can be completed in 5 to 30,minutes. Since array processing and data analysis are fully automated, array-MLPA should be easy to implement in a standard diagnostic laboratory. The universal array can be used to analyze any tag-modified MLPA probe set. Hum Mutat 29(1), 190,197, 2008. © 2007 Wiley-Liss, Inc. [source] Determination of genomic copy number with quantitative microsphere hybridization,,HUMAN MUTATION, Issue 4 2006Heather L. Newkirk Abstract We developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5, ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.59±0.02 &!ndash;fold in three different deletion patients and increased 1.42±0.01 &!ndash;fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context. Hum Mutat 27(4), 376,386, 2006. © 2006 Wiley-Liss, Inc. [source] DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications,HUMAN MUTATION, Issue 1 2004David Fredman Abstract Secondary structures in polymerase chain reaction (PCR) target sequences have a negative impact on amplification success rates and on downstream uses of PCR products. For example, signal strength and allele discrimination in single nucleotide polymorphism (SNP) genotyping methods can be compromised by allele-biased amplification and/or by PCR product folding that limits access of interrogating probes. To increase the fidelity and robustness of PCR, and to aid follow-on applications, we have developed DFold (http://dfold.cgb.ki.se),a generalized software solution that creates PCR oligonucleotide primer designs devoid of stable secondary structures. We demonstrate the effectiveness of the tool by applying it to a range of dynamic allele-specific hybridization (DASH) assay designs, many of which we evaluate in the laboratory. We further consider how the system throughput may be made sufficiently high for use upon millions of target sequences in order to support whole-genome analyses. Hum Mutat 24:1,8, 2004. © 2004 Wiley-Liss, Inc. [source] Comparison between two PCR-based bacterial identification methods through artificial neural network data analysisJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2008Jie Wen Abstract The 16S ribosomal ribonucleic acid (rRNA) and 16S-23S rRNA spacer region genes are commonly used as taxonomic and phylogenetic tools. In this study, two pairs of fluorescent-labeled primers for 16S rRNA genes and one pair of primers for 16S-23S rRNA spacer region genes were selected to amplify target sequences of 317 isolates from positive blood cultures. The polymerase chain reaction (PCR) products of both were then subjected to restriction fragment length polymorphism (RFLP) analysis by capillary electrophoresis after incomplete digestion by Hae III. For products of 16S rRNA genes, single-strand conformation polymorphism (SSCP) analysis was also performed directly. When the data were processed by artificial neural network (ANN), the accuracy of prediction based on 16S-23S rRNA spacer region gene RFLP data was much higher than that of prediction based on 16S rRNA gene SSCP analysis data(98.0% vs. 79.6%). This study proved that the utilization of ANN as a pattern recognition method was a valuable strategy to simplify bacterial identification when relatively complex data were encountered. J. Clin. Lab. Anal. 22:14,20, 2008. © 2008 Wiley-Liss, Inc. [source] High-affinity triplex-forming oligonucleotide target sequences in mammalian genomesMOLECULAR CARCINOGENESIS, Issue 1 2007Qi Wu Abstract Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only ,37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted. © 2006 Wiley-Liss, Inc. [source] Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNAMOLECULAR MICROBIOLOGY, Issue 4 2004Patrick A. Pribil Summary The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA bending. [source] The X philes: structure-specific endonucleases that resolve Holliday junctionsMOLECULAR MICROBIOLOGY, Issue 4 2001Gary J. Sharples Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication. It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four-way branched intermediate commonly referred to as the Holliday junction. DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division. This resolution reaction is mediated by structure-specific endonucleases that catalyse dual-strand incision across the point of strand cross-over. Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands. Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination. Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA. They use a metal-binding pocket to co-ordinate an activated water molecule for phosphodiester bond hydrolysis. In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences. Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R). Recent studies have brought about a reappraisal of the origins of junction-specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core. [source] The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 4 2000Hiroki Yamamoto citS and citT genes encoding a new two-component system were identified in the 71° region between the pel and citM loci on the Bacillus subtilis chromosome. citS- and citT- deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg-citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM,yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite-responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM,cre mutants. From the result of a citM,promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from ,62 to ,74 and from ,149 to ,189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from ,36 to ,84 and from ,168 to ,194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA. [source] | |||||||||||||||||||