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Sample records for acid dna isolated

  1. A Magnetic Nanoparticle Based Nucleic Acid Isolation and Purification Instrument for DNA Extraction of Escherichia Coli O157: H7.

    PubMed

    Chen, Yahui; Lin, Jianhan; Jiang, Qin; Chen, Qi; Zhang, Shengjun; Li, Li

    2016-03-01

    The objective of this study was to evaluate the performance of a nucleic acid isolation and purification instrument using Escherichia coli O157:H7 as the model. The instrument was developed with magnetic nanoparticles for efficiently capturing nucleic acids and an intelligent mechanical unit for automatically performing the whole nucleic acid extraction process. A commercial DNA extraction kit from Huier Nano Company was used as reference. Nucleic acids in 1 ml of E. coli O157: H7 at a concentration of 5 x 10(8) CFU/mL were extracted by using this instrument and the kit in parallel and then detected by an ultraviolet spectrophotometer to obtain A260 values and A260/A280 values for the determination of the extracted DNA's quantity and purity, respectively. The A260 values for the instrument and the kit were 0.78 and 0.61, respectively, and the A260/A280 values were 1.98 and 1.93. The coefficient of variations of these parallel tests ranged from 10.5% to 16.7%. The results indicated that this nucleic acid isolation and purification instrument could extract a comparable level of nucleic acid within 50 min compared to the commercial DNA extraction kit.

  2. Fatty acid and DNA analyses of Permian bacteria isolated from ancient salt crystals reveal differences with their modern relatives.

    PubMed

    Vreeland, Russell H; Rosenzweig, William D; Lowenstein, Tim; Satterfield, Cindy; Ventosa, Antonio

    2006-02-01

    The isolation of living microorganisms from primary 250-million-year-old (MYA) salt crystals has been questioned by several researchers. The most intense discussion has arisen from questions about the texture and age of the crystals used, the ability of organisms to survive 250 million years when exposed to environmental factors such as radiation and the close similarity between 16S rRNA sequences in the Permian and modern microbes. The data in this manuscript are not meant to provide support for the antiquity of the isolated bacterial strains. Rather, the data presents several comparisons between the Permian microbes and other isolates to which they appear related. The analyses include whole cell fatty acid profiling, DNA-DNA hybridizations, ribotyping, and random amplified polymorphic DNA amplification (RAPD). These data show that the Permian strains, studied here, differ significantly from their more modern relatives. These differences are accumulating in both phenotypic and molecular areas of the cells. At the fatty acid level the differences are approaching but have not reached separate species status. At the molecular level the variation appears to be distributed across the genome and within the gene regions flanking the highly conserved 16S rRNA itself. The data show that these bacteria are not identical and help to rule out questions of contamination by putatively modern strains.

  3. Nucleic acid isolation process

    DOEpatents

    Longmire, Jonathan L.; Lewis, Annette K.; Hildebrand, Carl E.

    1990-01-01

    A method is provided for isolating DNA from eukaryotic cell and flow sorted chromosomes. When DNA is removed from chromosome and cell structure, detergent and proteolytic digestion products remain with the DNA. These products can be removed with organic extraction, but the process steps associated with organic extraction reduce the size of DNA fragments available for experimental use. The present process removes the waste products by dialyzing a solution containing the DNA against a solution containing polyethylene glycol (PEG). The waste products dialyze into the PEG leaving isolated DNA. The remaining DNA has been prepared with fragments containing more than 160 kb. The isolated DNA has been used in conventional protocols without affect on the protocol.

  4. Nucleic acid isolation

    DOEpatents

    Longmire, J.L.; Lewis, A.K.; Hildebrand, C.E.

    1988-01-21

    A method is provided for isolating DNA from eukaryotic cell and flow sorted chromosomes. When DNA is removed from chromosome and cell structure, detergent and proteolytic digestion products remain with the DNA. These products can be removed with organic extraction, but the process steps associated with organic extraction reduces the size of DNA fragments available for experimental use. The present process removes the waste products by dialyzing a solution containing the DNA against a solution containing polyethylene glycol (PEG). The waste products dialyze into the PEG leaving isolated DNA. The remaining DNA has been prepared with fragments containing more than 160 kb. The isolated DNA has been used in conventional protocols without effect on the protocol.

  5. Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences.

    PubMed

    Suzuki, T; Inoue, N; Higashi, T; Mizobuchi, R; Sugimura, N; Yokouchi, K; Furukohri, T

    2000-12-01

    Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 micromole Pi min(-1) mg protein(-1) for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1,608 and 1,239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66-73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63-71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55-57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.

  6. Method for isolating nucleic acids

    SciTech Connect

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    2015-09-29

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids for a wide variety of applications including, sequencing or species population analysis.

  7. Functional characterization of an acidic SK(3) dehydrin isolated from an Opuntia streptacantha cDNA library.

    PubMed

    Ochoa-Alfaro, A E; Rodríguez-Kessler, M; Pérez-Morales, M B; Delgado-Sánchez, P; Cuevas-Velazquez, C L; Gómez-Anduro, G; Jiménez-Bremont, J F

    2012-03-01

    Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.

  8. Evaluation of deoxyribonucleic acid (DNA) isolated from human bloodstains exposed to ultraviolet light, heat, humidity, and soil contamination

    SciTech Connect

    McNally, L.; Shaler, R.C.; Baird, M.; Balazs, I.; De Forest, P.; Kobilinsky, L. )

    1989-09-01

    This study was designed to analyze the effects of common environmental insults on the ability to obtain deoxyribonucleic acid (DNA) restriction fragment-length polymorphisms (RFLP) patterns from laboratory prepared specimens. The environmental conditions studied include the exposure of dried bloodstains to varying amounts of relative humidity (0, 33, 67, and 98%), heat (37{degree}C), and ultraviolet light for periods of up to five days. In addition, the effect of drying over a four-day period in whole blood collected with and without ethylenediaminetetraacetate (EDTA) was examined. The results of the study showed that, under the conditions studied, the integrity of DNA is not altered such that false RFLP patterns are obtained. The only effect observed was that the overall RFLP pattern becomes weaker, but individual RFLP fragments are neither created nor destroyed.

  9. Lactation Affects Isolated Mitochondria and Its Fatty Acid Composition but Has No Effect on Tissue Protein Oxidation, Lipid Peroxidation or DNA-Damage in Laboratory Mice

    PubMed Central

    Valencak, Teresa G.; Raith, Johannes; Staniek, Katrin; Gille, Lars; Strasser, Alois

    2016-01-01

    Linking peak energy metabolism to lifespan and aging remains a major question especially when focusing on lactation in females. We studied, if and how lactation affects in vitro mitochondrial oxygen consumption and mitochondrial fatty acid composition. In addition, we assessed DNA damage, lipid peroxidation and protein carbonyls to extrapolate on oxidative stress in mothers. As model system we used C57BL/6NCrl mice and exposed lactating females to two ambient temperatures (15 °C and 22 °C) while they nursed their offspring until weaning. We found that state II and state IV respiration rates of liver mitochondria were significantly higher in the lactating animals than in non-lactating mice. Fatty acid composition of isolated liver and heart mitochondria differed between lactating and non-lactating mice with higher n-6, and lower n-3 polyunsaturated fatty acids in the lactating females. Surprisingly, lactation did not affect protein carbonyls, lipid peroxidation and DNA damage, nor did moderate cold exposure of 15 °C. We conclude that lactation increases rates of mitochondrial uncoupling and alters mitochondrial fatty acid composition thus supporting the “uncoupling to survive” hypothesis. Regarding oxidative stress, we found no impact of lactation and lower ambient temperature and contribute to growing evidence that there is no linear relationship between oxidative damage and lactation. PMID:26805895

  10. Silicon dioxide thin film mediated single cell nucleic acid isolation.

    PubMed

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube.

  11. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    PubMed Central

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  12. Isolation of DNA methyltransferase from plants

    SciTech Connect

    Ehrlich, K.; Malbroue, C.

    1987-05-01

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from (/sup 3/H)AdoMet incorporated into acid precipitable material per h at 30/sup 0/). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30/sup 0/.

  13. Simple & Safe Genomic DNA Isolation.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  14. Isolating DNA from Gram-Negative Bacteria.

    PubMed

    Green, Michael R; Sambrook, Joseph

    2017-01-03

    The isolation of DNA from bacteria, described in this protocol, relies upon the use of sodium dodecyl sulfate and proteinase K to lyse the cells. High-molecular-weight DNA is then sheared (to reduce its viscosity and make it more manageable), extracted with phenol:chloroform, and precipitated with isopropanol. DNA isolated according to this procedure ranges from 30 to 80 kb in length.

  15. Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples.

    PubMed

    Reyes-Escogido, Lourdes; Balam-Chi, Mario; Rodríguez-Buenfil, Ingrid; Valdés, Jesús; Kameyama, Luis; Martínez-Pérez, Francisco

    2010-11-01

    We established a Chelex 100-Microwave method for the purification of bacterial genomic DNA (gDNA) in less than 20 min with high yield and good quality, useful for multiple purposes. It combines Chelex 100, proteinase K, RNase A and heating in a microwave oven. The resulting gDNA was used directly to identify bacterial species of the Order Lactobacillales by means of PCR amplification of their 16S rDNA gene, isolated from sediments on the Yucatan Peninsula, Mexico. This method produced gDNA free of phenolic and protein residual contaminants from 100 of these isolated bacteria. 16S rDNA amplification and sequencing showed Pediococcus acidilactici to prevail in inland lagoons, and Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sp., and Lactobacillus fermentum to be most abundant in the soils of livestock farms. The combination of Chelex 100, enzymes and microwave heating used in the Chelex 100-Microwave method produced large amounts of highly pure gDNA from Gram-positive and Gram-negative bacteria, in less than 20 min.

  16. Isolation of high molecular weight plant nuclear DNA suitable for use in recombinant DNA technology.

    PubMed

    Kiss, T; Solymosy, F

    1987-01-01

    Nuclei isolated from leaf cells of broad bean (Vicia faba L) by a newly developed method based on the use of citric acid in the isolation medium and flotation on a Percoll cushion yielded high molecular weight plant nuclear DNA which was suitable for (i) analysis by restriction endonucleases, (ii) molecular cloning and (iii) genomic blot hybridization. Starting from nuclear preparations obtained by this method, U2 small nuclear RNA-specific DNA sequences were detected in Vicia faba L. This is the first report on the demonstration of small nuclear RNA-specific DNA sequences in plant material.

  17. Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

    PubMed Central

    Segel, G B; Boal, T R; Cardillo, T S; Murant, F G; Lichtman, M A; Sherman, F

    1992-01-01

    A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP. Images PMID:1352881

  18. Methods for isolation of cell-free plasma DNA strongly affect DNA yield.

    PubMed

    Fleischhacker, Michael; Schmidt, Bernd; Weickmann, Sabine; Fersching, Debora M I; Leszinski, Gloria S; Siegele, Barbara; Stötzer, Oliver J; Nagel, Dorothea; Holdenrieder, Stefan

    2011-11-20

    Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6 ng/mL and 28.1 ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used.

  19. Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

    PubMed

    Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

    2011-08-01

    Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both

  20. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

    PubMed

    Kapustin, D V; Prostyakova, A I; Alexeev, Ya I; Varlamov, D A; Zubov, V P; Zavriev, S K

    2014-04-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation.

  1. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis

    PubMed Central

    Kapustin, D. V.; Prostyakova, A. I.; Alexeev, Ya. I.; Varlamov, D. A.; Zubov, V. P.; Zavriev, S. K.

    2014-01-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  2. Phenol-stacked carbon nanotubes: A new approach to genomic DNA isolation from plants.

    PubMed

    Nazarian-Firouzabadi, Farhad; Ismaili, Ahmad; Zabeti, Sayed Mahmoud

    2014-09-01

    Extraction of intact quality DNA from plant tissues, especially those rich in secondary metabolites, is often challenging. Literally, hundreds of different DNA isolation protocols from various plant species have been published over the last decades. Although many commercial DNA isolation kits are convenient and designed to be safe, their cost and availability cause limitations in small molecular labs in many developing countries. In nearly all protocols and DNA isolation kits, phenol and chloroform are used to precipitate various classes of impurities. However, phenol is partially soluble in water, resulting in the co-existence of proteins in upper (aqueous) phases. This phenomenon results in the contamination of the nucleic acids and low quality DNA. Nanotechnology advances have helped many areas of molecular biology such as the development of new diagnosis and purification kits. In this study, for the first time, we report a different approach to isolate DNA from plants based on carbon nanotubes (CNTs). The results show that the phenol reagent stack on CNTs can effectively remove proteins, polysaccharides and other polyphenol constituents. The A260/A280nm absorbance ratios of isolated DNA samples were 1.9 and 1.8 for chamomile and opium plants, respectively, indicating the high purity of the isolated DNA. DNA yield was more than two times the standard Doyle and Doyle method. Furthermore, the isolated DNA proved amenable to PCR amplification, using Random Amplified Polymorphic DNA (RAPD) analysis.

  3. Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR)

    PubMed Central

    Kunnev, Dimiter; Freeland, Amy; Qin, Maochun; Wang, Jianmin; Pruitt, Steven C.

    2015-01-01

    Nascent strand capture and release (NSCR) is a method for isolation of short nascent strands to identify origins of DNA replication. The protocol provided involves isolation of total DNA, denaturation, size fractionation on a sucrose gradient, 5′-biotinylation of the appropriate size nucleic acids, binding to a streptavidin coated magnetic beads, intensive washing, and specific release of only the RNA-containing chimeric nascent strand DNA using ribonuclease I (RNase I). The method has been applied to mammalian cells derived from proliferative tissues and cell culture but could be used for any system where DNA replication is primed by a small RNA resulting in chimeric RNA-DNA molecules. PMID:26949711

  4. Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment.

    PubMed

    Gupta, Amit Kumar; Harish; Rai, Manoj Kumar; Phulwaria, Mahendra; Shekhawat, Narpat Singh

    2011-11-10

    Isolation of intact and pure genomic DNA (gDNA) is essential for many molecular biology applications. It is difficult to isolate pure DNA from mature trees of hot and dry desert regions because of the accumulation of high level of polysaccharides, phenolic compounds, tannins etc. We hereby report the standardized protocol for the isolation and purification of gDNA from seven ecologically and medically important tree species of Combretaceae viz. Anogeissus (Anogeissus sericea var. nummularia, Anogeissus pendula, and Anogeissus latifolia) and Terminalia (Terminalia arjuna, Terminalia bellirica, Terminalia catappa and Terminalia chebula). This method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin-column. gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is also critical. The quality of DNA was determined by the A(260)/A(280) absorbance ratio. gDNA was also observed for its intactness by running on 0.8% agarose gel. The suitability of extracted DNA for PCR was tested by amplification with RAPD primers, which was successful. Further, rbcLa (barcoding gene) was amplified and sequenced to check the quality of extracted gDNA for its downstream applications.

  5. Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.

    PubMed

    Lopez, Blanca R; Hernandez, Juan-Pablo; Bashan, Yoav; de-Bashan, Luz E

    2017-04-01

    Isolation of nucleic acids from Chlorella is difficult, given the chemically complex nature of their cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difficulty. Here, we modified, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgae C. sorokiniana. Isolation of nucleic acids from immobilized cells required two steps in dissolving the alginate matrix, releasing the cells, and mechanical disruption with glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins efficiently and had low levels of contamination from residual polysaccharides from the matrices and/or metabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR).

  6. Isolation of genomic DNA using magnetic cobalt ferrite and silica particles.

    PubMed

    Prodelalová, Jana; Rittich, Bohuslav; Spanová, Alena; Petrová, Katerina; Benes, Milan J

    2004-11-12

    Adsorption separation techniques as an alternative to laborious traditional methods (e.g., based on phenol extraction procedure) have been applied for DNA purification. In this work we used two types of particles: silica and cobalt ferrite (unmodified or modified with a reagent containing weakly basic aminoethyl groups, aminophenyl groups, or alginic acid). DNA from chicken erythrocytes and DNA isolated from bacteria Lactococcus lactis were used for testing of adsorption/desorption properties of particles. The cobalt ferrite particles modified with different reagents were used for isolation of PCR-ready bacterial DNA from different dairy products.

  7. [A composite polyaniline-containing silica sorbent for DNA isolation].

    PubMed

    Kapustin, D V; Iagudaeva, E Iu; Zavada, L L; Zhigis, L S; Zubov, V P; Iaroshevskaia, E M; Plobner, L; Leiser, R M; Brem, G

    2003-01-01

    A composite sorbent based on porous glass beads modified with thin polyaniline coating was prepared by precipitating aniline polymerization in the presence of carrier particles. It was shown that the modification ensures the uniform coating of the inner surface of the carrier pores with the polymer layer approximately 70 A thick. It was shown that the resulting material retains the initial porosity of the carrier and is selective in the separation of nucleic acids and proteins. The polyaniline-coated sorbents were shown to be efficient for both the preparative DNA isolation from bacterial lysates and for analytical purposes, in particular, for studying DNA fragmentation during apoptosis proceeding under UV irradiation of cell lysates of colon carcinoma. The morphological and chromatographic characteristics of the new sorbent were demonstrated to be similar to those of the polyfluorobutadiene sorbent.

  8. Isolation and characterization of deoxyribonucleic acid from tissue of the woolly mammoth, Mammuthus primigenius.

    PubMed

    Johnson, P H; Olson, C B; Goodman, M

    1985-01-01

    DNA was isolated from tissue samples of several mammoth specimens, radiocarbon dated between 10,000 and 53,000 years old. The DNA was purified by chromatography on hydroxyapatite at 60 degrees C and was characterized as a heterogeneous population of fragments ranging in size from 3000 to 200 base pairs. Thermal denaturation analysis demonstrated that approximately 25% of the DNA had a base composition similar to Asian elephant DNA calculated as 36% G + C. Preliminary analysis by nucleic acid hybridization indicated that only a small fraction of DNA isolated from mammoth tissue (2-5%) was homologous to DNA of Asian elephant, a close living relative of the mammoth. Our results provide the first definitive isolation and characterization of DNA from ancient tissue and suggest a purification strategy that will lead to preparations of DNA from mammoth tissue significantly enriched in elephant-related DNA sequences.

  9. Isolation and Characterization of Bacterial DNA.

    ERIC Educational Resources Information Center

    Wilson, W. David; Davidson, Michael W.

    1979-01-01

    An inexpensive DNA preparation is presented which starts with commercially available frozen packed bacterial cells. Methods for analyzing the DNA are also presented, along with physical chemical experiments which can be done using the purified DNA. (BB)

  10. Isolation and characterization of germ line DNA from mouse sperm.

    PubMed Central

    Shiurba, R; Nandi, S

    1979-01-01

    Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas. PMID:291053

  11. Method for isolating chromosomal DNA in preparation for hybridization in suspension

    DOEpatents

    Lucas, Joe N.

    2000-01-01

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. Chromosomal DNA in a sample containing cell debris is prepared for hybridization in suspension by treating the mixture with RNase. The treated DNA can also be fixed prior to hybridization.

  12. Isolation and characteristics of lactic acid bacteria isolated from ripe mulberries in Taiwan

    PubMed Central

    Chen, Yi-sheng; Wu, Hui-chung; Yanagida, Fujitoshi

    2010-01-01

    The objective of this study was to isolate, characterize, and identify lactic acid bacteria (LAB) from ripe mulberries collected in Taiwan. Ripe mulberry samples were collected at five mulberry farms, located in different counties of Taiwan. Eighty-eight acid-producing cultures were isolated from these samples, and isolates were divided into classes first by phenotype, then into groups by restriction fragment length polymorphism (RFLP) analysis and sequencing of 16S ribosomal DNA (rDNA). Phenotypic and biochemical characteristics led to identification of four bacterial groups (A to D). Weissella cibaria was the most abundant type of LAB distributed in four mulberry farms, and Lactobacillus plantarum was the most abundant LAB found in the remaining farm. Ten W. cibaria and one Lactococcus lactis subsp. lactis isolate produced bacteriocins against the indicator strain Lactobacillus sakei JCM 1157T. These results suggest that various LAB are distributed in ripe mulberries and W. cibaria was the most abundant LAB found in this study. PMID:24031571

  13. Isolation of genomic DNA from medicinal plants without liquid nitrogen.

    PubMed

    Sharma, Pratibha; Joshi, Neha; Sharma, Anubhuti

    2010-06-01

    Genomic DNA was extracted from eight medicinal plants using the present DNA extraction protocols (CTAB extraction method) with some modifications. Leaves were fixed in different fixing solutions containing absolute alcohol (99.99%), chloroform and EDTA, but without liquid nitrogen. DNA quality and quantity obtained were comparable to those isolated with liquid nitrogen, as the lambda260/lambda280 ratio with liquid nitrogen was in range 1.3-1.7 and with other fixing solutions it was 1.1-1.5. Absolute alcohol showed best results as fixing solution. Good quality of DNA was isolated without using liquid nitrogen from different medicinal plant species. DNA isolated by this method was suitable for various molecular biology applications.

  14. Rapid, Effective DNA Isolation from Osmanthus via Modified Alkaline Lysis

    PubMed Central

    2016-01-01

    Variability of leaf structure and presence of secondary metabolites in mature leaf tissue present a challenge for reliable DNA extraction from Osmanthus species and cultivars. The objective of this study was to develop a universal rapid, effective, and cost-efficient method of DNA isolation for Osmanthus mature leaf tissue. Four different methods were used to isolate DNA from 8 cultivars of Osmanthus. Absorbance spectra, DNA concentration, appearance on agarose gel, and performance in PCR were used to analyze quality, quantity, and integrity of isolated DNA. Methods were ranked in order, based on total quantity, quality, and performance points as the following: 1) solid-phase extraction (SPE), 2) modified alkaline lysis (SDS), 3) cetyltrimethylammonium bromide (CTAB) with chloroform (CHL), and 4) CTAB with phenol/chloroform (PHE). Total DNA, isolated via SPE, showed the least contamination but the lowest mean quantity (9.6 ± 3.4 μg) and highest cost. The highest quantity of DNA was isolated via SDS (117 ± 54.1 μg). SPE and SDS resolved the most individuals on agarose gel, whereas the 2 CTAB methods had poorly resolved gels. All methods except PHE performed well in PCR. Additions to the modified alkaline lysis method increased A260:A230 by up to 59% without affecting yield. With the use of SDS, an average of 1000 μg/g DNA was isolated from fresh leaf tissue of 18 samples in ∼1.5 h at a cost of 0.74 U.S. dollars (USD)/sample. We recommend improved alkaline lysis as a rapid, effective, and cost-efficient method of isolating DNA from Osmanthus species. PMID:26816495

  15. Rapid, Effective DNA Isolation from Osmanthus via Modified Alkaline Lysis.

    PubMed

    Alexander, Lisa

    2016-07-01

    Variability of leaf structure and presence of secondary metabolites in mature leaf tissue present a challenge for reliable DNA extraction from Osmanthus species and cultivars. The objective of this study was to develop a universal rapid, effective, and cost-efficient method of DNA isolation for Osmanthus mature leaf tissue. Four different methods were used to isolate DNA from 8 cultivars of Osmanthus. Absorbance spectra, DNA concentration, appearance on agarose gel, and performance in PCR were used to analyze quality, quantity, and integrity of isolated DNA. Methods were ranked in order, based on total quantity, quality, and performance points as the following: 1) solid-phase extraction (SPE), 2) modified alkaline lysis (SDS), 3) cetyltrimethylammonium bromide (CTAB) with chloroform (CHL), and 4) CTAB with phenol/chloroform (PHE). Total DNA, isolated via SPE, showed the least contamination but the lowest mean quantity (9.6 ± 3.4 μg) and highest cost. The highest quantity of DNA was isolated via SDS (117 ± 54.1 μg). SPE and SDS resolved the most individuals on agarose gel, whereas the 2 CTAB methods had poorly resolved gels. All methods except PHE performed well in PCR. Additions to the modified alkaline lysis method increased A260:A230 by up to 59% without affecting yield. With the use of SDS, an average of 1000 μg/g DNA was isolated from fresh leaf tissue of 18 samples in ∼1.5 h at a cost of 0.74 U.S. dollars (USD)/sample. We recommend improved alkaline lysis as a rapid, effective, and cost-efficient method of isolating DNA from Osmanthus species.

  16. Modified method for combined DNA and RNA isolation from peanut and other oil seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA...

  17. Acid soluble platelet aggregating material isolated from human umbilical cord

    SciTech Connect

    Schneider, M.D.

    1983-12-27

    An acid soluble, pepsin sensitive platelet aggregating material is isolated from human umbilical cord tissue by extraction with dilute aqueous acid. The method of isolation is disclosed and its use to control bleeding is described. 2 figs.

  18. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  19. Identification of lactic acid bacteria isolated from corn stovers.

    PubMed

    Pang, Huili; Zhang, Meng; Qin, Guangyong; Tan, Zhongfang; Li, Zongwei; Wang, Yanping; Cai, Yimin

    2011-10-01

    One hundred and twenty-six strains were isolated from corn stover in Henan Province, China, of which 105 isolates were considered to be lactic acid bacteria (LAB) according to Gram-positive, catalase-negative and mainly metabolic lactic acid product. Analysis of the 16S ribosomal DNA sequence of 21 representative strains was used to confirm the presence of the predominant groups and to determine the phylogenetic affiliation of isolates. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank type strains between 99.4% and 100%. The prevalent LAB, predominantly Lactobacillus (85.6%), consisted of L. plantarum (33.3%), L. pentosus (28.6%) and L. brevis (23.7%). Other LAB species as Leuconostoc lactis (4.8%), Weissella cibaria (4.8%) and Enterococcus mundtii (4.8%) also presented in corn stover. The present study is the first to fully document corn stover-associated LAB involved in the silage fermentation. The identification results revealed LAB composition inhabiting corn stover and enabling the future design of appropriate inoculants aimed at improving the fermentation quality of silage.

  20. Alicyclobacillus fodiniaquatilis sp. nov., isolated from acid mine water.

    PubMed

    Zhang, Bo; Wu, Yu-Fan; Song, Jin-Long; Huang, Zhong-Sheng; Wang, Bao-Jun; Liu, Shuang-Jiang; Jiang, Cheng-Ying

    2015-12-01

    Two novel, Gram-stain-variable, moderately thermophilic, acidophilic, rod-shaped, endospore-forming bacteria, G45-16T and G45-17, were isolated from acid mine water of Zijin copper mine in Fujian Province, China. Phylogenetic analysis of 16S rRNA gene sequences showed that they were closely related to Alicyclobacillus acidoterrestris ATCC 49025T with sequence similarities of 96.8 %. Cells grew aerobically at 20-45 °C (optimum, 40 °C), at pH 2.5-5.5(optimum, pH 3.5) and in the presence of 0-4.0 % (w/v) NaCl. Strains contained MK-7 as the major menaquinone and the major cellular fatty acids were ω-cyclohexane C19 : 0 and ω-cyclohexane C17 : 0. The DNA G+C content was 51.3 and 49.8 mol% (Tm) for G45-16T and G45-17, respectively. On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with their relatives and DNA-DNA relatedness values, it is concluded that strains G45-16T and G45-17 represent a novel species within the genus Alicyclobacillus, for which the name Alicyclobacillus fodiniaquatilis sp. nov. is proposed; the type strain is G45-16T(=CGMCC 1.15049T=NBRC 111483T).

  1. Paramagnetic cellulose DNA isolation improves DNA yield and quality among diverse plant taxa1

    PubMed Central

    Moeller, Jackson R.; Moehn, Nicholas R.; Waller, Donald M.; Givnish, Thomas J.

    2014-01-01

    • Premise of the study: The chemical diversity of land plants ensures that no single DNA isolation method results in high yield and purity with little effort for all species. Here we evaluate a new technique originally developed for forensic science, based on MagnaCel paramagnetic cellulose particles (PMC), to determine its efficacy in extracting DNA from 25 plant species representing 21 families and 15 orders. • Methods and Results: Yield and purity of DNA isolated by PMC, DNeasy Plant Mini Kit (silica column), and cetyltrimethylammonium bromide (CTAB) methods were compared among four individuals for each of 25 plant species. PMC gave a twofold advantage in average yield, and the relative advantage of the PMC method was greatest for samples with the lowest DNA yields. PMC also produced more consistent sample purity based on absorbance ratios at 260:280 and 260:230 nm. • Conclusions: PMC technology is a promising alternative for plant DNA isolation. PMID:25309836

  2. Antibiotic resistance of lactic acid bacteria isolated from Chinese yogurts.

    PubMed

    Zhou, N; Zhang, J X; Fan, M T; Wang, J; Guo, G; Wei, X Y

    2012-09-01

    The aim of this study was to evaluate the susceptibility of 43 strains of lactic acid bacteria, isolated from Chinese yogurts made in different geographical areas, to 11 antibiotics (ampicillin, penicillin G, roxithromycin, chloramphenicol, tetracycline, chlortetracycline, lincomycin, kanamycin, streptomycin, neomycin, and gentamycin). The 43 isolates (18 Lactobacillus bulgaricus and 25 Streptococcus thermophilus) were identified at species level and were typed by random amplified polymorphic DNA analysis. Thirty-five genotypically different strains were detected and their antimicrobial resistance to 11 antibiotics was determined using the agar dilution method. Widespread resistance to ampicillin, chloramphenicol, chlortetracycline, tetracyclines, lincomycin, streptomycin, neomycin, and gentamycin was found among the 35 strains tested. All of the Strep. thermophilus strains tested were susceptible to penicillin G and roxithromycin, whereas 23.5 and 64.7% of Lb. bulgaricus strains, respectively, were resistant. All of the Strep. thermophilus and Lb. bulgaricus strains were found to be resistant to kanamycin. The presence of the corresponding resistance genes in the resistant isolates was investigated through PCR, with the following genes detected: tet(M) in 1 Lb. bulgaricus and 2 Strep. thermophilus isolates, ant(6) in 2 Lb. bulgaricus and 2 Strep. thermophilus isolates, and aph(3')-IIIa in 5 Lb. bulgaricus and 2 Strep. thermophilus isolates. The main threat associated with these bacteria is that they may transfer resistance genes to pathogenic bacteria, which has been a major cause of concern to human and animal health. To our knowledge, the aph(3')-IIIa and ant(6) genes were found in Lb. bulgaricus and Strep. thermophilus for the first time. Further investigations are required to analyze whether the genes identified in Lb. bulgaricus and Strep. thermophilus isolates might be horizontally transferred to other species.

  3. Naturally Occurring Lactic Acid Bacteria Isolated from Tomato Pomace Silage

    PubMed Central

    Wu, Jing-jing; Du, Rui-ping; Gao, Min; Sui, Yao-qiang; Xiu, Lei; Wang, Xiao

    2014-01-01

    Silage making has become a significant method of forage conservation worldwide. To determine how tomato pomace (TP) may be used effectively as animal feed, it was ensilaged for 90 days and microbiology counts, fermentation characteristics and chemical composition of tomato pomace silage (TPS) were evaluated at the 30th, 60th, and 90th days, respectively. In addition, 103 lactic acid bacteria were isolated from TPS. Based on the phenotypic and chemotaxonomic characteristics, 16S rDNA sequence and carbohydrate fermentation tests, the isolates were identified as 17 species namely: Lactobacillus coryniformis subsp. torquens (0.97%), Lactobacillus pontis (0.97%), Lactobacillus hilgardii (0.97%), Lactobacillus pantheris (0.97%), Lactobacillus amylovorus (1.9%), Lactobacillus panis (1.9%), Lactobacillus vaginalis (1.9%), Lactobacillus rapi (1.9%), Lactobacillus buchneri (2.9%), Lactobacillus parafarraginis (2.9%), Lactobacillus helveticus (3.9%), Lactobacillus camelliae (3.9%), Lactobacillus fermentum (5.8%), Lactobacillus manihotivorans (6.8%), Lactobacillus plantarum (10.7%), Lactobacillus harbinensis (16.5%) and Lactobacillus paracasei subsp. paracasei (35.0%). This study has shown that TP can be well preserved for 90 days by ensilaging and that TPS is not only rich in essential nutrients, but that physiological and biochemical properties of the isolates could provide a platform for future design of lactic acid bacteria (LAB) inoculants aimed at improving the fermentation quality of silage. PMID:25049999

  4. Modification of extraction method for community DNA isolation from salt affected compact wasteland soil samples.

    PubMed

    Zaveri, Purvi; Patel, Rushika; Patel, Meghavi; Sarodia, Devki; Munshi, Nasreen S

    2017-01-01

    To overcome the issue of interferences by salt and compactness in release of bacterial cell required for lysis, method described by Yeates et al. (1998), was optimized for isolation of genomic material (Deoxyribo Nucleic Acid, DNA) from soil microbial community by addition of Al(NH4)SO4. Very low total viable count was observed in the samples tested and hence use of higher amount of soil is required primarily for DNA isolation from wasteland soils. The method proves itself efficient where commercially available bead beating and enzymatic lysis methods could not give isolation of any amount of community genomic DNA due to compact nature and salt concentrations present in soil. •The protocol was found efficient for soil samples with high clay content for microbial community DNA extraction.•Variation in lysis incubation and amount of soil may help with soil samples containing low microbial population.•Addition of Al(NH4)SO4 is crucial step in humic acid removal from extracted DNA samples for soil samples containing high salinity and clay particles.

  5. Amino Acid Racemization and the Preservation of Ancient DNA

    NASA Technical Reports Server (NTRS)

    Poinar, Hendrik N.; Hoss, Matthias

    1996-01-01

    The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber.

  6. Isolation of PCR quality microbial community DNA from heavily contaminated environments.

    PubMed

    Gunawardana, Manjula; Chang, Simon; Jimenez, Abraham; Holland-Moritz, Daniel; Holland-Moritz, Hannah; La Val, Taylor P; Lund, Craig; Mullen, Madeline; Olsen, John; Sztain, Terra A; Yoo, Jennifer; Moss, John A; Baum, Marc M

    2014-07-01

    Asphalts, biochemically degraded oil, contain persistent, water-soluble compounds that pose a significant challenge to the isolation of PCR quality DNA. The adaptation of existing DNA purification protocols and commercial kits proved unsuccessful at overcoming this hurdle. Treatment of aqueous asphalt extracts with a polyamide resin afforded genomic microbial DNA templates that could readily be amplified by PCR. Physicochemically distinct asphalt samples from five natural oil seeps successfully generated the expected 291 bp amplicons targeting a region of the 16S rRNA gene, illustrating the robustness of the method. DNA recovery yields were in the 50-80% range depending on how the asphalt sample was seeded with exogenous DNA. The scope of the new method was expanded to include soil with high humic acid content. DNA from soil samples spiked with a range of humic acid concentrations was extracted with a commercial kit followed by treatment with the polyamide resin. The additional step significantly improved the purity of the DNA templates, especially at high humic acid concentrations, based on qPCR analysis of the bacterial 16S rRNA genes. The new method has the advantages of being inexpensive, simple, and rapid and should provide a valuable addition to protocols in the field of petroleum and soil microbiology.

  7. Systematic biases in DNA copy number originate from isolation procedures

    PubMed Central

    2013-01-01

    Background The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal. Results While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing. Conclusion These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses. PMID:23618369

  8. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing

    PubMed Central

    Pérez-Barrios, Clara; Nieto-Alcolado, Irene; Torrente, María; Jiménez-Sánchez, Carolina; Calvo, Virginia; Gutierrez-Sanz, Lourdes; Palka, Magda; Donoso-Navarro, Encarnación; Provencio, Mariano

    2016-01-01

    Background The implementation of liquid biopsy for biomarker testing and response to treatment monitoring in cancer patients would presumable increase laboratory throughput, requiring the development of automated methods for circulating free DNA (cfDNA) isolation. Methods The present study compares the MagNA Pure Compact (MPC) Nucleic Acid Isolation Kit I and Maxwell® RSC (MR) ccfDNA Plasma Kit and the later with QIAamp Circulating Nucleid Acid (QCNA) Kit using 57 plasma samples from cancer patients. cfDNA concentration was measured using the Qubit fluorometer. DNA fragments lengt were assessed using the Agilent 2100 Bioanalyzer. Circulating tumor DNA (ctDNA) was quantified by digital PCR (dPCR). Results Firstly, we observed that MPC method significantly extracted less cfDNA than MR (P<0.0001). However, there were no significant differences in extraction yields of QCNA and MR kits. cfDNA isolation yield was also associated with tumor stage but not with tumor location. Secondly, an oligonucleosomal DNA ladder pattern was observed in 88% of the samples and significant differences in the recovery of mono-, di- and tri-nucleosomes DNA fragments were observed between MPC and MR methodologies. Finally, tumor mutation quantification on cfDNA was performed on 38 paired samples using digital PCR. Mutant allele fractions (MAFs) between paired samples were not significantly different. Conclusions Methods for isolation of cfDNA can affect DNA yield and molecular weight fractions recovery. These observations should be taken into account for cfDNA analysis in routine clinical practice. PMID:28149760

  9. Biotypes and randomly amplified polymorphic DNA (RAPD) profiles of subgingival Candida albicans isolates in HIV infection.

    PubMed

    Pizzo, Giuseppe; Giammanco, Giovanni M; Pecorella, Sonia; Campisi, Giuseppina; Mammina, Caterina; D'Angelo, Matteo

    2005-01-01

    A group of subgingival isolates of C. albicans recovered from Italian HIV-positive (HIV+) subjects were characterized both phenotypically and genotypically. Phenotyping of the isolates was carried out by a biotyping method based on the enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts. Genotyping was performed through randomly amplified polymorphic DNA (RAPD) analysis. Five biotypes were found among the 29 subgingival C. albicans strains examined. The predominant biotypes were A1R (55.17%), A1S (24.14%), and A2R (13.79%), while the biotypes A11R and A13R were represented by a single isolate each. RAPD profiles identified 15 genotypes among the 29 isolates. Almost every individual harboured his/her own specific isolate and in three out of the six subjects with multiple isolates (two to six each) more than one genotype (two to six) was found. The biotype distribution we found is consistent with previous reports on C. albicans isolates from other oral sources, whereas the resistance to boric acid was highly frequent in subgingival strains. RAPD analysis showed high genetic heterogeneity within subgingival isolates, also when isolates were phenotypically identical. These findings, obtained from HIV+ subjects living in Southern Italy, may be useful as baseline information on subgingival C. albicans colonization in the Mediterranean area.

  10. Magnetophoretic-based microfluidic device for DNA isolation.

    PubMed

    Hale, C; Darabi, J

    2014-07-01

    This paper presents a continuous flow microfluidic device for the separation of DNA from blood using magnetophoresis for biological applications and analysis. This microfluidic bio-separation device has several benefits, including decreased sample handling, smaller sample and reagent volumes, faster isolation time, and decreased cost to perform DNA isolation. One of the key features of this device is the use of short-range magnetic field gradients, generated by a micro-patterned nickel array on the bottom surface of the separation channel. In addition, the device utilizes an array of oppositely oriented, external permanent magnets to produce strong long-range field gradients at the interfaces between magnets, further increasing the effectiveness of the device. A comprehensive simulation is performed using COMSOL Multiphysics to study the effect of various parameters on the magnetic flux within the separation channel. Additionally, a microfluidic device is designed, fabricated, and tested to isolate DNA from blood. The results show that the device has the capability of separating DNA from a blood sample with a purity of 1.8 or higher, a yield of up to 33 μg of polymerase chain reaction ready DNA per milliliter of blood, and a volumetric throughput of up to 50 ml/h.

  11. Method for nucleic acid isolation using supercritical fluids

    SciTech Connect

    Nivens, David E.; Applegate, Bruce M.

    1999-01-01

    A method for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification.

  12. Method for nucleic acid isolation using supercritical fluids

    DOEpatents

    Nivens, D.E.; Applegate, B.M.

    1999-07-13

    A method is disclosed for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification. 4 figs.

  13. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    PubMed Central

    2011-01-01

    Background High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants. PMID:21599914

  14. Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

    PubMed Central

    Gianguzza, Fabrizio; Antonietta Ragusa, Maria; Roccheri, Maria Carmela; Liegro, Italia Di; Rinaldi, Anna Maria

    2000-01-01

    Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution. PMID:11147969

  15. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  16. 4-coumarate:coenzyme a ligase from loblolly pine xylem. Isolation, characterization, and complementary DNA cloning.

    PubMed Central

    Voo, K S; Whetten, R W; O'Malley, D M; Sederoff, R R

    1995-01-01

    4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) was purified from differentiating xylem of loblolly pine (Pinus taeda L.). The pine enzyme had an apparent molecular mass of 64 kD and was similar in size and kinetic properties to 4CL isolated from Norway spruce. The pine enzyme used 4-coumaric acid, caffeic acid, ferulic acid, and cinnamic acid as substrates but had no detectable activity using sinapic acid. 4CL was inhibited by naringenin and coniferin, products of phenylpropanoid metabolism. Although the lignin composition in compression wood is higher in p-hydroxyphenyl units than lignin from normal wood, there was no evidence for a different form of 4CL enzyme in differentiating xylem that was forming compression wood. cDNA clones for 4CL were obtained from a xylem expression library. The cDNA sequences matched pine xylem 4CL protein sequences and showed 60 to 66% DNA sequence identity with 4CL sequences from herbaceous angiosperms. There were two classes of cDNA obtained from pine xylem, and the genetic analysis showed that they were products of a single gene. PMID:7784527

  17. Gluconacetobacter kakiaceti sp. nov., an acetic acid bacterium isolated from a traditional Japanese fruit vinegar.

    PubMed

    Iino, Takao; Suzuki, Rei; Tanaka, Naoto; Kosako, Yoshimasa; Ohkuma, Moriya; Komagata, Kazuo; Uchimura, Tai

    2012-07-01

    Two novel acetic acid bacteria, strains G5-1(T) and I5-1, were isolated from traditional kaki vinegar (produced from fruits of kaki, Diospyros kaki Thunb.), collected in Kumamoto Prefecture, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains G5-1(T) and I5-1 formed a distinct subline in the genus Gluconacetobacter and were closely related to Gluconacetobacter swingsii DST GL01(T) (99.3% 16S rRNA gene sequence similarity). The isolates showed 96-100% DNA-DNA relatedness with each other, but <53% DNA-DNA relatedness with closely related members of the genus Gluconacetobacter. The isolates could be distinguished from closely related members of the genus Gluconacetobacter by not producing 2- and 5-ketogluconic acids from glucose, producing cellulose, growing without acetic acid and with 30% (w/v) d-glucose, and producing acid from sugars and alcohols. Furthermore, the genomic DNA G+C contents of strains G5-1(T) and I5-1 were a little higher than those of their closest phylogenetic neighbours. On the basis of the phenotypic characteristics and phylogenetic position, strains G5-1(T) and I5-1 are assigned to a novel species, for which the name Gluconacetobacter kakiaceti sp. nov. is proposed; the type strain is G5-1(T) (=JCM 25156(T)=NRIC 0798(T)=LMG 26206(T)).

  18. Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

    PubMed

    Radpour, Ramin; Sikora, Michal; Grussenmeyer, Thomas; Kohler, Corina; Barekati, Zeinab; Holzgreve, Wolfgang; Lefkovits, Ivan; Zhong, Xiao Yan

    2009-11-01

    Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.

  19. DNA Macroarray for Identification and Typing of Staphylococcus aureus Isolates

    PubMed Central

    Trad, Salim; Allignet, Jeanine; Frangeul, Lionel; Davi, Marilyne; Vergassola, Massimo; Couve, Elisabeth; Morvan, Anne; Kechrid, Amel; Buchrieser, Carmen; Glaser, Philippe; El Solh, Névine

    2004-01-01

    A DNA macroarray containing 465 intragenic amplicons was designed to identify Staphylococcus aureus at the species level and to type S. aureus isolates. The genes selected included those encoding (i) S. aureus-specific proteins, (ii) staphylococcal and enterococcal proteins mediating antibiotic resistance and factors involved in their expression, (iii) putative virulence proteins and factors controlling their expression, and (iv) proteins produced by mobile elements. The macroarray was hybridized with the cellular DNAs of 80 S. aureus clinical isolates that were previously typed by analyses of their antibiograms and SmaI patterns. The set selected contained unrelated, endemic, and outbreak-related isolates belonging to 45 SmaI genotypes. In a gene content dendrogram, the 80 isolates were distributed into 52 clusters. The outbreak-related isolates were linked in the same or a closely related cluster(s). Clustering based on gene content provided a better discrimination than SmaI pattern analysis for the tested mecA+ isolates that were endemic to Europe. All of the antibiotic resistance genes detected could be correlated with their corresponding phenotypes, except for one isolate which carried a mecA gene without being resistant. The 16 isolates responsible for bone infections were distinguishable from the 12 isolates from uninfected nasal carriers by a significantly higher prevalence of the sdrD gene coding for a putative SD (serine-aspartate) adhesin (in 15 and 7 isolates, respectively). In conclusion, the macroarray designed for this study offers an attractive and rapid typing method which has the advantage of providing additional information concerning the gene content of the isolate of interest. PMID:15131170

  20. A simple and efficient DNA isolation method for Salvia officinalis.

    PubMed

    Aleksić, Jelena M; Stojanović, Danilo; Banović, Bojana; Jančić, Radiša

    2012-12-01

    We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4 M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6 μg DNA g(-1) of dry leaf tissue, and the absorbance ratios 260/280 and 260/230 nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.

  1. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  2. Triterpenes acids and saponins isolated from Licania arianeae Prance (Chrysobalanaceae).

    PubMed

    de Carvalho, Mário Geraldo; Cândido, Lucilene Faustina de Oliveira; da Costa, Patrícia Miranda; do Nascimento, Ildomar Alves; Braz-Filho, Raimundo

    2008-07-01

    From the leaves and wood of Licania arianeae, ten known compounds were isolated and identified. They belong to pharmacological active triterpenes acids, including three oleanoic acids, five ursane acids, and two triterpenes saponin acids. Their structures were established by analysis of infrared, mass spectrometry, and nuclear magnetic resonance spectral data of natural triterpenes and of methyl acetyl derivatives.

  3. Isolation and characterization of pyrimidine-psoralen photoadducts from DNA

    SciTech Connect

    Straub, K.; Kanne, D.; Hearst, J.E.; Rapoport, H.

    1981-05-06

    We have examined the photoadducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and native DNA. Five nucleoside-HMT monoaddition products have been isolated and characterized, corresponding to three deoxythymidine-HMT and two deoxyuridine (derived from deoxycytidine)-HMT adducts. Structural assignments are based on high resolution mass spectrometry and /sup 1/H NMR studies, including homonuclear spin decoupling and nuclear Overhauser effect (NOE) experiments. The results of this study indicate that (1) a limited number of nucleoside-psoralen adducts are formed with native, double-stranded DNA, and (2) the sterochemistry of the adducts is apparently determined by the geometry of the noncovalent intercalative complex formed by HMT and DNA prior to irradiation. 8 figures, 8 tables.

  4. Isolation and characterization of pyrimidine-psoralen photoadducts from DNA

    SciTech Connect

    Straub, Kenneth; Kanne, David; Hearst, John E.; Rapoport, Henry

    1981-05-01

    In this study, we have examined the photoadducts of 4'-hydroxymethyl-4,5',8-trirnethylpsoralen (HMT) and native DNA. Five nucleoside-HMT monoaddition products have been isolated and characterized, corresponding to three deoxythymidine-HMT and two deoxyuridine (derived from deoxycytidine)-HMT adducts. Structural assignments are based on high resolution mass spectrometry and 1H NMR studies, including homonuclear spin decoupling and nuclear Overhauser effect (NOE) experiments. Our results indicate that (1) a limited number of nucleoside-psoralen adducts are formed with native, double-stranded DNA, and (2) the stereochemistry of the adducts is apparently determined by the geometry of the non-covalent intercalative complex formed by HMT and DNA prior to irradiation.

  5. Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

    PubMed Central

    Knutzon, Deborah S.; Bleibaum, Janice L.; Nelsen, Janet; Kridl, Jean C.; Thompson, Gregory A.

    1992-01-01

    Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34- and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50- to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16- and 18-carbon acyl-ACP substrates. Images Figure 1 Figure 4 PMID:16653193

  6. Isolation of fetal DNA from nucleated erythrocytes in maternal blood

    SciTech Connect

    Bianchi, D.W.; Knoll, J.H.M. Harvard Medical School, Boston, MA ); Flint, A.F. ); Pizzimenti, M.F. ); Latt, S.A. Harvard Medical School, Boston, MA )

    1990-05-01

    Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. The authors used monoclonal antibody against the transferrin receptor (TIR) to identify nucleated erythrocytes in the peripheral blood of pregnant women. Candidate fetal cells from 19 pregnancies were isolated by flow sorting at 12 1/2-17 weeks gestation. The DNA in these cells was amplified for a 222-base-pair (bp) sequence present on the short arm of the Y chromosome as proof that the cells were derived from the fetus. The amplified DNA was compared with standardized DNA concentrations. In the case of the female fetus, DNA prepared from samples at 32 weeks of gestation and cord blood at delivery also showed the presence of the Y chromosomal sequence, suggesting Y sequence mosaicism or translocation. In 10/12 cases where the 222-bp band was absent, the fetuses were female. Thus, they were successful in detecting the Y chromosomal sequence in 75% of the male-bearing pregnancies, demonstrating that it is possible to isolate fetal gene sequences from cells in maternal blood. Further refinement in methodology should increase sensitivity and facilitate noninvasive screening for fetal gene mutations.

  7. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu

    PubMed Central

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group. PMID:27335670

  8. Direct Isolation of Purines and Pyrimidines from Nucleic Acids Using Sublimation

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Schubert, Michael; Bada, Jeffrey L.

    2003-01-01

    A sublimation technique was developed to isolate purines and pyrimidines directly from lambda-deoxyribonucleic acid (lambda-DNA) and Escherichia coli cells. The sublimation of adenine, cytosine, guanine, and thymine from lambda-DNA was tested under reduced pressure (approx. 0.5 Torr) at temperatures of >150 C. With the exception of guanine, approximately 60 -75% of each base was sublimed directly from the lambda-DNA and recovered on a coldfinger of the sublimation apparatus after heating to 450 C. Several nucleobases including adenine, cytosine, thymine, and uracil were also recovered from E. coli bacteria after heating the cells to the same temperature, although some thermal decomposition of the bases also occurred. These results demonstrate the feasibility of using sublimation to isolate purines and pyrimidines from native E. coli DNA and RNA without any chemical treatment of the cells.

  9. Isolation and characterization of DNA probes for human chromosome 21.

    PubMed

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  10. Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.).

    PubMed

    Akkurt, M

    2012-08-13

    Various protocols have been developed and used for DNA extraction in grapevine. However, owing to the long duration of the isolation steps in previously developed protocols, researchers have preferred to use isolation kits for studies in recent years. In our study, the DNA yield and purity obtained using six methods--namely three DNA isolation protocols and three commercial DNA isolation kits--were compared. Modifications were made and the isolation steps were shortened in the previously developed DNA isolation protocols to achieve more rapid and practical protocols. The samples were taken from plants grown under vineyard and greenhouse conditions in two periods during spring and autumn. The best results among the six DNA isolation methods were discussed. The results were also supported with polymerase chain reaction analyses conducted with isolated DNAs.

  11. Assessment of environmental DNA for detecting presence of imperiled aquatic amphibian species in isolated wetlands

    USGS Publications Warehouse

    Mckee, Anna; Calhoun, Daniel L.; Barichivich, William J.; Spear, Stephen F.; Goldberg, Caren S.; Glenn, Travis C

    2015-01-01

    Environmental DNA (eDNA) is an emerging tool that allows low-impact sampling for aquatic species by isolating DNA from water samples and screening for DNA sequences specific to species of interest. However, researchers have not tested this method in naturally acidic wetlands that provide breeding habitat for a number of imperiled species, including the frosted salamander (Ambystoma cingulatum), reticulated flatwoods salamanders (Ambystoma bishopi), striped newt (Notophthalmus perstriatus), and gopher frog (Lithobates capito). Our objectives for this study were to develop and optimize eDNA survey protocols and assays to complement and enhance capture-based survey methods for these amphibian species. We collected three or more water samples, dipnetted or trapped larval and adult amphibians, and conducted visual encounter surveys for egg masses for target species at 40 sites on 12 different longleaf pine (Pinus palustris) tracts. We used quantitative PCRs to screen eDNA from each site for target species presence. We detected flatwoods salamanders at three sites with eDNA but did not detect them during physical surveys. Based on the sample location we assumed these eDNA detections to indicate the presence of frosted flatwoods salamanders. We did not detect reticulated flatwoods salamanders. We detected striped newts with physical and eDNA surveys at two wetlands. We detected gopher frogs at 12 sites total, three with eDNA alone, two with physical surveys alone, and seven with physical and eDNA surveys. We detected our target species with eDNA at 9 of 11 sites where they were present as indicated from traditional surveys and at six sites where they were not detected with traditional surveys. It was, however, critical to use at least three water samples per site for eDNA. Our results demonstrate eDNA surveys can be a useful complement to traditional survey methods for detecting imperiled pond-breeding amphibians. Environmental DNA may be particularly useful in situations

  12. Nucleic Acid Isolation and Enrichment on a Microchip.

    PubMed

    Kim, Jinho; Hilton, John P; Yang, Kyung A; Pei, Renjun; Stojanovic, Milan; Lin, Qiao

    2013-06-01

    This paper presents a microchip that isolates and enriches target-binding single-stranded DNA (ssDNA) from a randomized DNA mixture using a combination of solid-phase extraction and electrophoresis. Strands of ssDNA in a randomized mixture are captured via specific binding onto target-functionalized microbeads in a microchamber. The strands are further separated from impurities and enriched on-chip via electrophoresis. The microchip consists of two microchambers that are connected by a channel filled with agarose gel. In the isolation chamber, beads functionalized with human immunoglobulin E (IgE) are retained by a weir structure. An integrated heater elevates the temperature in the chamber to elute desired ssDNA from the beads, and electrophoretic transport of the DNA through the gel to the second chamber is accomplished by applying an electric potential difference between the two chambers. Experimental results show that ssDNA expressing binding affinity to IgE was captured and enriched from a sample of ssDNA with random sequences, demonstrating the potential of the microchip to enhance the sensitivity of ssDNA detection methods in dilute and complex biological samples.

  13. Scalable Isolation of Mammalian Mitochondria for Nucleic Acid and Nucleoid Analysis.

    PubMed

    Lee, Ken-Wing; Bogenhagen, Daniel F

    2016-01-01

    Isolation of mitochondria from cultured cells and animal tissues for analysis of nucleic acids and bona fide mitochondrial nucleic acid binding proteins and enzymes is complicated by contamination with cellular nucleic acids and their adherent proteins. Protocols presented here allow for quick isolation of mitochondria from a small number of cells and for preparation of highly purified mitochondria from a larger number of cells using nuclease treatment and high salt washing of mitochondria to reduce contamination. We further describe a method for the isolation of mitochondrial DNA-protein complexes known as nucleoids from these highly purified mitochondria using a combination of glycerol gradient sedimentation followed by isopycnic centrifugation in a non-ionic iodixanol gradient.

  14. [Efficiency of three commercial kits dedicated to DNA and RNA isolation from various clinical and forensic materials using the Janus automated workstation].

    PubMed

    Małodobra, Małgorzata; Jonkisz, Anna; Kowalczyk, Elzbieta; Lebioda, Arleta; Bartnik, Beata; Swiatek, Barbara

    2011-01-01

    Isolation of genetic material is a crucial stage in molecular biology. Increasing needs for DNA analysis cause continuous improving of genetic material isolation methods toward higher accuracy and output. Automatization in molecular biology is widely seen, especially in clinical and forensic medicine. The objective of this research was optimization of methods for automatic nucleic acid isolation using the Janus automated workstation, Perkin Elmer. The efficiency and purity of isolated DNA was satisfactory. Despite numerous attempts at achieving automatic RNA isolation, we did not succeed in obtaining RNA working in other applications, such as RT-PCR or Real-Time PCR.

  15. Isolation and Characterization of the DNA and Protein Binding Activities of Adenovirus Core Protein V

    PubMed Central

    Pérez-Vargas, Jimena; Vaughan, Robert C.; Houser, Carolyn; Hastie, Kathryn M.; Kao, C. Cheng

    2014-01-01

    ABSTRACT The structure of adenovirus outer capsid was revealed recently at 3- to 4-Å resolution (V. Reddy, S. Natchiar, P. Stewart, and G. Nemerow, Science 329:1071–1075, 2010, http://dx.doi.org/10.1126/science.1187292); however, precise details on the function and biochemical and structural features for the inner core still are lacking. Protein V is one the most important components of the adenovirus core, as it links the outer capsid via association with protein VI with the inner DNA core. Protein V is a highly basic protein that strongly binds to DNA in a nonspecific manner. We report the expression of a soluble protein V that exists in monomer-dimer equilibrium. Using reversible cross-linking affinity purification in combination with mass spectrometry, we found that protein V contains multiple DNA binding sites. The binding sites from protein V mediate heat-stable nucleic acid associations, with some of the binding sites possibly masked in the virus by other core proteins. We also demonstrate direct interaction between soluble proteins V and VI, thereby revealing the bridging of the inner DNA core with the outer capsid proteins. These findings are consistent with a model of nucleosome-like structures proposed for the adenovirus core and encapsidated DNA. They also suggest an additional role for protein V in linking the inner nucleic acid core with protein VI on the inner capsid shell. IMPORTANCE Scant knowledge exists of how the inner core of adenovirus containing its double-stranded DNA (dsDNA) genome and associated proteins is organized. Here, we report a purification scheme for a recombinant form of protein V that allowed analysis of its interactions with the nucleic acid core region. We demonstrate that protein V exhibits stable associations with dsDNA due to the presence of multiple nucleic acid binding sites identified both in the isolated recombinant protein and in virus particles. As protein V also binds to the membrane lytic protein VI molecules

  16. Characterization of IS6110 insertions in the dnaA-dnaN intergenic region of Mycobacterium tuberculosis clinical isolates.

    PubMed

    Turcios, L; Casart, Y; Florez, I; de Waard, J; Salazar, L

    2009-02-01

    Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to be clonally related. The presence of IS6110 in the dnaA-dnaN intergenic region, one preferential locus for the integration of IS6110, was evaluated in 125 M. tuberculosis isolates. Five isolates had IS6110 inserted in this region, and two consisted of a mix of isogenic strains that putatively have evolved during a single infection. Strains from the same isolate had identical spoligo and mycobacterial interspersed repetitive unit-variable-number tandem repeat profiles, but had slight variations in IS6110 RFLP patterns, due to the presence of IS6110 in the dnaA-dnaN intergenic region. Duplication of the dnaA-dnaN intergenic region was found in one isogenic strain.

  17. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

    PubMed Central

    Law, S W; Grant, S M; Higuchi, K; Hospattankar, A; Lackner, K; Lee, N; Brewer, H B

    1986-01-01

    Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias. PMID:3464946

  18. Toxicity of ethacrynic acid in isolated rat hepatocytes.

    PubMed

    Yamamoto, K; Masubuchi, Y; Narimatsu, S; Kobayashi, S; Horie, T

    2002-04-01

    Ethacrynic acid, a loop diuretic drug, caused lipid peroxidation in isolated rat hepatocytes. The thiobarbituric acid reactive substances (TBARS) formation showed a good correlation with the leakage of glutamic-oxaloacetic acid transaminase (GOT) from the hepatocytes. The addition of antioxidants such as N, N'-diphenyl-p-phenylenediamine (DPPD) and promethazine to the isolated rat hepatocyte suspension containing ethacrynic acid prevented the lipid peroxidation and decreased the GOT leakage to some extent. SKF-525A inhibited the oxidative metabolism of ethacrynic acid and decreased the TBARS formation, suggesting that the lipid peroxidation was caused by the oxidative metabolism. The intracellular reduced glutathione markedly decreased in the hepatocyte suspension containing ethacrynic acid and the hepatocellular protein sulfhydryls were decreased, which was negatively correlated with the GOT leakage. Thus the ethacrynic acid-induced hepatotoxicity was found to be related to the lipid peroxidation and the decrease of cellular protein sulfhydryls.

  19. Isolation and analysis of a cDNA clone encoding an S. guttatum alternataive oxidase protein

    SciTech Connect

    Rhoads, D.M.; McIntosh, L. Michigan State Univ., East Lansing )

    1990-05-01

    Antibodies that recognize the 35, 36, and 37 kilodalton (kDa) alternative oxidase proteins were used to isolate a cDNA proteins were used to isolate a cDNA clone of a nuclearly encoded protein of Sauromatum guttatum. The amino acid sequence deduced from clone pAOSG81 revealed a protein with a predicted molecular mass of 44 kDa, while a 42 kDa protein is immunoprecipitated from in vitro translation products made using S. guttatum poly A+ RNA. The protein contains a 60-65 amino acid transit peptide which is predicted to form amphiphilic helices. We have also identified regions of the mature 42 kDa protein which are likely to be membrane associated. Clone pAOSG81 is being used to screen a genomic library. The genomic clone encoding the 42 kDa protein will be used to investigate the salicylic-acid-controlled transcriptional regulation of the S. guttatum alternative oxidase proteins.

  20. Isolation of cDNA and genomic DNA clones encoding type II collagen.

    PubMed Central

    Young, M F; Vogeli, G; Nunez, A M; Fernandez, M P; Sullivan, M; Sobel, M E

    1984-01-01

    A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs. Images PMID:6203098

  1. Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism.

    PubMed Central

    Krokan, H; Wist, E; Krokan, R H

    1981-01-01

    Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation. PMID:6795595

  2. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    PubMed

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  3. Evolution of the major histocompatibility complex: isolation of class II A cDNA clones from the cartilaginous fish.

    PubMed Central

    Kasahara, M; Vazquez, M; Sato, K; McKinney, E C; Flajnik, M F

    1992-01-01

    Along with the T-cell receptor and immunoglobulin, the major histocompatibility complex (MHC) plays a key role in mounting immune responses to foreign antigen. To gain insights into the evolution of the MHC, class II A cDNA clones were isolated from nurse sharks, a member of the class of cartilaginous fish. Two closely related cDNA clones, which might encode allelic products, were identified; of the three amino acid substitutions found in the alpha 1 domain, two were located at positions postulated to interact with processed peptides. The deduced nurse shark MHC class II alpha chains showed conspicuous structural similarity to their mammalian counterparts. Isolation of cDNA clones encoding typical MHC class II alpha chains was unexpected since no direct evidence for T-cell-mediated immune responses has been obtained in the cartilaginous fish. The cartilaginous fish is phylogenetically the most primitive class of vertebrates from which any MHC gene has been isolated. PMID:1495958

  4. Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays.

    PubMed

    El-Adawy, Hosny; Ahmed, Marwa; Hotzel, Helmut; Monecke, Stefan; Schulz, Jochen; Hartung, Joerg; Ehricht, Ralf; Neubauer, Heinrich; Hafez, Hafez M

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding

  5. Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays

    PubMed Central

    El-Adawy, Hosny; Ahmed, Marwa; Hotzel, Helmut; Monecke, Stefan; Schulz, Jochen; Hartung, Joerg; Ehricht, Ralf; Neubauer, Heinrich; Hafez, Hafez M.

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding

  6. Isolation and characterization of monochloroacetic acid-degrading bacteria.

    PubMed

    Horisaki, Tadafumi; Yoshida, Eiko; Sumiya, Kaori; Takemura, Tetsuo; Yamane, Hisakazu; Nojiri, Hideaki

    2011-01-01

    Five Burkholderia strains (CL-1, CL-2, CL-3, CL-4, and CL-5) capable of degrading monochloroacetic acid (MCA) were isolated from activated sludge or soil samples gathered from several parts of Japan. All five isolates were able to grow on MCA as the sole source of carbon and energy, and argentometry and gas chromatography-mass spectroscopy analyses showed that these five strains consumed MCA completely and released chloride ions stoichiometrically within 25 h. The five isolates also grew on monobromoacetic acid, monoiodoacetic acid, and L-2-monochloropropionic acid as sole sources of carbon and energy. In addition, the five isolates could not grow with DCA but dehalogenate single chlorine from DCA. Because PCR analyses revealed that all five isolates have an identical group II dehalogenase gene fragment and no group I deh gene, only strain CL-1 was analyzed further. The partial amino acid sequence of the group II dehalogenase of strain CL-1, named DehCL1, showed 74.6% and 65.2% identities to corresponding regions of the two MCA dehalogenases, DehCI from Pseudomonas sp. strain CBS-3 and Hdl IVa from Burkholderia cepacia strain MBA4, respectively. The secondary-structure motifs of the haloacid dehalogenase (HAD) superfamily and the amino acid residues involved in substrate binding, catalysis, and hydrophobic pocket formation were conserved in the partial amino acid sequence of DehCL1.

  7. Isolation and characterisation of lactic acid bacteria from donkey milk.

    PubMed

    Soto Del Rio, Maria de Los Dolores; Andrighetto, Christian; Dalmasso, Alessandra; Lombardi, Angiolella; Civera, Tiziana; Bottero, Maria Teresa

    2016-08-01

    During the last years the interest in donkey milk has increased significantly mainly because of its compelling functional elements. Even if the composition and nutritional properties of donkey milk are known, its microbiota is less studied. This Research Communication aimed to provide a comprehensive characterisation of the lactic acid bacteria in raw donkey milk. RAPD-PCR assay combined with 16S rDNA sequencing analysis were used to describe the microbial diversity of several donkey farms in the North West part of Italy. The more frequently detected species were: Lactobacillus paracasei, Lactococcus lactis and Carnobacterium maltaromaticum. Less abundant genera were Leuconostoc, Enterococcus and Streptococcus. The yeast Kluyveromyces marxianus was also isolated. The bacterial and biotype distribution notably diverged among the farms. Several of the found species, not previously detected in donkey milk, could have an important probiotic activity and biotechnological potential. This study represents an important insight to the ample diversity of the microorganisms present in the highly selective ecosystem of raw donkey milk.

  8. Incorporation and/or adduction of formic acid with DNA in vivo studied by HPLC-AMS

    NASA Astrophysics Data System (ADS)

    Zhu, Jiadan; Cheng, Yan; Sun, Hongfang; Wang, Haifang; Li, Yuankai; Liu, Yuanfang; Ding, Xingfang; Fu, Dongpo; Liu, Kexin; Wang, Deqing; Deng, Xiaoyong

    2010-04-01

    The contribution of incorporation and/or adduction of formic acid with liver DNA in mouse was investigated using accelerator mass spectrometry (AMS) associated with high performance liquid chromatography (HPLC). Four kinds of 5'-formylated adducts, which were prepared by the reaction of formic acid and deoxyribonucleosides in vitro, were used as references for the HPLC-AMS analysis of in vivo adduction. After the administration of sodium 14C-formate to mice, the liver DNA pellets were isolated and enzymatically digested to deoxyribonucleosides. A precise analysis of the hydrolysate by HPLC-AMS indicates that a majority of formic acid incorporates directly into DNA, whereas less than 1.5% might form instable formylated DNA adducts in vivo. The results greatly support the important perspective that formic acid is not carcinogenic. Moreover, this study demonstrates that a combination of HPLC with AMS is an essential means for the evaluation of DNA adduction.

  9. Improved DNA hybridization parameters by Twisted Intercalating Nucleic Acid (TINA).

    PubMed

    Schneider, Uffe Vest

    2012-01-01

    This thesis establishes oligonucleotide design rules and applications of a novel group of DNA stabilizing molecules collectively called Twisted Intercalating Nucleic Acid - TINA. Three peer-reviewed publications form the basis for the thesis. One publication describes an improved and rapid method for determination of DNA melting points and two publications describe the effects of positioning TINA molecules in parallel triplex helix and antiparallel duplex helix forming DNA structures. The third publication establishes that TINA molecules containing oligonucleotides improve an antiparallel duplex hybridization based capture assay's analytical sensitivity compared to conventionel DNA oligonucleotides. Clinical microbiology is traditionally based on pathogenic microorganisms' culture and serological tests. The introduction of DNA target amplification methods like PCR has improved the analytical sensitivity and total turn around time involved in clinical diagnostics of infections. Due to the relatively weak hybridization between the two strands of double stranded DNA, a number of nucleic acid stabilizing molecules have been developed to improve the sensitivity of DNA based diagnostics through superior binding properties. A short introduction is given to Watson-Crick and Hoogsteen based DNA binding and the derived DNA structures. A number of other nucleic acid stabilizing molecules are described. The stabilizing effect of TINA molecules on different DNA structures is discussed and considered in relation to other nucleic acid stabilizing molecules and in relation to future use of TINA containing oligonucleotides in clinical diagnostics and therapy. In conclusion, design of TINA modified oligonucleotides for antiparallel duplex helixes and parallel triplex helixes follows simple purpose dependent rules. TINA molecules are well suited for improving multiplex PCR assays and can be used as part of novel technologies. Future research should test whether combinations of TINA

  10. Diffusion of isolated DNA molecules: dependence on length and topology.

    PubMed

    Robertson, Rae M; Laib, Stephan; Smith, Douglas E

    2006-05-09

    The conformation and dynamics of circular polymers is a subject of considerable theoretical and experimental interest. DNA is an important example because it occurs naturally in different topological states, including linear, relaxed circular, and supercoiled circular forms. A fundamental question is how the diffusion coefficients of isolated polymers scale with molecular length and how they vary for different topologies. Here, diffusion coefficients D for relaxed circular, supercoiled, and linear DNA molecules of length L ranging from approximately 6 to 290 kbp were measured by tracking the Brownian motion of single molecules. A topology-independent scaling law D approximately L(-nu) was observed with nu(L) = 0.571 +/- 0.014, nu(C) = 0.589 +/- 0.018, and nu(S) = 0.571 +/- 0.057 for linear, relaxed circular, and supercoiled DNA, respectively, in good agreement with the scaling exponent of nu congruent with 0.588 predicted by renormalization group theory for polymers with significant excluded volume interactions. Our findings thus provide evidence in support of several theories that predict an effective diameter of DNA much greater than the Debye screening length. In addition, the measured ratio D(Circular)/D(Linear) = 1.32 +/- 0.014 was closer to the value of 1.45 predicted by using renormalization group theory than the value of 1.18 predicted by classical Kirkwood hydrodynamic theory and agreed well with a value of 1.31 predicted when incorporating a recently proposed expression for the radius of gyration of circular polymers into the Zimm model.

  11. Identification of dairy lactic acid bacteria by tRNAAla-23S rDNA-RFLP.

    PubMed

    Mancini, Andrea; Lazzi, Camilla; Bernini, Valentina; Neviani, Erasmo; Gatti, Monica

    2012-12-01

    The aim of this study was to evaluate the potential of target tRNA(Ala)-23S ribosomal DNA for identification of lactic acid bacteria strains associated with dairy ecosystem. For this purpose tRNA(Ala)-23S ribosomal DNA Restriction Fragment Length Polymorphism (tRNA(Ala)-23S rDNA-RFLP) was compared with two widely used DNA fingerprinting methods - P1 Random Amplified Polymorphic DNA (RAPD), (GTG)5 repetitive extragenic palindromic PCR (rep-PCR) - for their ability to identify different species on a set of 10 type and 34 reference strains. Moreover, 75 unknown isolates collected during different stages of Grana Padano cheese production and ripening were identified using tRNA(Ala)-23S rDNA-RFLP and compared to the RFLP profiles of the strains in the reference database. This study demonstrated that the target tRNA(Ala)-23S rDNA has high potential in bacterial identification and tRNA(Ala)-23S rDNA-RFLP is a promising method for reliable species-level identification of lactic acid bacteria (LAB) in dairy products.

  12. Antifeedant activity of anticopalic acid isolated from Vitex hemsleyi.

    PubMed

    Villegas Gómez, Clarisa; Martínez-Vázquez, Mariano; Esquivel, Baldomero

    2009-01-01

    The known labdane-type diterpenoids anticopalic acid (1) and 3 beta-hydroxyanticopalic acid (2) were isolated from extracts of the aerial parts of Vitex hemsleyi Briq. (Labiatae). The acid 1 showed an antifeedant, dose-dependent activity against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae). To our knowledge this is the first report on the antifeedant activity of a labdane-type diterpene against S. frugiperda.

  13. Nucleic Acid Engineering: RNA Following the Trail of DNA.

    PubMed

    Kim, Hyejin; Park, Yongkuk; Kim, Jieun; Jeong, Jaepil; Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2016-02-08

    The self-assembly feature of the naturally occurring biopolymer, DNA, has fascinated researchers in the fields of materials science and bioengineering. With the improved understanding of the chemical and structural nature of DNA, DNA-based constructs have been designed and fabricated from two-dimensional arbitrary shapes to reconfigurable three-dimensional nanodevices. Although DNA has been used successfully as a building block in a finely organized and controlled manner, its applications need to be explored. Hence, with the myriad of biological functions, RNA has recently attracted considerable attention to further the application of nucleic acid-based structures. This Review categorizes different approaches of engineering nucleic acid-based structures and introduces the concepts, principles, and applications of each technique, focusing on how DNA engineering is applied as a guide to RNA engineering.

  14. Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans

    PubMed Central

    Dingley, Stephen D.; Polyak, Erzsebet; Ostrovsky, Julian; Srinivasan, Satish; Lee, Icksoo; Rosenfeld, Amy B.; Tsukikawa, Mai; Xiao, Rui; Selak, Mary A.; Coon, Joshua J.; Hebert, Alexander S.; Grimsrud, Paul A.; Kwon, Young Joon; Pagliarini, David J.; Gai, Xiaowu; Schurr, Theodore G.; Hüttemann, Maik; Nakamaru-Ogiso, Eiko; Falk, Marni J.

    2014-01-01

    Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20 °C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25 °C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856 > N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856 > N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear– mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism. PMID:24534730

  15. Isolating and evaluating lactic acid bacteria strains for effectiveness of Leymus chinensis silage fermentation.

    PubMed

    Zhang, Q; Li, X J; Zhao, M M; Yu, Z

    2014-10-01

    Five LAB strains were evaluated using the acid production ability test, morphological observation, Gram staining, physiological, biochemical and acid tolerance tests. All five strains (LP1, LP2, LP3, LC1 and LC2) grew at pH 4·0, and LP1 grew at 15°C. Strains LP1, LP2 and LP3 were identified as Lactobacillus plantarum, whereas LC1 and LC2 were classified as Lactobacillus casei by sequencing 16S rDNA. The five isolated strains and two commercial inoculants (PS and CL) were added to native grass and Leymus chinensis (Trin.) Tzvel. for ensiling. All five isolated strains decreased the pH and ammonia nitrogen content, increased the lactic acid content and LP1, LP2 and LP3 increased the acetic content and lactic/acetic acid ratio of L. chinensis silage significantly. The five isolated strains and two commercial inoculants decreased the butyric acid content of the native grass silage. LP2 treatment had lower butyric acid content and ammonia nitrogen content than the other treatments. The five isolated strains improved the quality of L. chinensis silage. The five isolated strains and the two commercial inoculants were not effective in improving the fermentation quality of the native grass silage, but LP2 performed better comparatively. Significance and impact of the study: Leymus chinensis is an important grass in China and Russia, being the primary grass of the short grassland 'steppe' regions of central Asia. However, it has been difficult to make high-quality silage of this species because of low concentration of water-soluble carbohydrates (WSC). Isolating and evaluating lactic acid bacteria strains will be helpful for improving the silage quality of this extensively grown species.

  16. DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction.

    PubMed

    Focke, Felix; Haase, Ilka; Fischer, Markus

    2011-01-26

    Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.

  17. Isolation and characterization of human defensin cDNA clones

    SciTech Connect

    Daher, K.A.; Lehrer, R.I.; Ganz, T.; Kronenberg, M. )

    1988-10-01

    Four clones that encode defensins, a group of microbicidal and cytotoxic peptides made by neutrophils, were isolated from an HL-60 human promyelocytic leukemia cDNA library. Analysis of these clones indicated that the defensins are made as precursor proteins, which must be cleaved to yield the mature peptides. Defensin mRNA was detected in normal bone marrow cells, but not in normal peripheral blood leukocytes. Defensin transcripts were also found in the peripheral leukocytes of some leukemia patients and in some lung and intestine tissues. Defensin mRNA content was augmented by treatment of HL-60 cells with dimethyl sulfoxide. These results define important aspects of the mechanism of synthesis and the tissue-specific expression of a major group of neutrophil granule proteins.

  18. Isolation of Shikimic Acid from Star Aniseed

    ERIC Educational Resources Information Center

    Payne, Richard; Edmonds, Michael

    2005-01-01

    A new undergraduate laboratory experiment suitable for demonstrating some key techniques used in natural products chemistry is described. A laboratory experiment is developed which in the process of extracting shikimic acid from star aniseed exposes students to a number of important experimental techniques.

  19. DNA isolation method is a source of global DNA methylation variability measured with LUMA. Experimental analysis and a systematic review.

    PubMed

    Soriano-Tárraga, Carolina; Jiménez-Conde, Jordi; Giralt-Steinhauer, Eva; Ois, Angel; Rodríguez-Campello, Ana; Cuadrado-Godia, Elisa; Fernández-Cadenas, Israel; Montaner, Joan; Lucas, Gavin; Elosua, Roberto; Roquer, Jaume

    2013-01-01

    In DNA methylation, methyl groups are covalently bound to CpG dinucleotides. However, the assumption that methyl groups are not lost during routine DNA extraction has not been empirically tested. To avoid nonbiological associations in DNA methylation studies, it is essential to account for potential batch effect bias in the assessment of this epigenetic mechanism. Our purpose was to determine if the DNA isolation method is an independent source of variability in methylation status. We quantified Global DNA Methylation (GDM) by luminometric methylation assay (LUMA), comparing the results from 3 different DNA isolation methods. In the controlled analysis (n = 9), GDM differed slightly for the same individual depending on extraction method. In the population analysis (n = 580) there were significant differences in GDM between the 3 DNA isolation methods (medians, 78.1%, 76.5% and 75.1%; p<0.001). A systematic review of published data from LUMA GDM studies that specify DNA extraction methods is concordant with our findings. DNA isolation method is a source of GDM variability measured with LUMA. To avoid possible bias, the method used should be reported and taken into account in future DNA methylation studies.

  20. A mitomycin-N6-deoxyadenosine adduct isolated from DNA.

    PubMed

    Palom, Y; Lipman, R; Musser, S M; Tomasz, M

    1998-03-01

    A minor N6-deoxyadenosine adduct of mitomycin C (MC) was isolated from synthetic oligonucleotides and calf thymus DNA, representing the first adduct of MC and a DNA base other than guanine. The structure of the adduct (8) was elucidated using submilligram quantities of total available material. UV difference spectroscopy, circular dichroism, and electrospray mass spectroscopy as well as chemical transformations were utilized in deriving the structure of 8. A series of synthetic oligonucleotides was designed to probe the specificities of the alkylation of adenine by MC. The nature and frequency of the oligonucleotide-MC adducts formed under conditions of reductive activation of MC were determined by their enzymatic digestion to the nucleoside level followed by quantitative analysis of the products by HPLC. The analyses indicated the following: (i) (A)n sequence is favored over (AT)n for adduct formation; (ii) the alkylation favors the duplex structure; (iii) at adenine sites only monofunctional alkylation occurs; (iv) the adenine-to-alkylation frequency in the model oligonucleotides was 0.3-0.6 relative to guanine alkylation at the 5'-ApG sequence but only 0.02-0.1 relative to guanine alkylation at 5'-CpG. The 5'-phosphodiester linkage of the MC-adenine adduct is resistant to snake venom diesterase. The overall ratio of adenine to guanine alkylation in calf thymus DNA was 0.03, indicating that 8 is a minor MC-DNA adduct relative to MC-DNA adducts at guanine residues in the present experimental residues in the present experimental system. However, the HPLC elution time of 8 coincides with that of a major, unknown MC adduct detected previously in mouse mammary tumor cells treated with radiolabeled MC [Bizanek, R., Chowdary, D., Arai, H., Kasai, M., Hughes, C. S., Sartorelli, A. C., Rockwell, S., and Tomasz, M. (1993) Cancer Res. 53, 5127-5134]. Thus, 8 may be identical or closely related to this major adduct formed in vivo. This possibility can now be tested by

  1. Bombella intestini gen. nov., sp. nov., an acetic acid bacterium isolated from bumble bee crop.

    PubMed

    Li, Leilei; Praet, Jessy; Borremans, Wim; Nunes, Olga C; Manaia, Célia M; Cleenwerck, Ilse; Meeus, Ivan; Smagghe, Guy; De Vuyst, Luc; Vandamme, Peter

    2015-01-01

    In the frame of a bumble bee gut microbiota study, acetic acid bacteria (AAB) were isolated using a combination of direct isolation methods and enrichment procedures. MALDI-TOF MS profiling of the isolates and a comparison of these profiles with profiles of established AAB species identified most isolates as Asaia astilbis or as 'Commensalibacter intestini', except for two isolates (R-52486 and LMG 28161(T)) that showed an identical profile. A nearly complete 16S rRNA gene sequence of strain LMG 28161(T) was determined and showed the highest pairwise similarity to Saccharibacter floricola S-877(T) (96.5%), which corresponded with genus level divergence in the family Acetobacteraceae. Isolate LMG 28161(T) was subjected to whole-genome shotgun sequencing; a 16S-23S rRNA internal transcribed spacer (ITS) sequence as well as partial sequences of the housekeeping genes dnaK, groEL and rpoB were extracted for phylogenetic analyses. The obtained data confirmed that this isolate is best classified into a new genus in the family Acetobacteraceae. The DNA G+C content of strain LMG 28161(T) was 54.9 mol%. The fatty acid compositions of isolates R-52486 and LMG 28161(T) were similar to those of established AAB species [with C18:1ω7c (43.1%) as the major component], but the amounts of fatty acids such as C19:0 cyclo ω8c, C14:0 and C14:0 2-OH enabled to differentiate them. The major ubiquinone was Q-10. Both isolates could also be differentiated from the known genera of AAB by means of biochemical characteristics, such as their inability to oxidize ethanol to acetic acid, negligible acid production from melibiose, and notable acid production from d-fructose, sucrose and d-mannitol. In addition, they produced 2-keto-d-gluconate, but not 5-keto-d-gluconate from d-glucose. Therefore, the name Bombella intestini gen nov., sp. nov. is proposed for this new taxon, with LMG 28161(T) ( =DSM 28636(T) =R-52487(T)) as the type strain of the type species.

  2. DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters.

    PubMed

    Hadi, Sámed I I A; Santana, Hugo; Brunale, Patrícia P M; Gomes, Taísa G; Oliveira, Márcia D; Matthiensen, Alexandre; Oliveira, Marcos E C; Silva, Flávia C P; Brasil, Bruno S A F

    2016-01-01

    This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences' using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker.

  3. DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

    PubMed Central

    Hadi, Sámed I. I. A.; Santana, Hugo; Brunale, Patrícia P. M.; Gomes, Taísa G.; Oliveira, Márcia D.; Matthiensen, Alexandre; Oliveira, Marcos E. C.; Silva, Flávia C. P.; Brasil, Bruno S. A. F.

    2016-01-01

    This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker. PMID:26900844

  4. Singlet oxygen oxidation of isolated and cellular DNA: product formation and mechanistic insights.

    PubMed

    Cadet, Jean; Ravanat, Jean-Luc; Martinez, Glaucia R; Medeiros, Marisa H G; Di Mascio, Paolo

    2006-01-01

    This survey focuses on recent aspects of the singlet oxygen oxidation of the guanine moiety of nucleosides, oligonucleotides, isolated and cellular DNA that has been shown to be the exclusive DNA target for this biologically relevant photogenerated oxidant. A large body of mechanistic data is now available from studies performed on nucleosides in both aprotic solvents and aqueous solutions. A common process to both reaction conditions is the formation of 8-oxo-7,8-dihydroguanine by reduction of 8-hydroperoxyguanine that arises from the rearrangement of initially formed endoperoxide across the 4,8-bond of the purine moiety. However, in organic solvent the hydroperoxide is converted as a major degradation pathway into a dioxirane that subsequently decomposes into a complex pattern of oxidation products. A different reaction that involved the formation of a highly reactive quinonoid intermediate consecutively to the loss of a water molecule from the 8-hydroperoxide has been shown to occur in aqueous solution. Subsequent addition of a water molecule at C5 leads to the generation of a spiroiminodihy-dantoin compound via a rearrangement that involves an acyl shift. However, in both isolated and cellular DNA the latter decomposition pathway is at the best a minor process, because only 8-oxo-7,8-dihydroguanine has been found to be generated. It is interesting to point out that singlet oxygen has been shown to contribute predominantly to the formation of 8-oxo-7,8-dihydroguanine in the DNA of bacterial and human cells upon exposure to UVA radiation. It may be added that the formation of secondary singlet-oxygen oxidation products of 8-oxo-7,8-dihydroguanine, including spiroiminodihydantoin and oxaluric acid that were characterized in nucleosides and oligonucleotide, respectively, have not yet been found in cellular DNA.

  5. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  6. Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.

    PubMed

    Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

    2017-03-22

    A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the

  7. One-stop genomic DNA extraction by salicylic acid-coated magnetic nanoparticles.

    PubMed

    Zhou, Zhongwu; Kadam, Ulhas S; Irudayaraj, Joseph

    2013-11-15

    Salicylic acid-coated magnetic nanoparticles were prepared via a modified one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by nonspecific binding of the particles as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared with traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally friendly.

  8. Isolation of lactic acid-forming bacteria from biogas plants.

    PubMed

    Bohn, Jelena; Yüksel-Dadak, Aytül; Dröge, Stefan; König, Helmut

    2017-02-20

    Direct molecular approaches provide hints that lactic acid bacteria play an important role in the degradation process of organic material to methanogenetic substrates in biogas plants. However, their diversity in biogas fermenter samples has not been analyzed in detail yet. For that reason, five different biogas fermenters, which were fed mainly with maize silage and manure from cattle or pigs, were examined for the occurrence of lactic acid-forming bacteria. A total of 197 lactic acid-forming bacterial strains were isolated, which we assigned to 21 species, belonging to the genera Bacillus, Clostridium, Lactobacillus, Pediococcus, Streptococcus and Pseudoramibacter-related. A qualitative multiplex system and a real-time quantitative PCR could be developed for most isolates, realized by the selection of specific primers. Their role in biogas plants was discussed on the basis of the quantitative results and on physiological data of the isolates.

  9. Characterization of virus isolates by particle-associated nucleic acid PCR.

    PubMed

    Stang, Alexander; Korn, Klaus; Wildner, Oliver; Uberla, Klaus

    2005-02-01

    Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3' end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it

  10. Interactions of carcinogens with DNA (deoxyribonucleic acid)

    SciTech Connect

    Broyde, S.; Shapiro, R.

    1989-10-01

    The principal goal of this research has been the determination of the conformational changes produced in DNA by the covalent binding of a carcinogenic aromatic amine, and the correlation of these changes with the mutations and carcinogenic effects initiated by the same substances. To this end, we have devised new synthetic methods for the preparation of oligonucleotides modified by derivatives af 4-aminobiphenyl and aniline. We have also performed potential energy minimization studies on the above substances and on single and double stranded DNA fragments bearing the above amines as well as acetylaminofluorene, aminofluorene, aminopyrene and the antibiotic mitomycin. Our computations have been carried out on DOE supercomputers using our program, DUPLEX. We have defined a number of novel structures for these modified DNAs, including Hoogsteen, wedge'' (see below) denatured, cross-linked and intercalated forms. Some suggestions have been made about the relation of these forms to mutagenesis. 7 refs.

  11. Isolation of a tannic acid-degrading Streptococcus sp. from an anaerobic shea cake digester.

    PubMed

    Nitiema, L W; Dianou, D; Simpore, J; Karou, S D; Savadogo, P W; Traore, A S

    2010-01-01

    An anaerobic digester fed with shea cake rich in tannins and phenolic compounds rich-shea cake and previously inoculated with anaerobic sludge from the pit of a slaughterhouse, enabled six months acclimatization of the bacteria to aromatic compounds. Afterwards, digester waste water samples were subject to successive culture on media with 1 g L(-1) tannic acid allowing the isolation of a bacterial strain coded AB. Strain AB was facultatively anaerobic, mesophilic, non-motile, non-sporulating, catalase and oxidase negative bacterium, namely strain AB, was isolated from an anaerobic digester fed with shea cake rich in tannins and phenolic compounds, after inoculation with anaerobic sludge from the pit of a slaughterhouse and enrichment on tannic acid. The coccoid cells occurred in pair, short or long chains and stained Gram-positive. Strain AB fermented a wide range of carbohydrates including glucose, fructose, galactose, raffinose, arabinose, sucrose, maltose, lactose, starch and cellulose. Optimum growth occurred with glucose and tannic acid at 37 degrees C and pH 8. The pH, temperature and salt concentration for growth ranged from 5 to 9, 20 to 45 degrees C and 0 to 15 g L(-1), respectively. Strain AB converted tannic acid to gallic acid. These features were similar to those of the Streptococcus genus. The determination of tannic acid hydrolysis end products, ability to utilize various organic acids, alcohols and peptides, GC% of the DNA, the sequencing of 16S rRNA gene and DNA-DNA hybridization will permit to confirm this affiliation and to determine the species.

  12. Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology.

    PubMed

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J; Walter, Nils G

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA 'staples'. This revolutionary approach has led to the creation of a multitude of two-dimensional and three-dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy.

  13. Isolation of cDNA clones specifying the fourth component of mouse complement and its isotype, sex-limited protein.

    PubMed Central

    Nonaka, M; Takahashi, M; Natsuume-Sakai, S; Nonaka, M; Tanaka, S; Shimizu, A; Honjo, T

    1984-01-01

    cDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the beta-alpha junction and a Cys-Ala-Glu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence. PMID:6208559

  14. Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation.

    PubMed

    Günal, Gülçin; Kip, Çiğdem; Eda Öğüt, S; İlhan, Hasan; Kibar, Güneş; Tuncel, Ali

    2017-03-22

    Monodisperse silica microspheres with bimodal pore-size distribution were proposed as a high performance sorbent for DNA isolation in batch fashion under equilibrium conditions. The proposed sorbent including both macroporous and mesoporous compartments was synthesized 5.1 μm in-size, by a "staged shape templated hydrolysis and condensation method". Hydrophilic polymer based sorbents were also obtained in the form of monodisperse-macroporous microspheres ca 5.5 μm in size, with different functionalities, by a developed "multi-stage microsuspension copolymerization" technique. The batch DNA isolation performance of proposed material was comparatively investigated using polymer based sorbents with similar morphologies. Among all sorbents tried, the best DNA isolation performance was achieved with the monodisperse silica microspheres with bimodal pore size distribution. The collocation of interconnected mesoporous and macroporous compartments within the monodisperse silica microspheres provided a high surface area and reduced the intraparticular mass transfer resistance and made easier both the adsorption and desorption of DNA. Among the polymer based sorbents, higher DNA isolation yields were achieved with the monodisperse-macroporous polymer microspheres carrying trimethoxysilyl and quaternary ammonium functionalities. However, batch DNA isolation performances of polymer based sorbents were significantly lower with respect to the silica microspheres.

  15. Ascorbic acid conjugates isolated from the phloem of Cucurbitaceae.

    PubMed

    Hancock, Robert D; Chudek, John A; Walker, Paul G; Pont, Simon D A; Viola, Roberto

    2008-06-01

    Analysis of phloem exudates from the fruit of Cucurbitaceae revealed the presence of several compounds with UV-visible absorption spectra identical to that of l-ascorbic acid. In Cucurbita pepo L. (zucchini), the compounds could be isolated from phloem exudates collected from aerial parts of the plant but were not detected in whole tissue homogenates. The compounds isolated from the phloem exudates of C. pepo fruit were eluted from strong anion exchange resin in the same fraction as l-ascorbic acid and were oxidised by ascorbate oxidase (E.C. 1.10.3.3). The major compound purified from C. pepo fruit exudates demonstrated similar redox properties to l-ascorbic acid and synthetic 6-O-glucosyl-l-ascorbic acid (6-GlcAsA) but differed from those of 2-O-glucosyl-l-ascorbic acid (2-GlcAsA) isolated from the fruit of Lycium barbarum L. Parent and fragment ion masses of the compound were consistent with hexosyl-ascorbate in which the hexose moiety was attached to C5 or C6 of AsA. Acid hydrolysis of the major C. pepo compound resulted in the formation of l-ascorbic acid and glucose. The purified compound yielded a proton NMR spectrum that was almost identical to that of synthetic 6-GlcAsA. A series of l-ascorbic acid conjugates have, therefore, been identified in the phloem of Cucurbitaceae and the most abundant conjugate has been identified as 6-GlcAsA. The potential role of such conjugates in the long-distance transport of l-ascorbic acid is discussed.

  16. Electricity-free, sequential nucleic acid and protein isolation.

    PubMed

    Pawlowski, David R; Karalus, Richard J

    2012-05-15

    Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification

  17. Lactobacillus formosensis sp. nov., a lactic acid bacterium isolated from fermented soybean meal.

    PubMed

    Chang, Chi-huan; Chen, Yi-sheng; Lee, Tzu-tai; Chang, Yu-chung; Yu, Bi

    2015-01-01

    A Gram-reaction-positive, catalase-negative, facultatively anaerobic, rod-shaped lactic acid bacterium, designated strain S215(T), was isolated from fermented soybean meal. The organism produced d-lactic acid from glucose without gas formation. 16S rRNA gene sequencing results showed that strain S215(T) had 98.74-99.60 % sequence similarity to the type strains of three species of the genus Lactobacillus (Lactobacillus farciminis BCRC 14043(T), Lactobacillus futsaii BCRC 80278(T) and Lactobacillus crustorum JCM 15951(T)). A comparison of two housekeeping genes, rpoA and pheS, revealed that strain S215(T) was well separated from the reference strains of species of the genus Lactobacillus. DNA-DNA hybridization results indicated that strain S215(T) had DNA related to the three type strains of species of the genus Lactobacillus (33-66 % relatedness). The DNA G+C content of strain S215(T) was 36.2 mol%. The cell walls contained peptidoglycan of the d-meso-diaminopimelic acid type and the major fatty acids were C18 : 1ω9c, C16 : 0 and C19 : 0 cyclo ω10c/C19 : 1ω6c. Phenotypic and genotypic features demonstrated that the isolate represents a novel species of the genus Lactobacillus, for which the name Lactobacillus formosensis sp. nov. is proposed. The type strain is S215(T) ( = NBRC 109509(T) = BCRC 80582(T)).

  18. Biodiversity and γ-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow's milk cheeses.

    PubMed

    Franciosi, Elena; Carafa, Ilaria; Nardin, Tiziana; Schiavon, Silvia; Poznanski, Elisa; Cavazza, Agostino; Larcher, Roberto; Tuohy, Kieran M

    2015-01-01

    "Nostrano-cheeses" are traditional alpine cheeses made from raw cow's milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of "Nostrano-cheeses" and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n = 97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was a Sc. thermophilus.

  19. Biodiversity and γ-Aminobutyric Acid Production by Lactic Acid Bacteria Isolated from Traditional Alpine Raw Cow's Milk Cheeses

    PubMed Central

    Nardin, Tiziana; Schiavon, Silvia; Cavazza, Agostino; Larcher, Roberto; Tuohy, Kieran M.

    2015-01-01

    “Nostrano-cheeses” are traditional alpine cheeses made from raw cow's milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n = 97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was a Sc. thermophilus. PMID:25802859

  20. Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein

    PubMed Central

    1988-01-01

    A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2- terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2- terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic. PMID:3199069

  1. Using FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) to isolate active regulatory DNA

    PubMed Central

    Simon, Jeremy M.; Giresi, Paul G.; Davis, Ian J.; Lieb, Jason D.

    2013-01-01

    Eviction or destabilization of nucleosomes from chromatin is a hallmark of functional regulatory elements of the eukaryotic genome. Historically identified by nuclease hypersensitivity, these regulatory elements are typically bound by transcription factors or other regulatory proteins. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) is an alternative approach to identify these genomic regions and has proven successful in a multitude of eukaryotic cell and tissue types. Cells or dissociated tissues are crosslinked briefly with formaldehyde, lysed, and sonicated. Sheared chromatin is subjected to phenol-chloroform extraction and the isolated DNA, typically encompassing 1–3% of the human genome, is purified. We provide guidelines for quantitative analysis by PCR, microarrays, or next-generation sequencing. Regulatory elements enriched by FAIRE display high concordance with those identified by nuclease hypersensitivity or ChIP, and the entire procedure can be completed in three days. FAIRE exhibits low technical variability, which allows its use in large-scale studies of chromatin from normal or diseased tissues. PMID:22262007

  2. Isolation of cDNA encoding a binding protein specific to 5'-phosphorylated single-stranded DNA with G-rich sequences.

    PubMed Central

    Mizuta, T R; Fukita, Y; Miyoshi, T; Shimizu, A; Honjo, T

    1993-01-01

    We have isolated the cDNA encoding a binding protein to the sequence motif of the immunoglobulin S mu region by the southwestern method. The binding protein designated S mu bp-2 specifically binds to 5'-phosphorylated single-stranded DNA containing 5'-G and GGGG stretches. The amino acid sequence deduced from the cDNA sequence showed that the S mu bp-2 belongs to the putative helicase superfamily which is involved in replication, recombination and repair. Expression of S mu bp-2 mRNA is ubiquitous and augmented in spleen cells stimulated with lipopolysaccharide and interleukin 4 which also induce class switching. The S mu bp-2 gene is conserved among vertebrates. Possible involvement of S mu bp-2 in class switching is discussed. Images PMID:8493094

  3. A nontoxic and versatile protein salting-out method for isolation of DNA.

    PubMed

    Laitinen, J; Samarut, J; Hölttä, E

    1994-08-01

    A pivotal technique in basic and applied molecular biology is the isolation of DNA. However, the present DNA extraction methods are either toxic, expensive, time-consuming and laborious or restricted to certain applications. Here we describe a nontoxic and versatile protein salting-out method for convenient and rapid extraction of large as well as small DNA molecules from vertebrate cells and plasmid DNA from bacteria. Easy and relatively imprecise manipulations of a large number of samples result in high yields of pure mammalian and plasmid DNA that are suitable for transformation of bacteria, restriction enzyme analyses, Southern blotting, end labeling of DNA, PCR and sequencing.

  4. Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids.

    PubMed

    Olson, Xiaoping; Kotani, Shohei; Yurke, Bernard; Graugnard, Elton; Hughes, William L

    2017-03-30

    Locked nucleic acids (LNAs) are conformationally restricted RNA nucleotides. Their increased thermal stability and selectivity toward their complements make them well-suited for diagnostic and therapeutic applications. Although the structural and thermodynamic properties of LNA-LNA, LNA-RNA, and LNA-DNA hybridizations are known, the kinetic effects of incorporating LNA nucleotides into DNA strand displacement systems are not. Here, we thoroughly studied the strand displacement kinetics as a function of the number and position of LNA nucleotides in DNA oligonucleotides. When compared to that of an all-DNA control, with an identical sequence, the leakage rate constant was reduced more than 50-fold, to an undetectable level, and the invasion rate was preserved for a hybrid DNA/LNA system. The total performance enhancement ratio also increased more than 70-fold when calculating the ratio of the invading rate to the leakage rate constants for a hybrid system. The rational substitution of LNA nucleotides for DNA nucleotides preserves sequence space while improving the signal-to-noise ratio of strand displacement systems. Hybrid DNA/LNA systems offer great potential for high-performance chemical reaction networks that include catalyzed hairpin assemblies, hairpin chain reactions, motors, walkers, and seesaw gates.

  5. Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses▿

    PubMed Central

    Siragusa, S.; De Angelis, M.; Di Cagno, R.; Rizzello, C. G.; Coda, R.; Gobbetti, M.

    2007-01-01

    The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg−1. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA. PMID:17890341

  6. Semisynthesis of the antiviral abietane diterpenoid jiadifenoic acid C from callitrisic acid (4-epidehydroabietic acid) isolated from sandarac resin.

    PubMed

    González, Miguel A; Zaragozá, Ramón J

    2014-09-26

    The semisynthesis of the antiviral abietane diterpenoid (+)-jiadifenoic acid C starting from the available methyl ester of callitrisic acid (4-epidehydroabietic acid) isolated from sandarac resin is reported. A protocol for the isolation of methyl callitrisate (methyl 4-epidehydroabietate) in gram quantities from sandarac resin is also described. Allylic C-17 oxygenation was introduced by regioselective dehydrogenation of the isopropyl group of methyl callitrisate with DDQ followed by selenium-catalyzed allylic oxidation. Ester hydrolysis afforded (+)-jiadifenoic acid C in 22% overall yield from methyl callitrisate. This semisynthetic route provides a convenient source of this anti-Coxsackie virus B natural product for further biological studies.

  7. Gluconacetobacter medellinensis sp. nov., cellulose- and non-cellulose-producing acetic acid bacteria isolated from vinegar.

    PubMed

    Castro, Cristina; Cleenwerck, Ilse; Trcek, Janja; Zuluaga, Robin; De Vos, Paul; Caro, Gloria; Aguirre, Ricardo; Putaux, Jean-Luc; Gañán, Piedad

    2013-03-01

    The phylogenetic position of a cellulose-producing acetic acid bacterium, strain ID13488, isolated from commercially available Colombian homemade fruit vinegar, was investigated. Analyses using nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rRNA gene internal transcribed spacer (ITS) sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated the micro-organism to the genus Gluconacetobacter, and more precisely to the Gluconacetobacter xylinus group. Moreover, the data suggested that the micro-organism belongs to a novel species in this genus, together with LMG 1693(T), a non-cellulose-producing strain isolated from vinegar by Kondo and previously classified as a strain of Gluconacetobacter xylinus. DNA-DNA hybridizations confirmed this finding, revealing a DNA-DNA relatedness value of 81 % between strains ID13488 and LMG 1693(T), and values <70 % between strain LMG 1693(T) and the type strains of the closest phylogenetic neighbours. Additionally, the classification of strains ID13488 and LMG 1693(T) into a single novel species was supported by amplified fragment length polymorphism (AFLP) and (GTG)5-PCR DNA fingerprinting data, as well as by phenotypic data. Strains ID13488 and LMG 1693(T) could be differentiated from closely related species of the genus Gluconacetobacter by their ability to produce 2- and 5-keto-d-gluconic acid from d-glucose, their ability to produce acid from sucrose, but not from 1-propanol, and their ability to grow on 3 % ethanol in the absence of acetic acid and on ethanol, d-ribose, d-xylose, sucrose, sorbitol, d-mannitol and d-gluconate as carbon sources. The DNA G+C content of strains ID13488 and LMG 1693(T) was 58.0 and 60.7 mol%, respectively. The major ubiquinone of LMG 1693(T) was Q-10. Taken together these data indicate that strains ID13488 and LMG 1693(T) represent a novel species of the genus Gluconacetobacter for which the name Gluconacetobacter

  8. The HIR protein family: isolation and characterization of a complete murine cDNA.

    PubMed

    Scamps, C; Lorain, S; Lamour, V; Lipinski, M

    1996-04-10

    A full-length cDNA has been isolated for the murine homolog of the human HIRA protein, a member of the HIR family of nuclear proteins that is encoded from the chromosome 22 region critical for the DiGeorge syndrome. This family also contains Hir1p and Hir2p, two proteins identified as regulators of histone gene transcription in yeast. The murine and human amino acid sequences are 95.3% identical, with a striking 99.2% identity in the N-terminal WD repeat domain that is characteristic of the family. The two cDNAs are highly conserved within the coding regions, but also in the entire 5' untranslated region and in a strikingly long stretch of nucleotides in the 3' untranslated region.

  9. Rapid isolation of DNA from chocolate and date palm tree crops.

    PubMed

    Haymes, Kenneth M; Ibrahim, Ibrahim A; Mischke, Sue; Scott, David L; Saunders, James A

    2004-08-25

    DNA isolation from plants is sometimes difficult due to the existence of high levels of endogenous phenolics, polysaccharides, or other substances that may interfere with DNA extraction. Theobroma cacao produces high levels of anthocyanins in young leaves. These plant polyphenols can interfere with DNA isolation. After examination of various procedures for DNA isolation, two commercial isolation procedures have proved to be repeatedly successful using these types of plants, the D(2) BioTechnologies DNA X-tract Plus kit and the Qiagen DNeasy Plant System DNA kit. These commercial kits were chosen for their speed and ease over the CTAB procedure, which is more labor intensive. All protocols assessed yielded DNA suitable for AFLP or SSR procedures. An additional factor in DNA extraction efficiency is the degree of cell breakage, which may be more difficult with the highly fibrous leaf tissue that is found in many monocots, including date palm. Two commercially produced pieces of equipment were tested and, for cacao, both resulted in template DNA yielding amplification product in AFLP or SSR fingerprinting. However, for the fibrous date palm leaf, the larger FastPrep homogenizer consistently yielded DNA that generated higher signals when amplified than did the smaller Disruptor Genie.

  10. Characterization of ascorbic acid uptake by isolated rat kidney cells

    SciTech Connect

    Bowers-Komro, D.M.; McCormick, D.B. )

    1991-01-01

    Isolated kidney cells accumulated L(1-14C)ascorbic acid in a time-dependent manner and reached a steady state after 15 min at 37 degrees C. Initial velocity for uptake was over 300 pmol/mg protein per min when cells were separated from the bathing solution using a density gradient established during centrifugation. The uptake process was saturable with an apparent concentration at half maximal uptake of 36 mumols/L. Ascorbate uptake was reduced by metabolic inhibitors and was temperature dependent. Although ascorbic acid is an acid anion at pH 7.4, uptake did not appear to be inhibited by other acid anions such as p-aminohippurate and probenecid; however, involvement of the ion gradient established by Na+, H(+)-adenosine triphosphatase could not be confirmed. Replacing the sodium ion with other monovalent ions reduced the accumulation of ascorbate significantly. Isoascorbic and dehydroascorbic acids inhibited ascorbate uptake (34 and 13 mmol/L, respectively), whereas high concentrations of glucose showed some stimulation. These findings indicated that ascorbic acid is reabsorbed by the kidney in a sodium-dependent active transport process that is not common to other acid anions and has some specificity for the ascorbic acid structure.

  11. Evaluation of DNA typing as a positive identification method for soft and hard tissues immersed in strong acids.

    PubMed

    Robino, C; Pazzi, M; Di Vella, G; Martinelli, D; Mazzola, L; Ricci, U; Testi, R; Vincenti, M

    2015-11-01

    Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes.

  12. ARISTOLOCHIC ACID I METABOLISM IN THE ISOLATED PERFUSED RAT KIDNEY

    PubMed Central

    Priestap, Horacio A.; Torres, M. Cecilia; Rieger, Robert A.; Dickman, Kathleen G.; Freshwater, Tomoko; Taft, David R.; Barbieri, Manuel A.; Iden, Charles R.

    2012-01-01

    Aristolochic acids are natural nitro-compounds found globally in the plant genus Aristolochia that have been implicated in the severe illness in humans termed aristolochic acid nephropathy (AAN). Aristolochic acids undergo nitroreduction, among other metabolic reactions, and active intermediates arise that are carcinogenic. Previous experiments with rats showed that aristolochic acid I (AA-I), after oral administration or injection, is subjected to detoxication reactions to give aristolochic acid Ia, aristolactam Ia, aristolactam I and their glucuronide and sulfate conjugates that can be found in urine and faeces. Results obtained with whole rats do not clearly define the role of liver and kidney in such metabolic transformation. In this study, in order to determine the specific role of the kidney on the renal disposition of AA-I and to study the biotransformations suffered by AA-I in this organ, isolated kidneys of rats were perfused with AA-I. AA-I and metabolite concentrations were determined in perfusates and urines using HPLC procedures. The isolated perfused rat kidney model showed that AA-I distributes rapidly and extensively in kidney tissues by uptake from the peritubular capillaries and the tubules. It was also established that the kidney is able to metabolize AA-I into aristolochic acid Ia, aristolochic acid Ia O-sulfate, aristolactam Ia, aristolactam I and aristolactam Ia O-glucuronide. Rapid demethylation and sulfation of AA-I in the kidney generate aristolochic acid Ia and its sulfate conjugate that are voided to the urine. Reduction reactions to give the aristolactam metabolites occur to a slower rate. Renal clearances showed that filtered AA-I is reabsorbed at the tubules whereas the metabolites are secreted. The unconjugated metabolites produced in the renal tissues are transported to both urine and perfusate whereas the conjugated metabolites are almost exclusively secreted to the urine. PMID:22118289

  13. QIAamp spin columns as a method of DNA isolation for forensic casework.

    PubMed

    Greenspoon, S A; Scarpetta, M A; Drayton, M L; Turek, S A

    1998-09-01

    The Detroit Police Crime Lab has historically used Chelex as a method to isolate DNA for amplification and typing of bloodstains at the HLADQA1, PM and D1S80 loci. However, preliminary validation of several STR systems for casework has demonstrated that the Chelex procedure is not the best method of DNA isolation for STR amplifications for our purposes. Long term storage at -20 degrees C in the presence of unbuffered Chelex beads (approximately 1 year), combined with multiple freeze thaws, resulted in signal loss at a locus for many database samples. Therefore, we have employed the QIAamp spin column as an alternative method of DNA isolation for amplification and typing of STR loci currently being validated for use in the laboratory. Moreover, we determined that QIAamp isolated DNA is also suitable for HLADQA1, PM and D1S80 typing. A matrix study was performed to determine if the QIAamp DNA procedure would give better results on bloodstains deposited on "problem surfaces" such as leather, dirt and various dyed fabrics. Again, QIAamp isolated DNA was more readily typeable than Chelex isolated DNA. We successfully replaced the phenol/chloroform extraction steps utilized in our laboratory for differential extractions, a commonly used method for separating sperm and non-sperm fractions of sexual assault evidence, with the QIAamp spin columns. The QIAamp extracted DNA performed as well in all PCR amplification and typing procedures tested (PM, HLADQA1, D1S80, and STR (PowerPlex)) as the phenol/chloroform Centricon isolated or EtOH precipitated DNAs. Thus we concluded that QIAamp spin columns are a superior method for isolating DNA to be typed for a variety of loci.

  14. An Improved Protocol for Intact Chloroplasts and cpDNA Isolation in Conifers

    PubMed Central

    Vieira, Leila do Nascimento; Faoro, Helisson; Fraga, Hugo Pacheco de Freitas; Rogalski, Marcelo; de Souza, Emanuel Maltempi; de Oliveira Pedrosa, Fábio; Nodari, Rubens Onofre; Guerra, Miguel Pedro

    2014-01-01

    Background Performing chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement. Methodology/Principal Findings We developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula. Conclusion Results show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences. PMID:24392157

  15. Enterococcus bulliens sp. nov., a novel lactic acid bacterium isolated from camel milk.

    PubMed

    Kadri, Zaina; Spitaels, Freek; Cnockaert, Margo; Praet, Jessy; El Farricha, Omar; Swings, Jean; Vandamme, Peter

    2015-11-01

    Four lactic acid bacteria isolates obtained from fresh dromedary camel milk produced in Dakhla, a city in southern Morocco, were characterised in order to determine their taxonomic position. The four isolates had highly similar MALDI-TOF MS and RAPD fingerprints and identical 16S rRNA gene sequences. Comparative sequence analysis revealed that the 16S rRNA gene sequence of the four isolates was most similar to that of Enterococcus sulfureus ATCC 49903(T) and Enterococcus italicus DSM 15952(T) (99.33 and 98.59% similarity, respectively). However, sequence analysis of the phenylalanyl-tRNA synthase (pheS), RNA polymerase (rpoA) and ATP synthase (atpA) genes revealed that the taxon represented by strain LMG 28766(T) was well separated from E. sulfureus LMG 13084(T) and E. italicus LMG 22039(T), which was further confirmed by DNA-DNA hybridization values that were clearly below the species demarcation threshold. The novel taxon was easily differentiated from its nearest neighbour species through sequence analysis of protein encoding genes, MALDI-TOF mass spectrometry and multiple biochemical tests, but had a similar percentage G+C content of about 39%. We therefore propose to formally classify these isolates as Enterococcus bulliens sp. nov., with LMG 28766(T) (=CCMM B1177(T)) as the type strain.

  16. DNA Tetrominoes: The Construction of DNA Nanostructures Using Self-Organised Heterogeneous Deoxyribonucleic Acids Shapes

    PubMed Central

    Ong, Hui San; Rahim, Mohd Syafiq; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

    2015-01-01

    The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner. PMID:26258940

  17. Isolation of Lactic Acid Bacteria Showing Antioxidative and Probiotic Activities from Kimchi and Infant Feces.

    PubMed

    Ji, Keunho; Jang, Na Young; Kim, Young Tae

    2015-09-01

    The purpose of this study was to investigate lactic acid bacteria with antioxidative and probiotic activities isolated from Korean healthy infant feces and kimchi. Isolates A1, A2, S1, S2, and S3 were assigned to Lactobacillus sp. and isolates A3, A4, E1, E2, E3, and E4 were assigned to Leuconostoc sp. on the basis of their physiological properties and 16S ribosomal DNA sequence analysis. Most strains were confirmed as safe bioresources through nonhemolytic activities and non-production of harmful enzymes such as β-glucosidase, β- glucuronidase and tryptophanase. The 11 isolates showed different resistance to acid and bile acids. In addition, they exhibited antibacterial activity against foodborne bacteria, especially Bacillus cereus, Listeria monocytogenes, and Escherichia coli. Furthermore, all strains showed significantly high levels of hydrophobicity. The antioxidant effects of culture filtrates of the 11 strains included 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging capacity, 2.2'- azino-bis (2-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity, and superoxide dismutase activity. The results revealed that most of the culture filtrates have effective scavenging activity for DPPH and ABTS radicals. All strains appeared to have effective superoxide dismutase activity. In conclusion, the isolated strains A1, A3, S1, and S3 have significant probiotic activities applicable to the development of functional foods and health-related products. These strains might also contribute to preventing and controlling several diseases associated with oxidative stress, when used as probiotics.

  18. Biological responses to the simulated Martian UV radiation of bacteriophages and isolated DNA.

    PubMed

    Fekete, Andrea; Kovács, Gáspár; Hegedüs, Márton; Módos, Károly; Lammer, Helmut

    2008-08-21

    Mars is considered as a main target for astrobiologically relevant exploration programmes. In this work the effect of simulated Martian solar UV radiation was examined on bacteriophage T7 and on isolated T7 DNA. A decrease of the biological activity of phages, characteristic changes in the absorption spectrum and in the electrophoretic pattern of isolated DNA/phage and the decrease of the amount of PCR products were detected indicating damage of isolated and intraphage T7 DNA by UV radiation. Further mechanistic insights into the UV-induced formation of intraphage/isolated T7 DNA photoproducts were gained from the application of appropriate enzymatic digestion and neutral/alkaline agarose gel electrophoresis. Our results showed that intraphage DNA was about ten times more sensitive to simulated Martian UV radiation than isolated T7 DNA indicating the role of phage proteins in the DNA damage. Compared to solar UV radiation the total amount of DNA damage determined by QPCR was about ten times larger in isolated DNA and phage T7 as well, and the types of the DNA photoproducts were different, besides cyclobutane pyrimidine dimers (CPD), double-strand breaks (dsb), and single-strand breaks (ssb), DNA-protein cross-links were produced as well. Surprisingly, energy deposition as low as 4-6 eV corresponding to 200-400 nm range could induce significant amount of ssb and dsb in phage/isolated DNA (in phage the ratio of ssb/dsb was approximately 23%/12% and approximately 32%/19% in isolated DNA). 5-8% of the CPD, 3-5% of the AP (apurinic/apyrimidinic) sites were located in clusters in DNA/phage, suggesting that clustering of damage occur in the form of multiple damaged sites and these can have a high probability to produce strand breaks. The amount of total DNA damage in samples which were irradiated in Tris buffer was reduced by a factor approximately 2, compared to samples in phosphate buffer, suggesting that some of the photoproducts were produced via radicals.

  19. A glutamic acid-producing lactic acid bacteria isolated from Malaysian fermented foods.

    PubMed

    Zareian, Mohsen; Ebrahimpour, Afshin; Bakar, Fatimah Abu; Mohamed, Abdul Karim Sabo; Forghani, Bita; Ab-Kadir, Mohd Safuan B; Saari, Nazamid

    2012-01-01

    l-glutamaic acid is the principal excitatory neurotransmitter in the brain and an important intermediate in metabolism. In the present study, lactic acid bacteria (218) were isolated from six different fermented foods as potent sources of glutamic acid producers. The presumptive bacteria were tested for their ability to synthesize glutamic acid. Out of the 35 strains showing this capability, strain MNZ was determined as the highest glutamic-acid producer. Identification tests including 16S rRNA gene sequencing and sugar assimilation ability identified the strain MNZ as Lactobacillus plantarum. The characteristics of this microorganism related to its glutamic acid-producing ability, growth rate, glucose consumption and pH profile were studied. Results revealed that glutamic acid was formed inside the cell and excreted into the extracellular medium. Glutamic acid production was found to be growth-associated and glucose significantly enhanced glutamic acid production (1.032 mmol/L) compared to other carbon sources. A concentration of 0.7% ammonium nitrate as a nitrogen source effectively enhanced glutamic acid production. To the best of our knowledge this is the first report of glutamic acid production by lactic acid bacteria. The results of this study can be further applied for developing functional foods enriched in glutamic acid and subsequently γ-amino butyric acid (GABA) as a bioactive compound.

  20. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

    PubMed

    Graham, A; Hopkins, B; Powell, S J; Danks, P; Briggs, I

    1991-05-31

    Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

  1. Isolation and characterization of hyaluronic acid from marine organisms.

    PubMed

    Giji, Sadhasivam; Arumugam, Muthuvel

    2014-01-01

    Hyaluronic acid (HA) being a viscous slippery substance is a multifunctional glue with immense therapeutic applications such as ophthalmic surgery, orthopedic surgery and rheumatology, drug delivery systems, pulmonary pathology, joint pathologies, and tissue engineering. Although HA has been isolated from terrestrial origin (human umbilical cord, rooster comb, bacterial sources, etc.) so far, the increasing interest on this polysaccharide significantly aroused the alternative search from marine sources since it is at the preliminary level. Enthrallingly, marine environments are considered more biologically diverse than terrestrial environments. Although numerous methods have been described for the extraction and purification of HA, the hitch on the isolation methods which greatly influences the yield as well as the molecular weight of the polymer still exists. Adaptation of suitable method is essential in this venture. Stimulated by the developed technology, to sketch the steps involved in isolation and analytical techniques for characterization of this polymer, a brief report on the concerned approach has been reviewed.

  2. Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi

    PubMed Central

    Hong, Yeon-Chul; Hwang, Mi-Yul; Yun, Ho-Cheol; Yu, Hak-Sun; Kong, Hyun-Hee; Yong, Tai-Soon

    2002-01-01

    We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues, Cys25, His159, and Asn175. Deduced amino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue. PMID:11949209

  3. Isolation of Discrete Nanoparticle-DNA Conjugates for Plasmonic Applications

    SciTech Connect

    Alivisatos, Paul; Claridge, Shelley A.; Liang, Huiyang W.; Basu, Sourav Roger; Frechet, Jean M.J.; Alivisatos, A. Paul

    2008-04-11

    Discrete DNA-gold nanoparticle conjugates with DNA lengths as short as 15 bases for both 5 nm and 20 nm gold particles have been purified by anion-exchange HPLC. Conjugates comprising short DNA (<40 bases) and large gold particles (>_ 20 nm) are difficult to purify by other means, and are potential substrates for plasmon coupling experiments. Conjugate purity is demonstrated by hybridizing complementary conjugates to form discrete structures, which are visualized by TEM.

  4. [A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture].

    PubMed

    Vardevanian, P O; Davtian, A M; Tiratsuian, S G; Vardevanian, A O

    1990-01-01

    A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.

  5. A bone sample cleaning method using trypsin for the isolation of DNA.

    PubMed

    Li, Richard; Liriano, Lidissy

    2011-11-01

    Cleaning the surface of bone samples is a necessary step to remove contaminants prior to isolating DNA for forensic DNA analysis. In this study, a simple trypsin method for cleaning bone samples prior to DNA isolation was developed. Cleaning the surface of human bone samples was achieved by the application of trypsin solution. Light microscopy and scanning electron microscopy results indicated that trypsin treatment was effective in removing the outer surface of bone samples. The yield of DNA isolated from trypsin-treated bone samples was sufficient for subsequent short tandem repeat (STR) analysis. STR analysis revealed no adverse effect on the DNA profile after the trypsin treatment. The data suggest that this trypsin method can potentially be an alternative cleaning method to mechanical cleaning methods.

  6. Flexibility of nucleic acids: From DNA to RNA

    NASA Astrophysics Data System (ADS)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  7. Genomic fingerprinting of "Haemophilus somnus" isolates by using a random-amplified polymorphic DNA assay.

    PubMed Central

    Myers, L E; Silva, S V; Procunier, J D; Little, P B

    1993-01-01

    The random-amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints for 16 isolates of "Haemophilus somnus," and one isolate each of "Haemophilus agni," "Histophilus ovis," "Actinobacillus seminis," Pasteurella haemolytica, and Escherichia coli. The RAPD assay differentiated among "H. somnus" isolates, which shared similarity coefficients of 0.46 to 1.00 on the basis of pairwise comparisons of RAPD markers produced with nine random decamer primers. Three virulent encephalitic "H. somnus" isolates exhibited identical banding patterns, suggesting a common clonal ancestry. The RAPD assay clearly distinguished between the "H. somnus"-"H. agni"-"H. ovis" group and the other bacterial species tested. The results of the present study suggest that DNA fingerprinting of "H. somnus" isolates by the RAPD assay could be valuable in revealing subspecific divisions within this largely unexplored species. Images PMID:8458944

  8. Effects of lactic acid bacteria isolated from fermented mustard on lowering cholesterol

    PubMed Central

    Wang, Shu Chen; Chang, Chen Kai; Chan, Shu Chang; Shieh, Jiunn Shiuh; Chiu, Chih Kwang; Duh, Pin-Der

    2014-01-01

    Objective To evaluate the ability of lactic acid bacteria (LAB) strains isolated from fermented mustard to lower the cholesterol in vitro. Methods The ability of 50 LAB strains isolated from fermented mustard on lowering cholesterol in vitro was determined by modified o-phtshalaldehyde method. The LAB isolates were analyzed for their resistance to acid and bile salt. Strains with lowering cholesterol activity, were determined adherence to Caco-2 cells. Results Strain B0007, B0006 and B0022 assimilated more cholesterol than BCRC10474 and BCRC 17010. The isolated strains showed tolerance to pH 3.0 for 3 h despite variations in the degree of viability and bile-tolerant strains, with more than 108 CFU/mL after incubation for 24 h at 1% oxigall in MRS. In addition, strain B0007 and B0022 identified as Lactobacillus plantarum with 16S rDNA sequences were able to adhere to the Caco-2 cell lines. Conclusions These strains B0007 and B0022 may be potential functional sources for cholesterol-lowering activities as well as adhering to Caco-2 cell lines. PMID:25183271

  9. Phenotypic and genotypic characterization of lactic acid bacteria isolated from Artisanal Italian goat cheese.

    PubMed

    Colombo, E; Franzetti, L; Frusca, M; Scarpellini, M

    2010-04-01

    The lactic acid bacteria community in traditional goat cheese produced in three dairies in Valsesia (Piemonte, Italy) was studied at different steps of the manufacturing process. These cheeses were produced from raw milk without starter bacteria, and no protocol was followed during the manufacturing process. Three hundred thirty-two isolates were characterized and grouped by results of both morphophysiological tests and random amplification of polymorphic DNA plus PCR analysis. Bacteria were identified by partial sequencing of the 16S rRNA gene. Lactococci were the dominant lactic acid bacteria in raw milk. Their initial numbers ranged from 5 to 7 log CFU ml(-1). Their levels increased during manufacturing and decreased during ripening. The growth trend for enterococci was comparable to that of lactococci, although enterococci counts were lower. Lactococcus lactis subsp. cremoris, Lactococcus garviae, and Enterococcus faecalis were the most frequently isolated species during goat cheese manufacturing, whereas the highest numbers of Enterococcus (E. faecium, E. durans, E. gilvus, and E. casseliflavus) were isolated with the greatest frequency from ripened cheese samples. Occasionally, Leuconostoc mesenteroides, Leuconostoc lactis, and Lactobacillus paraplantarum also were isolated.

  10. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  11. Isolation and characterization of two cDNA clones of anaerobically induced lactate dehydrogenase from barley roots

    SciTech Connect

    Hondred, D.; Hanson, A.D. )

    1990-05-01

    In barley roots during hypoxia, five lactate dehydrogenase (LDH) isozymes accumulate with a concomitant increase in enzyme activity ({approximately}20-fold). These isozymes are thought to be tetramers resulting from the random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH have been isolated from a {lambda}gt11 library using antiserum raised against barley LDH purified {approximately}3,000-fold and using nucleic acid probes synthesized by the polymerase chain reaction. Two cDNA clones were obtained (1,305 and 1,166 bp). The deduced amino acid sequences of the two barley LDHs are 96% identical to each other, and 50% and 40% identical to vertebrate and bacterial LDHs, respectively. Northern blots identified a single mRNA band ({approximately}1.5 kb) whose level rose 8-fold during hypoxia.

  12. Influence of DNA isolation method on the investigation of archaeal diversity and abundance in biogas plants.

    PubMed

    Theiss, Juliane; Rother, Michael; Röske, Kerstin

    2016-09-01

    Various methods are available for DNA isolation from environmental samples. Because the chemical and biological composition of samples such as soil, sludge, or plant material is different, the effectiveness of DNA isolation can vary depending on the method applied and thus, have a substantial effect on the results of downstream analysis of the microbial community. Although the process of biogas formation is being intensely investigated, a systematic evaluation of kits for DNA isolation from material of biogas plants is still lacking. Since no DNA isolation kit specifically tailored for DNA isolation from sludge of biogas plants is available, this study compares five commercially available kits regarding their influence on downstream analyses such denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR). The results show that not all kits are equally suited for the DNA isolation from samples of different biogas plants, but highly reproducible DGGE fingerprints as well as qPCR results across the tested samples from biogas reactors using different substrate compositions could be produced using selected kits.

  13. Isolated nucleic acids encoding antipathogenic polypeptides and uses thereof

    DOEpatents

    Altier, Daniel J.; Crane, Virginia C.; Ellanskaya, Irina; Ellanskaya, Natalia; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K.; Schepers, Eric J.; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-04-20

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from fungal fermentation broths. Nucleic acids that encode the antipathogenic polypeptides are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  14. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family.

    PubMed

    Preston, G M; Agre, P

    1991-12-15

    CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.

  15. How-to-Do-It: A Simple DNA Isolation Technique Using Halophilic Bacteria.

    ERIC Educational Resources Information Center

    Guilfoile, Patrick

    1989-01-01

    Described is a simple technique for isolating DNA from halophilic bacteria. Materials, procedure, and additional experiments are outlined. It is stated that the DNA obtained will be somewhat contaminated with cellular proteins and RNA. Offers a procedure for greater purification. (RT)

  16. Application of real-time PCR to postharvest physiology – DNA isolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time PCR technology has been widely used in the postharvest plant physiology research. One of the difficulties to isolate DNA from plant martial and pathogen cells is the presence of rigid polysaccharide cell walls and capsules, which physically protect DNA from cell lysis. Many materials requi...

  17. High-throughput DNA isolation method for detection of Xylella fastidiosa in plant and insect samples.

    PubMed

    Brady, Jeff A; Faske, Jennifer B; Castañeda-Gill, Jessica M; King, Jonathan L; Mitchell, Forrest L

    2011-09-01

    We report an inexpensive, high-throughput method for isolating DNA from insect and plant samples for the purpose of detecting Xylella fastidiosa infection. Existing methods often copurify inhibitors of DNA polymerases, limiting their usefulness for PCR-based detection assays. When compared to commercially available kits, the method provides enhanced pathogen detection at a fraction of the cost.

  18. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...

  19. Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin.

    PubMed

    Goffin, C; Verly, W G

    1982-10-01

    Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.

  20. DNA analysis of temperate bacteriophage Aa(phi)23 isolated from actinobacillus actinomycetemcomitans.

    PubMed

    Willi, K; Meyer, J

    1998-05-01

    The DNA of the temperate bacteriophage Aaphi23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44 kb linear molecules with a terminal redundancy of 1.6 kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaphi23 prophage is integrated into the host chromosome.

  1. Serotypes and DNA fingerprint profiles of Pasteurella multocida isolated from raptors

    USGS Publications Warehouse

    Wilson, M.A.; Duncan, R.M.; Nordholm, G.E.; Berlowski, B.M.

    1995-01-01

    Pasteurella multocida isolates from 21 raptors were examined by DNA fingerprint profile and serotyping methods. Isolates were obtained from noncaptive birds of prey found in 11 states from November 28, 1979, through February 10, 1993. Nine isolates were from bald eagles, and the remaining isolates were from hawks, falcons, and owls. Seven isolates were members of capsule group A, and 14 were nonencapsulated. One isolate was identified as somatic type 3, and another was type 3,4,7; both had unique HhaI DNA fingerprint profiles. Nineteen isolates expressed somatic type 1 antigen; HhaI profiles of all type 1 isolates were identical to each other and to the HhaI profile of the reference somatic type 1, strain X-73. The 19 type 1 isolates were differentiated by sequential digestion of DNA with HpaII; four HpaII fingerprint profiles were obtained. The HpaII profile of one isolate was identical to the HpaII profile of strain X-73. Incidence of P. multocida somatic type 1 in raptors suggests that this type may be prevalent in other wildlife or wildlife environments.

  2. Inhibition of N-nitrosamine carcinogenesis and aflatoxin DNA damage by ellagic acid

    SciTech Connect

    Mandal-Chaudhuri, S.

    1988-01-01

    The effect of ellagic acid (EA), on the tumorigenicity of N-nitrosobenzylmethylamine (NBMA) in the rat esophagus was investigated. Groups of 30 male F-344 rats were fed a semipurified diet containing EA for 27 weeks. N-nitrosobenzylmethylamine was administered subcutaneously, once a week for 18 weeks. Ellagic acid produced a significant inhibition in the average number of esophageal tumors at both 20 weeks and 27 weeks. To investigate the mechanism(s) of this inhibition, EA was tested for its effect on the metabolism, DNA-binding and DNA-adduct formation of NBMA in cultured explants of rat esophagus. Explants were incubated in medium containing EA at concentrations of 10, 50, and 100 {mu}M for 16 hours, followed by the addition of 1{mu}M ({sup 3}H)NBMA and EA for 12 hours. Explant DNA was isolated by phenol extraction and hydroxylapatite chromatography, and benzaldehyde formation was determined by h.p.l.c. analysis of the culture medium. Finally, EA was examined for its ability to inhibit DNA damage induced by aflatoxin B{sub 1} (AFB{sub 1}) in cultured explants of rat trachea and esophagus, and human tracheobronchus.

  3. Antifungal Activity of Lactic Acid Bacteria Strains Isolated from Natural Honey against Pathogenic Candida Species

    PubMed Central

    Bulgasem, Bulgasem Y.; Lani, Mohd Nizam; Wan Yusoff, Wan Mohtar; Fnaish, Sumaya G.

    2016-01-01

    The role of lactic acid bacteria (LAB) in honey as antifungal activity has received little attention and their mechanism of inhibitory of fungi is not fully understood. In this study, LAB were isolated from honey samples from Malaysia, Libya, Saudi Arabia, and Yemen. Twenty-five isolates were confirmed LAB by catalase test and Gram staining, and were screened for antifungal activity. Four LAB showed inhibitory activity against Candida spp. using the dual agar overlay method. And they were identified as Lactobacillus plantarum HS isolated from Al-Seder honey, Lactobacillus curvatus HH isolated from Al-Hanon honey, Pediococcus acidilactici HC isolated from Tualang honey and Pediococcus pentosaceus HM isolated from Al-Maray honey by the 16S rDNA sequence. The growth of Candida glabrata ATCC 2001 was strongly inhibited (>15.0 mm) and (10~15 mm) by the isolates of L. curvatus HH and P. pentosaceus HM, respectively. The antifungal activity of the crude supernatant (cell free supernatant, CFS) was evaluated using well diffusion method. The CFS showed high antifungal activity against Candida spp. especially The CFS of L. curvatus HH was significantly (p < 0.05) inhibited growth of C. glabrata ATCC 2001, C. parapsilosis ATCC 2201, and C. tropicalis ATCC 750 with inhibitory zone 22.0, 15.6, and 14.7 mm, respectively. While CFS of P. pentosaceus HM was significantly (p < 0.05) effective against C. krusei, C. glabrata, and C. albicans with inhibition zone 17.2, 16.0, and 13.3 mm, respectively. The results indicated that LAB isolated from honey produced compounds which can be used to inhibit the growth of the pathogenic Candida species. PMID:28154488

  4. Antifungal Activity of Lactic Acid Bacteria Strains Isolated from Natural Honey against Pathogenic Candida Species.

    PubMed

    Bulgasem, Bulgasem Y; Lani, Mohd Nizam; Hassan, Zaiton; Wan Yusoff, Wan Mohtar; Fnaish, Sumaya G

    2016-12-01

    The role of lactic acid bacteria (LAB) in honey as antifungal activity has received little attention and their mechanism of inhibitory of fungi is not fully understood. In this study, LAB were isolated from honey samples from Malaysia, Libya, Saudi Arabia, and Yemen. Twenty-five isolates were confirmed LAB by catalase test and Gram staining, and were screened for antifungal activity. Four LAB showed inhibitory activity against Candida spp. using the dual agar overlay method. And they were identified as Lactobacillus plantarum HS isolated from Al-Seder honey, Lactobacillus curvatus HH isolated from Al-Hanon honey, Pediococcus acidilactici HC isolated from Tualang honey and Pediococcus pentosaceus HM isolated from Al-Maray honey by the 16S rDNA sequence. The growth of Candida glabrata ATCC 2001 was strongly inhibited (>15.0 mm) and (10~15 mm) by the isolates of L. curvatus HH and P. pentosaceus HM, respectively. The antifungal activity of the crude supernatant (cell free supernatant, CFS) was evaluated using well diffusion method. The CFS showed high antifungal activity against Candida spp. especially The CFS of L. curvatus HH was significantly (p < 0.05) inhibited growth of C. glabrata ATCC 2001, C. parapsilosis ATCC 2201, and C. tropicalis ATCC 750 with inhibitory zone 22.0, 15.6, and 14.7 mm, respectively. While CFS of P. pentosaceus HM was significantly (p < 0.05) effective against C. krusei, C. glabrata, and C. albicans with inhibition zone 17.2, 16.0, and 13.3 mm, respectively. The results indicated that LAB isolated from honey produced compounds which can be used to inhibit the growth of the pathogenic Candida species.

  5. Method for the isolation of citric acid and malic acid in Japanese apricot liqueur for carbon stable isotope analysis.

    PubMed

    Akamatsu, Fumikazu; Hashiguchi, Tomokazu; Hisatsune, Yuri; Oe, Takaaki; Kawao, Takafumi; Fujii, Tsutomu

    2017-02-15

    A method for detecting the undeclared addition of acidic ingredients is required to control the authenticity of Japanese apricot liqueur. We developed an analytical procedure that minimizes carbon isotope discrimination for measurement of the δ(13)C values of citric and malic acid isolated from Japanese apricot liqueur. Our results demonstrated that freeze-drying is preferable to nitrogen spray-drying, because it does not significantly affect the δ(13)C values of citric acid and results in smaller isotope discrimination for malic acid. Both 0.1% formic acid and 0.2% phosphoric acid are acceptable HPLC mobile phases for the isolation of citric and malic acid, although the δ(13)C values of malic acid exhibited relatively large variation compared with citric acid following isolation using either mobile phase. The developed procedure allows precise δ(13)C measurements of citric and malic acid isolated from Japanese apricot liqueur.

  6. Epidemiological investigation of Pseudomonas aeruginosa nosocomial bacteraemia isolates by PCR-based DNA fingerprinting analysis.

    PubMed

    Liu, Y; Davin-Regli, A; Bosi, C; Charrel, R N; Bollet, C

    1996-11-01

    Between July 1994 and March 1995, 64 isolates of Pseudomonas aeruginosa were implicated in bacteraemia in 25 cancer patients in five wards of two hospitals. These, together with 24 environmental isolates and one isolate from a bacteraemia in a non-cancer patient were examined by three PCR-based DNA fingerprinting methods: random amplified polymorphic DNA (RAPD), enterobacterial-repetitive intergenic consensus (ERIC)-PCR, and 16S-23S spacer region-based RAPD. These methods were reproducible, discriminatory and showed close agreement; all indicated that 47 isolates that had caused bacteraemia in 19 cancer patients were indistinguishable. Seventeen other isolates that had caused bacteraemia in 10 cancer patients were discriminated into eight further groups, and the 24 environmental and non-cancer patient isolates into further distinct groups. No environmental source of the epidemic strain was found, but it was suspected that the outbreak was related to infusion implants.

  7. Simple method of isolating humic acids from organic soils

    NASA Astrophysics Data System (ADS)

    Ahmed, O.

    2009-04-01

    Humic substances particularly humic acids (HA) play a major role in soil conditioning e.g. erosion control, soil cation exchange capacity, complexation of heavy metal ions and pesticides, carbon and nitrogen cycles, plant growth and reduction of ammonia volatilization from urea. Humified substances such as coal, composts, and peat soils have substantial amounts of HA but the isolation of these acids is expensive, laborious, and time consuming. Factors that affect the quality and yield of HA isolated from these materials include extraction, fractionation, and purification periods. This work developed a simple, rapid, and cost effective method of isolating HA from peat soils. There was a quadratic relationship between extraction period and HA yield. Optimum extraction period was estimated at 4 h instead of the usual range of 12 to 48 h. There was no relationship between fractionation period and HA yield. As such 2 h instead of the usual range of 12 to 24 h fractionation period could be considered optimum. Low ash content (5%), remarkable reduction in K, coupled with the fact that organic C, E4/E6, carboxylic COOH, phenolic OH, and total acidity values of the HA were consistent with those reported by other authors suggest that the HA dealt with were free from mineral matter. This was possible because the distilled water used to purify the HA served as Bronsted-Lowry acid during the purification process of the HA. Optimum purification period using distilled waster was 1 h instead of the usual range of 1 and 7 days (uses HF and HCl and dialysis). Humic acids could be isolated from tropical peat soils within 7 h (i.e. 4 h extraction, 2 h fractionation, and 1 h purification) instead of the existing period of 2 and 7 days. This could facilitate the idea of producing organic fertilizers such as ammonium-humate and potassium-humate from humified substances since techniques devised in this study did not alter the true nature of the HA. Besides, the technique is rapid, simple

  8. Isolation of a sex-linked DNA sequence in cranes.

    PubMed

    Duan, W; Fuerst, P A

    2001-01-01

    A female-specific DNA fragment (CSL-W; crane sex-linked DNA on W chromosome) was cloned from female whooping cranes (Grus americana). From the nucleotide sequence of CSL-W, a set of polymerase chain reaction (PCR) primers was identified which amplify a 227-230 bp female-specific fragment from all existing crane species and some other noncrane species. A duplicated versions of the DNA segment, which is found to have a larger size (231-235 bp) than CSL-W in both sexes, was also identified, and was designated CSL-NW (crane sex-linked DNA on non-W chromosome). The nucleotide similarity between the sequences of CSL-W and CSL-NW from whooping cranes was 86.3%. The CSL primers do not amplify any sequence from mammalian DNA, limiting the potential for contamination from human sources. Using the CSL primers in combination with a quick DNA extraction method allows the noninvasive identification of crane gender in less than 10 h. A test of the methodology was carried out on fully developed body feathers from 18 captive cranes and resulted in 100% successful identification.

  9. Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt.

    PubMed

    Yu, Jin-Ju; Oh, Suk-Heung

    2010-08-01

    Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO(2) production. The isolates grew well on MRS broth at 30-37 degrees C and a pH of 6.5-8.0. The optimum temperature and pH for growth are 37 degrees C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0-6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.

  10. Diversity of bacteriocinogenic lactic acid bacteria isolated from Mediterranean fish viscera.

    PubMed

    Migaw, Sarra; Ghrairi, Taoufik; Belguesmia, Yanath; Choiset, Yvan; Berjeaud, Jean-Marc; Chobert, Jean-Marc; Hani, Khaled; Haertlé, Thomas

    2014-04-01

    Nine lactic acid bacteria strains showing bacteriocin-like activity were isolated from various fresh fish viscera. The following species were identified based on 16S rDNA sequences: Enterococcus durans (7 isolates), Lactococcus lactis (1) and Enterococcus faecium (1). These strains were active against Listeria innocua and other LAB. Random amplified polymorphic DNA analyses showed four major patterns for the E. durans species. PCR analyses revealed a nisin gene in the genome of the Lc. lactis strain. Genes coding enterocins A, B and P were found in the genome of the E. faecium isolate. Enterocins A and B genes were also present in the genome of E. durans GM19. Hence, this is the first report describing E. durans strains producing enterocins A and B. Electrospray ionization mass spectrometry revealed that the purified bacteriocin produced by the E. durans GMT18 strain had an exact molecular mass of 6,316.89 Da. This bacteriocin was designated as durancin GMT18. Edman sequencing failed to proceed; suggesting that durancin GTM18 may contain terminal lanthionine residues. Overall, the results obtained revealed the presence of a variety of enterococci in Mediterranean fish viscera, as evidenced by their genetic profiles and abilities to produce different bacteriocins. These strains could be useful for food biopreservation or as probiotics.

  11. Developing a simple method to process bone samples prior to DNA isolation.

    PubMed

    Li, Richard; Chapman, Sandra; Thompson, Mary; Schwartz, Michal

    2009-03-01

    Bone tissue is often used for recovering DNA samples for the purpose of human identification. However, the initial cleaning and sampling of the bone specimen is a labor-intensive and time-consuming step, which must be completed prior to isolating DNA. Thus, it is difficult to adapt the current method for automation. To address this issue, we have developed a simple processing method using a trypsin treatment prior to DNA isolation. The use of the trypsin-based procedure potentially reduces the amount of labor required by a physical method such as sanding. By incubating samples with the trypsin solution, the soft tissue and outer surface of the bone fragment samples are removed. The processed bone fragment or a portion of the fragment can then be used for DNA isolation.

  12. Isolation of chicken alpha ENaC splice variants from a cochlear cDNA library.

    PubMed

    Killick, R; Richardson, G

    1997-01-03

    Three splice variants of the alpha subunit of the amiloride-sensitive epithelial sodium channel (alpha ENaC) have been isolated from a chicken cochlear cDNA library. A PCR product, generated from the cochlear library using degenerate primers to regions of homology between the rat alpha ENaC and the degenerin Mec-4, was used as a probe. The three splice variant cDNAs with sizes of 2321, 3399 and 3845 bp correspond to transcripts of 2.5, 3.5 and 3.9 kb as detected by Northern blot analysis. The 3399 bp clone differs from the 2321 clone solely by the addition of 1079 bases in the 3'-non-coding region. Both these cDNAs code for an identical predicted protein of 637 amino acids which has 68% similarity to the rat alpha ENaC, and is probably the chicken homologue of alpha ENaC. The third cDNA of 3845 bp is similar to the 3399 bp clone but includes two exons within the open reading frame. The first of these exons introduces a premature stop codon resulting in a truncated predicted protein of 434 amino acids. Northern blot analysis shows expression of the 2.5 and 3.5 kb transcripts in cochlea and colon, the 2.5 kb transcript in cartilage, whilst the 3.9 kb transcript is only detected in cochlea. No expression is detected in brain, liver, and heart nor, most notably, in lung or kidney.

  13. Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo L-(+)-lactic acid.

    PubMed

    Abdel-Rahman, Mohamed Ali; Tashiro, Yukihiro; Zendo, Takeshi; Shibata, Keisuke; Sonomoto, Kenji

    2011-02-01

    Effective utilisation of cellulosic biomasses for economical lactic acid production requires a microorganism with potential ability to utilise efficiently its major components, glucose and cellobiose. Amongst 631 strains isolated from different environmental samples, strain QU 25 produced high yields of l-(+)-lactic acid of high optical purity from cellobiose. The QU 25 strain was identified as Enterococcus mundtii based on its sugar fermentation pattern and 16S rDNA sequence. The production of lactate by fermentation was optimised for the E. mundtii QU25 strain. The optimal pH and temperature for batch culturing were found to be 7.0°C and 43°C, respectively. E. mundtii QU 25 was able to metabolise a mixture of glucose and cellobiose simultaneously without apparent carbon catabolite repression. Moreover, under the optimised culture conditions, production of optically pure l-lactic acid (99.9%) increased with increasing cellobiose concentrations. This indicates that E. mundtii QU 25 is a potential candidate for effective lactic acid production from cellulosic hydrolysate materials.

  14. Comparison of DNA fingerprint patterns of isolates of Mycobacterium africanum from east and west Africa.

    PubMed Central

    Haas, W H; Bretzel, G; Amthor, B; Schilke, K; Krommes, G; Rüsch-Gerdes, S; Sticht-Groh, V; Bremer, H J

    1997-01-01

    Mycobacterium africanum is a pathogen found in tuberculosis patients in certain parts of Africa and is a member of the Mycobacterium tuberculosis complex. Biochemically, strains of M. africanum exhibit a high degree of variability, with some tendency to cluster according to their geographical origin. To investigate whether this phenotypic variability is reflected at the genetic level, we performed DNA fingerprint analysis of strains isolated from patients with pulmonary tuberculosis in Uganda and Sierra Leone. IS6110 DNA fingerprinting was carried out by the mixed-linker PCR method. A total of 138 strains of M. africanum were analyzed: 42 isolates from Uganda and 96 isolates from Sierra Leone. With few exceptions, the resulting DNA fingerprint patterns grouped together according to their country of origin. A striking lack of variability of DNA fingerprints was found for strains from Sierra Leone, where 70 of 96 isolates (61.5%) fell into clusters. The two largest clusters accounted for 41.7% of all isolates and differed by only one band, as confirmed by standard DNA fingerprinting. In contrast, only two clusters (7.1%) with two and three isolates, respectively, were found for M. africanum isolates collected in Uganda, and three of the DNA fingerprints contained fewer than seven bands. Strains of M. tuberculosis collected and processed during the same time period were highly variable in both countries. Our results support the concept of geographically defined subtypes of M. africanum. In addition, they demonstrate that natural geographic differences in the variability of IS6110 DNA fingerprints within the M. tuberculosis complex must be considered if this technique is used for epidemiologic studies. PMID:9041408

  15. Isolation and identification of fatty acid amides from Shengli coal

    SciTech Connect

    Ming-Jie Ding; Zhi-Min Zong; Ying Zong; Xiao-Dong Ou-Yang; Yao-Guo Huang; Lei Zhou; Feng Wang; Jiang-Pei Cao; Xian-Yong Wei

    2008-07-15

    Shengli coal, a Chinese brown coal, was extracted with carbon disulfide and the extract was gradiently eluted with n-hexane and ethyl acetate (EA)/n-hexane mixed solvents with different concentrations of EA in a silica gel-filled column. A series of fatty acid amides, including fourteen alkanamides (C{sub 15}-C{sub 28}) and three alkenamides (C{sub 18} and C{sub 22}), were isolated from the coal by this method and analyzed with a gas chromatography/mass spectrometry. 26 refs., 2 figs., 2 tabs.

  16. An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

    PubMed

    Ghosh, Raju; Paul, Sujay; Ghosh, Subrata Kumar; Roy, Anirban

    2009-07-01

    A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

  17. Interaction of photosensitive surfactant with DNA and poly acrylic acid

    SciTech Connect

    Zakrevskyy, Yuriy Paasche, Jens; Lomadze, Nino; Santer, Svetlana; Cywinski, Piotr; Cywinska, Magdalena; Reich, Oliver; Löhmannsröben, Hans-Gerd

    2014-01-28

    In this paper, we investigate interactions and phase transitions in polyelectrolyte-surfactant complexes formed between a cationic azobenzene-containing surfactant and two types of polyelectrolytes: natural (DNA) or synthetic (PAA: poly acrylic acid). The construction of a phase diagram allowed distancing between four major phases: extended coil conformation, colloidally stable compacted globules, colloidal instability range, and surfactant-stabilized compact state. Investigation on the complexes’ properties in different phases and under irradiation with UV light provides information about the role of the surfactant's hydrophobic trans isomers both in the formation and destruction of DNA and PAA globules as well as in their colloidal stabilization. The trans isomer shows much stronger affinity to the polyelectrolytes than the hydrophilic cis counterpart. There is no need for complete compensation of the polyelectrolyte charges to reach the complete compaction. On contrary to the findings previously reported in the literature, we demonstrate – for the first time – complete polyelectrolyte compaction which occurs already at 20% of DNA (and at 50% of PAA) charge compensation. The trans isomer plays the main role in the compaction. The aggregation between azobenzene units in the photosensitive surfactant is a driving force of this process. The decompaction can be realized during UV light irradiation and is strongly influenced by the interplay between surfactant-surfactant and surfactant-DNA interactions in the compacted globules.

  18. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.

    PubMed

    Mittra, Indraneel; Khare, Naveen Kumar; Raghuram, Gorantla Venkata; Chaubal, Rohan; Khambatti, Fatema; Gupta, Deepika; Gaikwad, Ashwini; Prasannan, Preeti; Singh, Akshita; Iyer, Aishwarya; Singh, Ankita; Upadhyay, Pawan; Nair, Naveen Kumar; Mishra, Pradyumna Kumar; Dutt, Amit

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  19. Amino Acid Composition of an Organic Brown Rice Protein Concentrate and Isolate Compared to Soy and Whey Concentrates and Isolates.

    PubMed

    Kalman, Douglas S

    2014-06-30

    A protein concentrate (Oryzatein-80™) and a protein isolate (Oryzatein-90™) from organic whole-grain brown rice were analyzed for their amino acid composition. Two samples from different batches of Oryzatein-90™ and one sample of Oryzatein-80™ were provided by Axiom Foods (Los Angeles, CA, USA). Preparation and analysis was carried out by Covance Laboratories (Madison, WI, USA). After hydrolysis in 6-N hydrochloric acid for 24 h at approximately 110 °C and further chemical stabilization, samples were analyzed by HPLC after pre-injection derivitization. Total amino acid content of both the isolate and the concentrate was approximately 78% by weight with 36% essential amino acids and 18% branched-chain amino acids. These results are similar to the profiles of raw and cooked brown rice except in the case of glutamic acid which was 3% lower in the isolate and concentrate. The amino acid content and profile of the Oryzatein-90™ isolate was similar to published values for soy protein isolate but the total, essential, and branched-chain amino acid content of whey protein isolate was 20%, 39% and 33% greater, respectively, than that of Oryzatein-90™. These results provide a valuable addition to the nutrient database of protein isolates and concentrates from cereal grains.

  20. Comparative study of Microsporum canis isolates by DNA fingerprinting.

    PubMed

    Shafiee, Shabnam; Khosravi, Ali Reza; Ashrafi Tamai, Iradj

    2014-08-01

    Microsporum canis is a zoophilic fungus and it is an important agent of dermatophytosis. Cats act as important reservoirs. Clinically, it is too difficult to differentiate dermatophytosis caused by various species, also this fungus loses its morphological characteristics easily because of subculture; so using of rapid and accurate laboratory techniques for identifying the dermatophytes is important, therefore, RAPD-PCR was applied for the differentiation of the isolates. In this study, 10 M. canis isolates were detected in cats, dog, human, fox and rabbit at the Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran. For running the RAPD-PCR, PCR set system and three random primers OPU 15, OPU 13 and OPA 04 were used. Then phylogenetic tree and similarity coefficient table were drawn. The results showed that there were some common bands between M. canis isolates. There were some specific bands for each isolates, as well. Our study showed, despite the typical morphology of the whole isolates, they were placed in different branches in molecular typing.

  1. High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes.

    PubMed

    Igoudjil, Anissa; Massart, Julie; Begriche, Karima; Descatoire, Véronique; Robin, Marie-Anne; Fromenty, Bernard

    2008-06-01

    The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c

  2. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates

    PubMed Central

    Nitschke, Heike; Slickers, Peter; Müller, Elke; Ehricht, Ralf

    2014-01-01

    Streptococcus agalactiae frequently colonizes the urogenital tract, and it is a major cause of bacterial septicemia, meningitis, and pneumonia in newborns. For typing purposes, a microarray targeting group B streptococcus (GBS) virulence-associated markers and resistance genes was designed and validated with reference strains, as well as clinical and veterinary isolates. Selected isolates were also subjected to multilocus sequence typing. It was observed that putative typing markers, such as alleles of the alpha-like protein or capsule types, vary independently of each other, and they also vary independently from the affiliation to their multilocus sequence typing (MLST)-defined sequence types. Thus, it is not possible to assign isolates to sequence types based on the identification of a single distinct marker, such as a capsule type or alp allele. This suggests the occurrence of frequent genomic recombination. For array-based typing, a set of 11 markers (bac, alp, pil1 locus, pepS8, fbsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2, and rgfC/A/D/B) was defined that provides a framework for splitting the tested 448 S. agalactiae isolates into 76 strains that clustered mainly according to MLST-defined clonal complexes. There was evidence for region- and host-specific differences in the population structure of S. agalactiae, as well as an overrepresentation of strains related to sequence type 17 among the invasive isolates. The arrays and typing scheme described here proved to be a convenient tool for genotyping large numbers of clinical/veterinary isolates and thus might help obtain insight into the epidemiology of S. agalactiae. PMID:25165085

  3. Development of a highly efficient expression cDNA cloning system: application to oncogene isolation.

    PubMed Central

    Miki, T; Fleming, T P; Crescenzi, M; Molloy, C J; Blam, S B; Reynolds, S H; Aaronson, S A

    1991-01-01

    We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest. Images PMID:2052597

  4. Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

    PubMed

    Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

    2014-05-01

    The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

  5. DNA isolation protocol for the medicinal plant lemon balm (Melissa officinalis, Lamiaceae).

    PubMed

    Ghaffariyan, S; Mohammadi, S A; Aharizad, S

    2012-04-27

    Lemon balm (Melissa officinalis) is a medicinal plant that is widely used as a sedative or calmant, spasmolytic and antibacterial agent and sleep aid. This has led to a high demand for lemon balm products, resulting in the extinction of this species in some of its natural habitats. Molecular techniques have increasingly been used in plant diversity conservation and isolation of PCR amplifiable genomic DNA is an important pre-requisite. Lemon balm contains high levels of polyphenols and polysaccharides, which pose a major challenge for the isolation of high-quality DNA. We compared different genomic DNA extraction protocols, including traditional phenol-chloroform DNA extraction protocols and two commercial kits for DNA purification for their ability to produce good-quality DNA from fresh leaves of five lemon balm genotypes. Quality and quantity of the DNA samples were determined using 0.8% agarose gel electrophoresis and a spectrophotometer. The DNA purity was further confirmed by PCR amplification using barley retrotransposon LTR base primers. The spectral quality of DNA as measured by the A(260)/A(280) ratio ranged from 1.46 to 2.37. The Fermentase genomic DNA purification kit and the CTAB extraction protocol using PVP and ammonium acetate to overcome the high levels of polyphenols and polysaccharides yielded high-quality DNA with a mean A(260)/A(280) ratio of 1.87. The quantity of DNA and its PCR purity were similar with all the protocols, but considering the time and cost required for extraction of DNA from a large number of samples, the CTAB protocol using PVP and ammonium acetate is suitable for lemon balm.

  6. Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB.

    PubMed

    Springer, Nathan M

    2010-11-01

    A general difficulty in isolation of DNA from plant cells is the presence of a cell wall. It is necessary to degrade plant cell walls, either physically or enzymatically, in order to effectively isolate plant DNA. Additionally, some tissues (such as endosperm) or some species contain high levels of starches or phenolic compounds that can complicate DNA isolation. A number of plant DNA isolation protocols are designed to overcome species-specific difficulties. This is a relatively simple protocol that uses an extraction buffer containing cetyltrimethylammonium bromide (CTAB); it can be used for many plant species. It provides a substantial amount of high-quality DNA that is suitable for polymerase chain reaction (PCR) procedures and is stable for long periods of time. The cost per sample is very low. In addition, this protocol is relatively robust and can be performed by individuals who have had relatively little training. A typical undergraduate student can perform ~200-300 isolations in a day using this protocol. The disadvantages are that it requires a freeze-dryer and a mill or paint-shaker-like device and that it utilizes an organic extraction step, requiring the use of a fume hood.

  7. A rapid method for isolation of total DNA from pathogenic filamentous plant fungi.

    PubMed

    González-Mendoza, D; Argumedo-Delira, R; Morales-Trejo, A; Pulido-Herrera, A; Cervantes-Díaz, L; Grimaldo-Juarez, O; Alarcón, A

    2010-02-02

    DNA isolation from some fungal organisms of agronomic importance is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. We have developed a fast DNA isolation protocol for Fusarium oxysporum, which causes fusarium wilt disease in more than 100 plant species, and for Pyrenochaeta terrestris, which causes pink root in onions. This protocol was based on the sodium dodecyl sulfate/phenol method, without beta-mercaptoethanol and without maceration in liquid nitrogen; it uses phenol/chloroform extraction to remove proteins and co-precipitated polysaccharides. The A(260/280) absorbance ratios of isolated DNA were around 1.9, suggesting that the DNA fraction was pure and may be used for further analysis. Additionally, the A(260/230) values were higher than 1.8, suggesting negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction.

  8. Production of conjugated linoleic acids by Lactobacillus plantarum strains isolated from naturally fermented Chinese pickles*

    PubMed Central

    Liu, Pei; Shen, Sheng-rong; Ruan, Hui; Zhou, Qian; Ma, Liu-liu; He, Guo-qing

    2011-01-01

    Naturally fermented pickles harbour many lactic acid bacteria (LAB). Forty-three LAB strains with conjugated linoleic acid (CLA)-producing ability were isolated from three naturally fermented pickle brines. Of these isolates, lp15 identified as Lactobacillus plantarum by API 50 CHL system and full-length 16S rDNA sequence analysis exhibited the highest CLA-producing ability (26.1% conversion) at 48 h in de Man Rogosa Sharpe (MRS) broth in the presence of 100 µg/ml of linoleic acid (LA). Compared to other strains, L. plantarum strain lp15 showed the highest tolerance upon increased levels of LA in the medium, i.e., up to 600 µg/ml. This strain converted about 25% of LA into CLA isomers [predominantly cis-9, trans-11 CLA (9-CLA) and trans-10, cis-12 CLA (10-CLA)], of which 75% was 9-CLA. Interestingly, though the conversion rate of LA into CLA by lp15 remained stable between 100 to 600 µg/ml LA levels in the medium, it dropped sharply at 1000 µg/ml. Taken together, the lp15 strain displayed relatively high LA tolerance with higher conversion rate, which implies that this strain is a valuable candidate for enhancing the CLA content in food-sources like pickles. PMID:22042657

  9. Isolation and characterization of Aurantiochytrium species: high docosahexaenoic acid (DHA) production by the newly isolated microalga, Aurantiochytrium sp. SD116.

    PubMed

    Gao, Mang; Song, Xiaojin; Feng, Yingang; Li, Wenli; Cui, Qiu

    2013-01-01

    A heterotrophic microalga, strain SD116, with the ability to produce high concentrations of docosahexaenoic acid (DHA, C22:6n-3) was isolated from Shuidong Bay, Guangdong Province, China. Nucleotide sequence analysis of the 18S rDNA of SD116 showed that the strain has a close phylogenetic relationship to Aurantiochytrium species. The highest rates for growth and DHA accumulation for SD116 were obtained in 6.0% glucose, 2.0% yeast extract, and 50% artificial seawater (ASW) at a pH of 7 at 28°C. The maximum total lipid content reached 56.3% of the dry cell weight (DCW), and the maximum DHA content accounted for 50.9% of the total fatty acid (TFA) content. It was further found that urea may be a potential nitrogen source for industrial fermentation because of its cheap price and ability to induce a relatively high biomass and lipid production capacity. Using 5 L fermenters, the DCW, total lipid content, and DHA yield were found to be 70.43 g L(-1), 71.09% of the DCW, and 17.42 g L(-1) (34.79% of the TFA), respectively. The results show that Aurantiochytrium sp. SD116 is a promising candidate for commercial DHA production and could be useful for the synthesis of biomass-related products.

  10. Isolation and Identification of Lactic Acid Bacteria Isolated from a Traditional Jeotgal Product in Korea

    NASA Astrophysics Data System (ADS)

    Cho, Gyu Sung; Do, Hyung Ki

    2006-06-01

    Seventeen lactic acid bacterial strains (LAB) were isolated using MRS agar medium from Jeotgal, a Korean fermented food, purchased at the Jukdo market of Pohang. To identify the strains isolated, they were tested by examining their cell morphologies, gram-staining, catalase activity, arginine hydrolase activity, D-L lactate form and carbohydrate fermentation. According to the phenotypic characteristics, three strains were tent atively identified as Lactobacillus spp., ten were Enterococcus spp. (or Streptococcus spp., or Pediococcus spp.) and the rest were Leuconostoc spp. (or Weissella spp.). Five strains among 17 were chosen by preliminary bacteriocin activity test. Four bacterial strains which inhibited both indicator microorganisms were identified by 16S rRNA sequencing. The results are as follows; Leuconostoc mesenteroides (HK 4), Leuconostoc mesenteroides (HK 5), Leuconostoc mesenteroides(HK 11), Streptococcus salivarius(HK 8). In order to check LAB which are showing a high survival rate in gut, we investigated three strains inhibiting both indicator microorganisms in artificial gastric acid and bile juice -all except HK8. The three strains mentioned above grew in extreme low acid conditions.

  11. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    PubMed

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

  12. Amino acid substitutions in gyrA and parC associated with quinolone resistance in nalidixic acid-resistant Salmonella isolates

    PubMed Central

    2013-01-01

    This study was undertaken to identify and characterize amino acid substitutions in gyrA and parC related with quinolone resistance of 27 nalidixic acid-resistant (NaR) Salmonella isolates collected in poultry slaughterhouses in Korea. A total of 51 Salmonella isolates were detected from 44.8% (47/105) of the total samples from 15 poultry slaughterhouses examined, among which 27 (52.9%) NaR isolates were detected while ciprofloxacin (Cip) resistance was not present in the isolates. These 27 NaR isolates of DNA sequencing revealed that it contained three types of gyrA mutations in only D87 codon. Mutations in the D87 codon resulted in substitutions to G in most of the isolates, but D87Y and D87N exchanges were also detected. Although Cip resistance was absent, reduced susceptibility characterized by mutations in gyrA was apparent among Salmonella isolates from poultry slaughterhouses in Korea. PMID:24237626

  13. Fatty Acid-Binding Protein in Small Intestine IDENTIFICATION, ISOLATION, AND EVIDENCE FOR ITS ROLE IN CELLULAR FATTY ACID TRANSPORT

    PubMed Central

    Ockner, Robert K.; Manning, Joan A.

    1974-01-01

    A soluble fatty acid-binding protein (FABP), mol wt ∼ 12,000 is present in intestinal mucosa and other tissues that utilize fatty acids, including liver, myocardium, adipose, and kidney. This protein binds long chain fatty acids both in vivo and in vitro. FABP was isolated from rat intestine by gel filtration and isoelectric focusing. It showed a reaction of complete immunochemical identity with proteins in the 12,000 mol wt fatty acid-binding fractions of liver, myocardium, and adipose tissue supernates. (The presence of immunochemically nonidentical 12,000 mol wt FABP in these tissues is not excluded.) By quantitative radial immunodiffusion, supernatant FABP concentration in mucosa from proximal and middle thirds of jejuno-ileum significantly exceeded that in distal third, duodenum, and liver, expressed as micrograms per milligram soluble protein, micrograms per gram DNA, and micrograms per gram tissue. FABP concentration in villi was approximately three times greater than in crypts. Small quantities of FABP were present in washed nuclei-cell membrane, mitochondrial and microsomal fractions. However, the amount of FABP solubilized per milligram membrane protein was similar for all particulate fractions, and total membrane-associated FABP was only about 16% of supernatant FABP. Intestinal FABP concentration was significantly greater in animals maintained on high fat diets than on low fat; saturated and unsaturated fat diets did not differ greatly in this regard. The preponderance of FABP in villi from proximal and middle intestine, its ability to bind fatty acids in vivo as well as in vitro, and its response to changes in dietary fat intake support the concept that this protein participates in cellular fatty acid transport during fat absorption. Identical or closely related 12,000 mol wt proteins may serve similar functions in other tissues. Images PMID:4211161

  14. Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food.

    PubMed

    El-Shafei, H A; Abd-El-Sabour, H; Ibrahim, N; Mostafa, Y A

    2000-03-01

    100 lactic acid bacterial strains isolated from traditional fermented foods (yoghurt, milk cream, sour dough and milk) were screened for bacteriocin production. Twenty six strains producing a nisin-like bacteriocin were selected. Most of these isolates gave only a narrow inhibitory spectrum, although one showed a broad inhibitory spectrum against the indicator strains tested, this strain was determined as Lactococcus lactis. The influence of several parameters on the fermentative production of nisin by Lactococcus lactis was studied. Production of nisin was optimal at 30 degrees C and in the pH range 5.5-6.3. The effect of different sulphur and nitrogen sources on Lactococcus lactis growth and nisin production was studied. Magnesium sulfate and manganese sulfate were found to be the best sulphur sources while triammonium citrate was the best inorganic nitrogen source and meat extract, peptone and yeast extract were the best organic nitrogen source for nisin production.

  15. DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

    PubMed

    Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

    2014-01-01

    Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

  16. Variation in Ribosomal DNA among Isolates of the Mycorrhizal Fungus Cenococcum Geophilum FR.

    NASA Astrophysics Data System (ADS)

    Lobuglio, Katherine Frances

    1990-01-01

    Cenococcum geophilum Fr., a cosmopolitan mycorrhizal fungus, is well-known for its extremely wide host and habitat range. The ecological diversity of C. geophilum sharply contrasts its present taxonomic status as a monotypic form -genus. Restriction fragment length polymorphisms (RFLPs) in nuclear ribosomal DNA (rDNA) was used to assess the degree of genetic variation among 72 isolates of C. geophilum. The probe used in this study was the rDNA repeat cloned from C. geophilum isolate A145 (pCG15). Length of the rDNA repeat was approximately 9 kb. The rDNA clone was mapped for 5 restriction endonucleases. Hybridization with cloned Saccharomyces cerevisiae rDNA (pSR118, and pSR125 containing the 18S, and 5.8-25S rRNA genes respectively), and alignment of restriction endonuclease sites conserved in the rDNA genes of other fungi, were used to position the corresponding rDNAs of C. geophilum. Southern hybridizations with EcoRI, HindIII, XhoI, and PstI digested DNAs indicated extensive variation among the C. geophilum isolates, greater than has been previously reported to occur within a fungal species. Most of the rDNA polymorphisms occurred in the IGS region. Restriction endonuclease site and length polymorphisms were also observed in the 5.8S-26S genic regions. Sixteen size categories of length mutations, 6 restriction endonuclease site additions, and 4 restriction endonuclease site deletions were determined using isolate A145 as a reference. The rDNA repeat length among the isolates varied from approximately 8.5 to 10.2 kb. RFLPs were also observed in the mitochondrial (mt) 24S rRNA gene and flanking regions of HindIII digested DNAs of C. geophilum isolates representing both geographically distinct and similar origins. Among the C. geophilum isolates analyzed there were fewer RFLPs in mt-DNA than in nuclear rDNA. EcoRI rDNA phenotypes between C. geophilum and Elaphomyces anthracinus, its proposed teleomorph or sexual state, did not correspond. In addition, the four

  17. Bioaugmentation of bromoamine acid degradation with Sphingomonas xenophaga QYY and DNA fingerprint analysis of augmented systems.

    PubMed

    Qu, Yuanyuan; Zhou, Jiti; Wang, Jing; Song, Zhiyong; Xing, Linlin; Fu, Xiang

    2006-02-01

    One high-effective bromoamine acid (1-amino-4-bromoanthraquinone-2-sulfonic acid, BAA) degrading strain was isolated previously with the ability to use BAA as sole source of carbon and nitrogen. It was identified as Sphingomonas xenophaga QYY by 16S rDNA sequence analysis and physio-biochemical tests. In this study, bioaugmentation of BAA degradation with suspended and immobilized cells of strain QYY was investigated. The optimal degradation conditions were as follows: temperature 30 degrees C, pH 6.0-7.0, 150 rev min(-1) and the immobilized cells maintained degradation activity to BAA after 60 days storage at 4 degrees C. The structure of BAA was evidently changed according to the analysis of total organic carbon removal of BAA (about 50%) and the UV-VIS spectra changes during the biodegradation. Bioaugmented systems exhibited stronger abilities degrading BAA than the non-bioaugmented control ones. And microbial community dynamics of augmented systems was revealed by amplified ribosomal DNA restriction analysis (ARDRA), a modern DNA fingerprint technique. The results indicated that the microbial community dynamics was substantially changed throughout the augmentation process. This study suggests that it is feasible and potentially useful to enhance BAA degradation using bioaugmentation with the immobilized cells of BAA-degrading bacterium.

  18. Isolation and characterization of a cDNA clone encoding wheat germ agglutinin

    SciTech Connect

    Raikhel, N.V.; Wilkins, T.A.

    1987-10-01

    Two sets of synthetic oligonucleotides coding for amino acids in the amino- and carboxyl-terminal portions of wheat germ agglutinin were synthesized and used as hybridization probes to screen cDNA libraries derived from developing embryos of tetraploid wheat. The nucleotide sequence for a cDNA clone recovered from the cDNA library was determined by dideoxynucleotide chain-termination sequencing in vector M13. The amino acid sequence deduced from the DNA sequence indicated that this cDNA clone (pNVR1) encodes isolectin 3 of wheat germ agglutinin. Comparison of the deduced amino acid sequence of clone pNVR1 with published sequences indicates isolectin 3 differs from isolectins 1 and 2 by 10 and 8 amino acid changes, respectively. In addition, the protein encoded by pNVR1 extends 15 amino acids beyond the carboxyl terminus of the published amino acid sequence for isolectins 1 and 2 and includes a potential site for N-linked glycosylation. Utilizing the insert of pNVR1 as a hybridization probe, the authors have demonstrated that the expression of genes for wheat germ agglutinin is modulated by exogenous abscisic acid. Striking homology is observed between wheat germ agglutinin and chitinase, both of which are proteins that bind chitin.

  19. Decarboxylation of Substituted Cinnamic Acids by Lactic Acid Bacteria Isolated during Malt Whisky Fermentation

    PubMed Central

    van Beek, Sylvie; Priest, Fergus G.

    2000-01-01

    Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, and L. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation. PMID:11097909

  20. Isolation and characterization of a full length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) gene

    SciTech Connect

    Oyake, M.; Onodera, O.; Ikeuchi, T.

    1994-09-01

    Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant spinocerebellar degeneration characterized by anticipation and variable combination of symptoms including myoclonus, epilepsy, cerebellar ataxia, choleoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a B37 gene on chromosome 12. To characterize functions of the DRPLA gene product, we isolated several cDNA clones for the DRPLA gene from human adult and fetus brain cDNA libraries, using an oligonucleotide flanking the CAG repeat. The cDNA spans 4247 bp in length and there is only an open reading frame coding for 986 amino acids. The CAG repeat, which is expanded in DRPLA, is located 291 bp downstream from the initiation methionine and encodes a polyglutamine tract. The deduced amino acid sequence from amino acids residues 582 to 707 has a high homology to published human hippocampus derived expressed sequence (M78755) located at chromosome 1p (63.8% identity), and 3{prime}-untranslated region of the DRPLA cDNA revealed homology to the mouse small nuclear RNA U7 gene (X54165). Northern blot analysis revealed a 4.7 knt transcript which is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. In human adult brain, the transcript was broadly expressed including amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus and thalamus, and was not specific to the dentatorubral-pallidoluysian system. The availability of a full length cDNA will be highly useful for analyzing the pathogenesis of this unique neurodegenerative disease as well as for analyzing other CAG repeat related neurodegenerative diseases.

  1. Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.).

    PubMed

    Valárik, M; Simková, H; Hribová, E; Safár, J; Dolezelová, M; Dolezel, J

    2002-01-01

    Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radkal and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in

  2. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    PubMed

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process.

  3. Identification of Clinical Isolates of Actinomyces Species by Amplified 16S Ribosomal DNA Restriction Analysis

    PubMed Central

    Hall, Val; Talbot, P. R.; Stubbs, S. L.; Duerden, B. I.

    2001-01-01

    Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA), using enzymes HaeIII and HpaII, was applied to 176 fresh and 299 stored clinical isolates of putative Actinomyces spp. referred to the Anaerobe Reference Unit of the Public Health Laboratory Service for confirmation of identity. Results were compared with ARDRA results obtained previously for reference strains and with conventional phenotypic reactions. Identities of some strains were confirmed by analysis of partial 16S rDNA sequences. Of the 475 isolates, 331 (70%) were clearly assigned to recognized Actinomyces species, including 94 isolates assigned to six recently described species. A further 52 isolates in 12 ARDRA profiles were designated as apparently resembling recognized species, and 44 isolates, in 18 novel profiles, were confirmed as members of genera other than Actinomyces. The identities of 48 isolates in nine profiles remain uncertain, and they may represent novel species of Actinomyces. For the majority of species, phenotypic results, published reactions for the species, and ARDRA profiles concurred. However, of 113 stored isolates originally identified as A. meyeri or resembling A. meyeri by phenotypic tests, only 21 were confirmed as A. meyeri by ARDRA; 63 were reassigned as A. turicensis, 7 as other recognized species, and 22 as unidentified actinomycetes. Analyses of incidence and clinical associations of Actinomyces spp. add to the currently sparse knowledge of some recently described species. PMID:11574572

  4. Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column.

    PubMed Central

    Park, E M; Shigenaga, M K; Degan, P; Korn, T S; Kitzler, J W; Wehr, C M; Kolachana, P; Ames, B N

    1992-01-01

    An immunoaffinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures of Escherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG;8-hydroxy-2'-deoxyguanosine), a bio-marker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8-dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 10(5) oxidative lesions to DNA being formed and excised in an average rat cell per day. PMID:1565629

  5. Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen.

    PubMed

    Yun, Byeong Hwa; Yao, Lihua; Jelaković, Bojan; Nikolić, Jovan; Dickman, Kathleen G; Grollman, Arthur P; Rosenquist, Thomas A; Turesky, Robert J

    2014-09-01

    DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected.

  6. Identification and Antimicrobial Activity Detection of Lactic Acid Bacteria Isolated from Corn Stover Silage

    PubMed Central

    Li, Dongxia; Ni, Kuikui; Pang, Huili; Wang, Yanping; Cai, Yimin; Jin, Qingsheng

    2015-01-01

    A total of 59 lactic acid bacteria (LAB) strains were isolated from corn stover silage. According to phenotypic and chemotaxonomic characteristics, 16S ribosomal DNA (rDNA) sequences and recA gene polymerase chain reaction amplification, these LAB isolates were identified as five species: Lactobacillus (L.) plantarum subsp. plantarum, Pediococcus pentosaceus, Enterococcus mundtii, Weissella cibaria and Leuconostoc pseudomesenteroides, respectively. Those strains were also screened for antimicrobial activity using a dual-culture agar plate assay. Based on excluding the effects of organic acids and hydrogen peroxide, two L. plantarum subsp. plantarum strains ZZU 203 and 204, which strongly inhibited Salmonella enterica ATCC 43971T, Micrococcus luteus ATCC 4698T and Escherichia coli ATCC 11775T were selected for further research on sensitivity of the antimicrobial substance to heat, pH and protease. Cell-free culture supernatants of the two strains exhibited strong heat stability (60 min at 100°C), but the antimicrobial activity was eliminated after treatment at 121°C for 15 min. The antimicrobial substance remained active under acidic condition (pH 2.0 to 6.0), but became inactive under neutral and alkaline condition (pH 7.0 to 9.0). In addition, the antimicrobial activities of these two strains decreased remarkably after digestion by protease K. These results preliminarily suggest that the desirable antimicrobial activity of strains ZZU 203 and 204 is the result of the production of a bacteriocin-like substance, and these two strains with antimicrobial activity could be used as silage additives to inhibit proliferation of unwanted microorganism during ensiling and preserve nutrients of silage. The nature of the antimicrobial substances is being investigated in our laboratory. PMID:25924957

  7. A DNA origami nanorobot controlled by nucleic acid hybridization.

    PubMed

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

    2014-07-23

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

  8. Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

    PubMed Central

    Munk, Patrick; Lukjancenko, Oksana; Priemé, Anders; Aarestrup, Frank M.

    2016-01-01

    ABSTRACT Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial resistance from different reservoirs. Here, we compare in a stepwise approach a total of eight commercially available DNA extraction kits and 16 procedures based on these for three specimen types (human feces, pig feces, and hospital sewage). We assess DNA extraction using spike-in controls and different types of beads for bead beating, facilitating cell lysis. We evaluate DNA concentration, purity, and stability and microbial community composition using 16S rRNA gene sequencing and for selected samples using shotgun metagenomic sequencing. Our results suggest that inferred community composition was dependent on inherent specimen properties as well as DNA extraction method. We further show that bead beating or enzymatic treatment can increase the extraction of DNA from Gram-positive bacteria. Final DNA quantities could be increased by isolating DNA from a larger volume of cell lysate than that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10.6084/m9.figshare.3475406. IMPORTANCE Sequencing-based analyses of microbiomes may lead to a breakthrough in our understanding of the microbial worlds associated with

  9. Double proton transfer in the isolated and DNA-embedded guanine-cytosine base pair

    NASA Astrophysics Data System (ADS)

    Zoete, Vincent; Meuwly, Markus

    2004-09-01

    The energetics and dynamics of double proton transfer (DPT) is investigated theoretically for the Watson-Crick conformation of the guanine-cytosine (GC) base pair. Using semiempirical density functional theory the isolated and DNA-embedded GC pair is considered. Differences in the energetics and dynamics of DPT thus addresses the question of how relevant studies of isolated base pairs are for the understanding of processes occurring in DNA. Two-dimensional potential energy surfaces involving the transferring hydrogen atoms and the proton donors and acceptors are presented for both systems. The DPT reaction is accompanied by a contraction of the distance between the two bases with virtually identical energetic barriers being 18.8 and 18.7 kcal/mol for the isolated and DNA-embedded system, respectively. However, the transition state for DPT in the DNA-embedded GC pair is offset by 0.1 Å to larger N-H separation compared to the isolated GC pair. Using activated ab initio molecular dynamics, DPT is readily observed for the isolated base pair with a minimal amount of 21.4 kcal/mol of initial average kinetic energy along the DPT normal mode vector. On a time scale of ≈100 fs DPT has occurred and the excess energy is redistributed. For the DNA-embedded GC pair considerably more kinetic energy is required (30.0 kcal/mol) for DPT and the process is completed within one hydrogen vibration. The relevance of studies of isolated base pairs and base pair analogs in regard of reactions or properties involving DNA is discussed.

  10. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  11. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation.

    PubMed

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-08-11

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice.

  12. Amycolatopsis thailandensis sp. nov., a poly(L-lactic acid)-degrading actinomycete, isolated from soil.

    PubMed

    Chomchoei, Atchareeya; Pathom-Aree, Wasu; Yokota, Akira; Kanongnuch, Chartchai; Lumyong, Saisamorn

    2011-04-01

    A novel actinomycete that was capable of degrading poly(l-lactic acid), strain CMU-PLA07(T), was isolated from soil in northern Thailand. Strain CMU-PLA07(T) had biochemical, chemotaxonomic, morphological and physiological properties that were consistent with its classification in the genus Amycolatopsis. 16S rRNA gene sequence analysis showed that the isolate formed a phyletic line within the genus Amycolatopsis. Strain CMU-PLA07(T) was most similar to Amycolatopsis coloradensis IMSNU 22096(T) (99.5 % 16S rRNA gene sequence similarity) and Amycolatopsis alba DSM 44262(T) (99.4 %). However, strain CMU-PLA07(T) was distinguishable from the type strains of species of the genus Amycolatopsis on the basis of DNA-DNA relatedness and phenotypic data. Therefore, strain CMU-PLA07(T) is considered to represent a novel species of the genus Amycolatopsis, for which the name Amycolatopsis thailandensis sp. nov. is proposed. The type strain is CMU-PLA07(T) ( = JCM 16380(T) = BCC 38279(T)).

  13. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation

    PubMed Central

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M.; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-01-01

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice. PMID:26216949

  14. Quantitative Field Testing Heterodera glycines from Metagenomic DNA Samples Isolated Directly from Soil under Agronomic Production

    PubMed Central

    Li, Yan; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2014-01-01

    A quantitative PCR procedure targeting the Heterodera glycines ortholog of the Caenorhabditis elegans uncoordinated-78 gene was developed. The procedure estimated the quantity of H. glycines from metagenomic DNA samples isolated directly from field soil under agronomic production. The estimation of H. glycines quantity was determined in soil samples having other soil dwelling plant parasitic nematodes including Hoplolaimus, predatory nematodes including Mononchus, free-living nematodes and biomass. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from field soil. PMID:24587100

  15. A novel antimicrobial protein isolated from potato (Solanum tuberosum) shares homology with an acid phosphatase.

    PubMed Central

    Feng, Jie; Yuan, Fenghua; Gao, Yin; Liang, Chenggang; Xu, Jin; Zhang, Changling; He, Liyuan

    2003-01-01

    The nucleotide and amino acids sequences for AP(1) will appear in the GenBank(R) and NCBI databases under accession number AY297449. A novel antimicrobial protein (AP(1)) was purified from leaves of the potato ( Solanum tuberosum, variety MS-42.3) with a procedure involving ammonium sulphate fractionation, molecular sieve chromatography with Sephacryl S-200 and hydrophobic chromatography with Butyl-Sepharose using a FPLC system. The inhibition spectrum investigation showed that AP(1) had good inhibition activity against five different strains of Ralstonia solanacearum from potato or other crops, and two fungal pathogens, Rhizoctonia solani and Alternaria solani from potato. The full-length cDNA encoding AP(1) has been successfully cloned by screening a cDNA expression library of potato with an anti-AP(1) antibody and RACE (rapid amplification of cDNA ends) PCR. Determination of the nucleotide sequences revealed the presence of an open reading frame encoding 343 amino acids. At the C-terminus of AP(1) there is an ATP-binding domain, and the N-terminus exhibits 58% identity with an/the acid phosphatase from Mesorhizobium loti. SDS/PAGE and Western blotting analysis suggested that the AP(1) gene can be successfully expressed in Escherichia coli and recognized by an antibody against AP(1). Also the expressed protein showed an inhibition activity the same as original AP(1) protein isolated from potato. We suggest that AP(1) most likely belongs to a new group of proteins with antimicrobial characteristics in vitro and functions in relation to phosphorylation and energy metabolism of plants. PMID:12927022

  16. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    PubMed

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  17. Sorption behavior of polyaramides in relation to isolation of nucleic acids and proteins.

    PubMed

    Liaw, Der-Jang; Yagudaeva, Elena; Prostyakova, Anna; Lazov, Michael; Zybin, Dmitry; Ischenko, Anatoly; Zubov, Vitaly; Chang, Cheng-Hung; Huang, Ying-Chi; Kapustin, Dmitry

    2016-09-01

    The effect of chemical composition and morphology of the surface layers of new polyaramide-containing sorbents on the mechanism of selective sorption of nucleic acids and proteins was investigated as compared to the previously studied sorbents modified with fluoropolymers and polyaniline (high-throughput materials providing one-step isolation of DNA from biological mixtures). A series of silica-based sorbents modified with polyaramides having consistently varying structure and containing the set of "key" structural elements (aromatic units and nitrogen atoms in the backbone, fluorinated groups), and various donor and acceptor moieties was prepared. The chemical composition of the polymer coatings was evaluated by X-ray photoelectron spectroscopy. The surface morphology was studied by scanning probe microscopy. The sorption properties were investigated by passing the mixtures containing DNA, RNA and proteins of different nature through the cartridges containing the obtained sorbents. All the investigated materials weakly retain double-stranded DNA but effectively retain RNA and proteins. The sorption capacity of the sorbents depends on the protein nature. The observed sorption behavior was shown to be determined by the chemical structure and not by the morphology of the polymer coating. It was proposed that similarity of the sorption properties of the series of chemically different polymers could be determined by similar total input of different sorption mechanisms.

  18. Identification of Hortaea werneckii Isolated from mangrove plant Aegiceras comiculatum based on morphology and rDNA sequences.

    PubMed

    Chen, Juan; Xing, Xiao-Ke; Zhang, Li-Chun; Xing, Yong-Mei; Guo, Shun-Xing

    2012-12-01

    Hortaea werneckii is a black yeast-like ascomycetous fungi associated with the human superficial infection tinea nigra, which commonly occurs in tropical and subtropical countries. Now, this fungus has been found in the halophilic environment all over the world and recognized as a new model organism in exploring the mechanisms of salt tolerance in eukaryotes. During a survey of endophytic fungi of mangrove forest at South China Sea, two isolates of H. werneckii were recovered from medicinal plant of Aegiceras comiculatum. The isolates were identified by morphological characters and phylogenetic analyses (e.g., ITS rDNA, LSU rDNA and translation elongation factor EF1α). Some physiological tests such as thermotolerance, acid tolerance (pH) and NaCl tolerance as well as pathogenicity test in vitro for the strains of Hortaea were performed. It is the first report that H. werneckii was isolated from medicinal plant of A. comiculatum in south sea of China as the endophytic fungi.

  19. Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes.

    PubMed

    Narayana, Kolla J P; Prabhakar, Peddikotla; Vijayalakshmi, Muvva; Venkateswarlu, Yenamandra; Krishna, Palakodety S J

    2007-01-01

    The strain ANU 6277 was isolated from laterite soil and identified as Streptomyces sp. closely related to Streptomyces albidoflavus cluster by 16S rRNA analysis. The cultural, morphological and physiological characters of the strain were recorded. The strain exhibited resistance to chloramphenicol, penicillin and streptomycin. It had the ability to produce enzymes such as amylase and chitinase. A bioactive compound was isolated from the strain at stationary phase of culture and identified as 3-phenylpropionic acid (3-PPA) by FT-IR, EI-MS, 1H NMR and 13C NMR spectral studies. It exhibited antimicrobial activity against different bacteria like Bacillus cereus, B. subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, P. flourescens, Staphylococcus aureus and some fungi including Aspergillus flavus, A. niger, Candida albicans, Fusarium oxysporum, F. udum and Penicillium citrinum. The antifungal activity of 3-PPA of the strain was evaluated in in vivo and in vitro conditions against Fusarium udum causing wilt disease in pigeon pea. The compound 3-PPA is an effective antifungal agent when compared to tricyclozole (fungicide) to control wilt caused by F. udum, but it exhibited less antifungal activity than carbendazim.

  20. Shewanella olleyana sp. nov., a marine species isolated from a temperate estuary which produces high levels of polyunsaturated fatty acids.

    PubMed

    Skerratt, Jennifer H; Bowman, John P; Nichols, Peter D

    2002-11-01

    Two polyunsaturated fatty acid (PUFA) producing strains (ACEM 6 and ACEM 9(T)) isolated from a temperate, humic-rich river estuary in Tasmania, Australia, were found to be members of the genus Shewanella. These strains were able to utilize humic compounds (tannic acid) and derivatives (2,6-anthraquinone disulfonate) as sole carbon sources and as electron acceptors for anaerobic respiration. The major fatty acids were typical of the genus Shewanella; however, PUFAs mostly made up of eicosapentaenoic acid were produced at high levels (10.2-23.6% of total fatty acids) and at relatively high incubation temperatures (10.2% at 24 degrees C). Sequence analysis indicated that ACEM 6 and ACEM 9(T) had identical 16S rDNA sequences and were most closely related to Shewanella japonica (sequence similarity 97.1%). DNA hybridization and phenotypic characteristics confirmed that the isolates constituted a novel species of the genus Shewanella, which is designated Shewanella olleyana sp. nov. (type strain ACEM 9(T) = ACAM 644(T) = LMG 21437(T)).

  1. High-yield noninvasive human genomic DNA isolation method for genetic studies in geographically dispersed families and populations

    SciTech Connect

    Meulenbelt, I.; Droog, S.; Slagboom, P.E.

    1995-11-01

    Human genomic DNA is commonly isolated from peripheral blood samples for genetic studies of families and populations. Blood sampling, however, is expensive and an invasive procedure to which, for ethical reasons, objections may be raised, especially in studies involving older individuals and babies. We have developed a new noninvasive DNA sampling and isolation method involving oral samples taken with cotton swabs. Participants can take mouth swabs themselves, and can send these by mail to the research center, where DNA can be isolated at least up to 3 wks after sampling. DNA isolation from 20 cotton swabs resulted in an average yield of 40{mu}g of high-molecular-weight DNA per individual, sufficient for complete genome searches with {approximately}800 polymorphic DNA markers when using PCR. Compared with blood sampling, which involves clinically trained personnel, this procedure is fast, less expensive, and suitable especially for DNA collection from geographically scattered subjects. 8 refs., 1 fig.

  2. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

  3. Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates.

    PubMed

    Oliveira, Caio Fernando de; Paim, Thiago Galvão da Silva; Reiter, Keli Cristine; Rieger, Alexandre; D'Azevedo, Pedro Alves

    2014-01-01

    Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

  4. Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants.

    PubMed

    Alatar, A A; Mahmoud, M A; Al-Sohaibani, S A; Abd-Elsalam, K A

    2012-02-13

    Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 μg (in 100-μL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.

  5. Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet.

    PubMed

    Wedrowicz, Faye; Karsa, Mawar; Mosse, Jennifer; Hogan, Fiona E

    2013-07-01

    The koala, an Australian icon, has been added to the threatened species list. Rationale for the listing includes proposed declines in population size, threats to populations (e.g. disease) and loss and fragmentation of habitat. There is now an urgent need to obtain accurate data to assess the status of koala populations in Australia, to ensure the long-term viability of this species. Advances in genetic techniques have enabled DNA analysis to study and inform the management of wild populations; however, sampling of individual koalas is difficult in tall, often remote, eucalypt forest. The collection of faecal pellets (scats) from the forest floor presents an opportunistic sampling strategy, where DNA can be collected without capturing or even sighting an individual. Obtaining DNA via noninvasive sampling can be used to rapidly sample a large proportion of a population; however, DNA from noninvasively collected samples is often degraded. Factors influencing DNA quality and quantity include environmental exposure, diet and methods of sample collection, storage and DNA isolation. Reduced DNA quality and quantity can introduce genotyping errors and provide inaccurate DNA profiles, reducing confidence in the ability of such data to inform management/conservation strategies. Here, we present a protocol that produces a reliable individual koala genotype from a single faecal pellet and highlight the importance of optimizing DNA isolation and analysis for the species of interest. This method could readily be adapted for genetic studies of mammals other than koalas, particularly those whose diet contains high proportions of volatile materials that are likely to induce DNA damage.

  6. Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods.

    PubMed

    Liu, Chao; Li, Bin; Du, Haishun; Lv, Dong; Zhang, Yuedong; Yu, Guang; Mu, Xindong; Peng, Hui

    2016-10-20

    In this work, nanocellulose was extracted from bleached corncob residue (CCR), an underutilized lignocellulose waste from furfural industry, using four different methods (i.e. sulfuric acid hydrolysis, formic acid (FA) hydrolysis, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and pulp refining, respectively). The self-assembled structure, morphology, dimension, crystallinity, chemical structure and thermal stability of prepared nanocellulose were investigated. FA hydrolysis produced longer cellulose nanocrystals (CNCs) than the one obtained by sulfuric acid hydrolysis, and resulted in high crystallinity and thermal stability due to its preferential degradation of amorphous cellulose and lignin. The cellulose nanofibrils (CNFs) with fine and individualized structure could be isolated by TEMPO-mediated oxidation. In comparison with other nanocellulose products, the intensive pulp refining led to the CNFs with the longest length and the thickest diameter. This comparative study can help to provide an insight into the utilization of CCR as a potential source for nanocellulose production.

  7. Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's milk cheese.

    PubMed

    Nikolic, Milica; Terzic-Vidojevic, Amarela; Jovcic, Branko; Begovic, Jelena; Golic, Natasa; Topisirovic, Ljubisa

    2008-02-29

    The Bukuljac cheese is traditionally homemade cheese, produced from heat-treated goat's milk without the addition of any bacterial starter culture. The presence of lactic acid bacteria (LAB) in Bukuljac cheese has been analyzed by using a polyphasic approach including microbiological and molecular methods such as rep-PCR with (GTG)5 primer. Lactobacillus paracasei subsp. paracasei represents a dominant strain in the microflora of analyzed cheese. Out of 55 Gram-positive and catalase-negative isolates, 48 belonged to L. paracasei subsp. paracasei species. Besides lactobacilli, five Lactococcus lactis subsp. lactis and two Enterococcus faecalis were found. Results of PCR-denaturing gradient gel electrophoresis (DGGE) of DNA extracted directly from the fresh cheese revealed the presence of Leuconostoc mesenteroides. Only lactobacilli showed a high proteolytic activity and hydrolyzed alpha(s1)- and beta-caseins. They are also producers of diacetyl. In addition, 34 out of 55 isolates, all determined as lactobacilli, showed the ability of auto-aggregation. Among 55 isolates, 50 also exhibited antimicrobial activity.

  8. Assessment of the in vitro bioactive properties of lactic acid bacteria isolated from native ecological niches of Ecuador.

    PubMed

    Benavides, Ana B; Ulcuango, Mario; Yépez, Lucía; Tenea, Gabriela N

    Lactic acid bacteria are known for their biotechnological potential. In various regions of Ecuador numerous indigenous biological resources are largely undocumented. In this study, we evaluated the potential probiotic characteristics and antagonistic in vitro properties of some lactic acid bacteria from native niches of the subtropical rain forests of Ecuador. These isolates were identified according to their morphological properties, standard API50CH fermentation profile and RAPD-DNA polymorphism pattern. The selected isolates were further evaluated for their probiotic potential. The isolates grew at 15°C and 45°C, survived at a pH ranging from 2.5 to 4.5 in the presence of 0.3% bile (>90%) and grew under sodium chloride conditions. All selected isolates were sensitive to ampicillin, amoxicillin and cefuroxime and some showed resistance to gentamicin, kanamycin and tetracycline. Moreover, the agar well diffusion assay showed that the supernatant of each strain at pH 3.0 and pH 4.0, but not at pH 7.0 exhibited increased antimicrobial activity (inhibition zone >15mm) against two foodborne pathogens, Escherichia coli and Salmonella spp. To our knowledge, this is the first report describing the antagonistic activity against two foodborne pathogens and the probiotic in vitro potential of lactic acid bacteria isolated from native biota of Ecuador.

  9. [Methodological features of DNA isolation for nested PCR for the differential diagnosis of malaria].

    PubMed

    Grinev, A B; Tokmalaev, A K; Chebyshev, N B; Rabinovich, S A; Isaeva, O V; Sadykova, V D; Begunova, S V; Galanin, N

    2013-01-01

    Current PCR assays for the differential diagnosis of malaria need to be further developed and certified in the Russian Federation. PCR may serve as a control diagnostic method. These investigations have demonstrated that an erythrocytic clot may be used as an adjunct to microscopy and the described procedure can be employed to isolate DNA for PRC in an inpatient setting.

  10. Acidiphilium iwatense sp. nov., isolated from an acid mine drainage treatment plant, and emendation of the genus Acidiphilium.

    PubMed

    Okamura, Keiko; Kawai, Akiko; Wakao, Norio; Yamada, Takeshi; Hiraishi, Akira

    2015-01-01

    Several strains of aerobic, acidophilic, chemo-organotrophic bacteria belonging to the genus Acidiphilium were isolated from an acid mine drainage (AMD) (pH 2.2) treatment plant. 16S rRNA gene sequence comparisons showed that most of the novel isolates formed a phylogenetically coherent group (designated Group Ia) distinguishable from any of the previously established species of the genus Acidiphilium at <98% similarity. This was supported by genomic DNA-DNA hybridization assays. The Group Ia isolates were characterized phenotypically by an oval cell morphology, non-motility, growth in the range pH 2.0-5.5 (optimum pH 3.5), lack of photosynthetic pigment and the presence of C19:0 cyclo ω8c as the main component of the cellular fatty acids and ubiquinone-10 as the major quinone. On the basis of these data, the name Acidiphilium iwatense sp. nov. is proposed to accommodate the Group Ia isolates, and the description of the genus Acidiphilium is emended. The type strain of Acidiphilium iwatense sp. nov. is MS8(T) ( =NBRC 107608(T)=KCTC 23505(T)).

  11. Aureispira marina gen. nov., sp. nov., a gliding, arachidonic acid-containing bacterium isolated from the southern coastline of Thailand.

    PubMed

    Hosoya, Shoichi; Arunpairojana, Vullapa; Suwannachart, Chatrudee; Kanjana-Opas, Akkharawit; Yokota, Akira

    2006-12-01

    Three strains of gliding bacteria, 24(T), 62 and 71, isolated from a marine sponge and algae from the southern coastline of Thailand, were studied using a polyphasic approach to clarify their taxonomic positions. A phylogenetic analysis based on 16S rRNA gene sequences showed that the three isolates formed a distinct lineage within the family 'Saprospiraceae' of the phylum Bacteroidetes and were related to members of the genus Saprospira. The G+C contents of the isolates were in the range 38-39 mol%. The major respiratory quinone was MK-7. The predominant cellular fatty acids were 20 : 4omega6c (arachidonic acid), 16 : 0 and iso-17 : 0. On the basis of morphological, physiological and chemotaxonomic characteristics, together with DNA-DNA hybridization data and 16S rRNA gene sequences, the isolates represent a novel species of a novel genus, for which the name Aureispira marina gen. nov., sp. nov. is proposed. The type strain of Aureispira marina is 24(T) (=IAM 15389(T)=TISTR 1719(T)).

  12. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support

    NASA Astrophysics Data System (ADS)

    Saiyed, Z. M.; Ramchand, C. N.; Telang, S. D.

    2008-05-01

    In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

  13. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  14. 18S ribosomal DNA genotypes of Acanthamoeba species isolated from contact lens cases in the Philippines.

    PubMed

    Rivera, Windell L; Adao, Davin Edric V

    2009-10-01

    This study was carried out to document the genotypes of Acanthamoeba present in contact lens cases from 50 randomly selected contact lens wearers living in Quezon City, Metro Manila, Philippines. Acanthamoeba species were isolated from eight (16%) in 50 contact lens cases examined. We analyzed partial 18S ribosomal DNA (Rns) sequences of the eight isolates and found that the sequence differences were sufficient to distinguish the genotypes. After the isolates were genotyped, using the Basic Local Alignment Search Tool program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. The model-based (GTR+Gamma+Iota) neighbor-joining, maximum likelihood, and Bayesian inference analyses, as well as the non-model-based maximum parsimony analysis were used. Results showed that of the eight isolates, six were Rns genotype T5 while two were Rns genotype T4. This present study indicates that genotype T5 is also a common contaminant in contact lens storage cases.

  15. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    PubMed

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

  16. Prooxidant action of chebulinic acid and tellimagrandin I: causing copper-dependent DNA strand breaks.

    PubMed

    Yi, Zong-Chun; Liu, Yan-Ze; Li, Hai-Xia; Wang, Zhao

    2009-04-01

    The prooxidant activity of two hydrolysable tannins, chebulinic acid and tellimagrandin I, on plasmid DNA and genomic DNA of cultured MRC-5 human embryo lung fibroblasts was assessed. The results revealed that both hydrolysable tannins in combination with Cu(II) induced DNA strand breaks in pBR322 plasmid DNA in a concentration-dependent manner. Chebulinic acid and tellimagrandin I also induced genomic DNA strand breaks of MRC-5 human embryo lung fibroblasts in the presence of Cu(II). After treatment with chebulinic acid or tellimagrandin I alone, the pBR322 plasmid DNA and genomic DNA in MRC-5 cells kept intact. In addition, addition of Cu(I) reagent bathocuproinedisulfonic acid or catalase markedly inhibited the copper-dependent DNA strand breaks by both tannins. However, three typical hydroxyl radical scavengers, DMSO, ethanol and mannitol, did not inhibit the DNA strand breaks. Both tannins were able to reduce Cu(II) to Cu(I). These results indicated that chebulinic acid and tellimagrandin I induced the copper-dependent strand breaks of pBR322 plasmid DNA and MRC-5 genomic DNA with prooxidant action, in which Cu(II)/Cu(I) redox cycle and H(2)O(2) were involved and hydroxyl radical formation is important in the hypothetical mechanism by which DNA strand breaks are formed.

  17. Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase.

    PubMed Central

    Grant, P M; Tellam, J; May, V L; Strauss, A W

    1986-01-01

    We have determined the complete sequence of the rat mitochondrial malate dehydrogenase (mMDH) precursor derived from nucleotide sequence of the cDNA. A single synthetic oligodeoxynucleotide probe was used to screen a rat atrial cDNA library constructed in lambda gt10. A 1.2 kb full-length cDNA clone provided the first complete amino acid sequence of pre-mMDH. The 1014 nucleotide-long open reading frame encodes the 314 residue long mature mMDH protein and a 24 amino acid NH2-terminal extension which directs mitochondrial import and is cleaved from the precursor after import to generate mature mMDH. The amino acid composition of the transit peptide is polar and basic. The pre-mMDH transit peptide shows marked homology with those of two other enzymes targeted to the rat mitochondrial matrix. Images PMID:3755817

  18. Gene activation properties of a mouse DNA sequence isolated by expression selection.

    PubMed Central

    von Hoyningen-Huene, V; Norbury, C; Griffiths, M; Fried, M

    1986-01-01

    The MES-1 element was previously isolated from restricted total mouse cellular DNA by "expression selection"--the ability to reactivate expression of a test gene devoid of its 5' enhancer sequences. Mes-1 has been tested in long-term transformation and short-term CAT expression assays. In both assays MES-1 is active independent of orientation and at a distance when placed 5' to the test gene. The element is active with heterologous promoters and functions efficiently in both rat and mouse cells. MES-1 activates expression by increasing transcription from the test gene's own start (cap) site. Thus the expression selection technique can be used for the isolation of DNA sequences with enhancer-like properties from total cellular DNA. Images PMID:3016657

  19. Mitochondrial DNA assessment of Phytophthora infestans isolates from potato and tomato in Ethiopia reveals unexpected diversity.

    PubMed

    Shimelash, Daniel; Hussien, Temam; Fininsa, Chemeda; Forbes, Greg; Yuen, Jonathan

    2016-08-01

    Mitochondrial DNA (mtDNA) haplotypes were determined using restriction fragment length polymorphism (RFLP) for P. infestans sampled from 513 foliar lesions of late blight found on potato and tomato in different regions of Ethiopia. Among the four reported mitochondrial haplotypes of Phytophthora infestans, Ia, Ib and IIb were detected in 93 % of the samples analyzed but the vast majority of these were Ia. The remaining 7 % represented a previously unreported haplotype. DNA sequencing of this new haplotype also confirmed a single base nucleotide substitution that resulted in loss of EcoRI restriction site and gain of two additional MspI sites in cox1 and atp1 genes, respectively. There were 28 polymorphic sites among all nucleotide sequences including five reference isolates. Sites with alignment gaps were observed in P4 with one nucleotide deletion in 11 Ethiopian isolates. None of the reference sequence produced frame-shifts, with the exception of the 3-nucleotide deletion in the P4 region by Phytophthora andina, a feature that can be used to distinguish the new Ethiopian isolates from P. andina. While a distinguishing molecular data presented here clearly separated them from P. infestans, 7 % of the isolates that share this feature formed an important component of the late blight pathogen causing disease on Solanum tuberosum in Ethiopia. Thus, these Ethiopian isolates could represent a novel Phytophthora species reported for the first time here.

  20. Reproductive Fitness and Random Amplified Polymorphic DNA Variation among Isolates of Pratylenchus vulnus

    PubMed Central

    Pinochet, P.; Cenis, J. L.; Fernández, C.; Doucet, M.; Maruli, J.

    1994-01-01

    The reproductive fitness of seven isolates of Pratylenchus vulnus from different geographical areas and hosts was assessed in monoxenic cultures (carrot), and greenhouse cultures (plum, sour orange, and quince). The genetic makeup of the different isolates was compared by Random Amplified Polymorphic DNA (RAPD-PCR). The apple (PvAP-S) and apricot (PvAT-F) isolates reproduced less in monoxenic cultures than the rose (PvRO-S) and walnut (PvWA-A and PvWA-U) isolates. On plum, the rose isolate (PvRO-S) reproduced better than the apple (PvAP-S) and walnut isolate from the United States (PvWA-U). On sour orange, the apple (PvAP-S), unknown origin (PvU-UK), and walnut isolate from Argentina (PvWA-A) multiplied well, whereas the walnut isolate from the United States (PvWA-U), apricot (PvAT-F), and rose (PvRO-S) did not. On quince, the apple (PvAP-S) and walnut (PvWA-U) isolates showed a higher reproduction than the one from unknown origin (PvU-UK). RAPD-PCR patterns among the seven P. vulnus isolates were similar, although high intraspecific varibility was detected. Very few bands of P. neglectus were shared by any population of P. vulnus. A high degree of similarity was found among the patterns corresponding to the rose (PvRO-S), apple (PvAP-S), walnut from the United States (PvWA-U), and unknown origin (PvUK-U) isolates. The apricot isolate (PvAT-F) was the most dissimilar among the seven isolates. No correlation could be established between the genetic variation of P. vulnus detected by RAPD-PCR and reproductive fitness. Results demonstrate high genetic varibility between geographically separated populations of P. vulnus. PMID:19279892

  1. Simple and efficient method for isolating cDNA fragments of lea3 genes with potential for wide application in the grasses (Poaceae).

    PubMed

    Yu, L; Wu, X; Tang, X; Yan, B

    2010-07-06

    cDNA fragments of lea3 genes with a high GC content (from 68 to 77%) were found in several Poaceae, including Sorghum vulgare, Saccharum officinarum, Oryza officinalis, Oryza meyeriana, Ampelocalamus calcareus, Cynodon dactylon, and Zizania latifoli. They were successfully isolated by means of optimal experimental parameters, which included dimethyl sulfoxide as additive and degenerate primers "AGETKAS" and "AGKDKTG", and their sequences were analyzed. Compared to the method of isolating genes by screening of a cDNA library using abscisic acid- and other stress-responsive cDNA clones, which is time-consuming and costly, this method is relatively easy and inexpensive. Using this new method, many new homologue lea3 genes were rapidly determined.

  2. DNA adsorption to and elution from silica surfaces: influence of amino acid buffers.

    PubMed

    Vandeventer, Peter E; Mejia, Jorge; Nadim, Ali; Johal, Malkiat S; Niemz, Angelika

    2013-09-19

    Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers. Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction.

  3. Isolation of "Caenorhabditis elegans" Genomic DNA and Detection of Deletions in the "unc-93" Gene Using PCR

    ERIC Educational Resources Information Center

    Lissemore, James L.; Lackner, Laura L.; Fedoriw, George D.; De Stasio, Elizabeth A.

    2005-01-01

    PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the…

  4. Random Amplified Polymorphic DNA Analysis to Differentiate Strains of Vibrio Vulnificus Isolated from Cockles and Shrimps.

    PubMed Central

    Ahbrizal, Tg.; Radu, Son; Latif, Abdul Reezal Abdul; Mutalib, Abdul Rahim; Rusul, Gulam; Elhadi, Nasreldin; Yuherman

    2000-01-01

    A random amplified polymorphic DNA (RAPD) fingerprinting method has been developed to differentiate Vibrio vulnificus strains isolated. Twenty-nine strains isolated from cockles and twenty-one strains isolated from shrimps were analyzed. A total of 10 primers were screened with Vibrio vulnificus strains to identify those capable of generating DNA polymorphisms and two primers were selected. Primer GEN 1-50-01 and GEN 1-50-08 produced polymorphisms in most strains tested, with the band sizes ranging from 10.0 to 0.25 kb pair. Dendrogram analysis showed that primer GEN 1-50-01 produced 10 clusters and 24 single strains at a 40% similarity, whereas primer GEN 1-50-08 produced 11 clusters and 20 single strains at a 40% similarity. This study revealed the potential use of PCR fingerprinting in epidemiological studies. PMID:22844214

  5. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed Central

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

  6. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  7. Comparison of two methods for isolating DNA from human skeletal remains for STR analysis.

    PubMed

    Rucinski, Cynthia; Malaver, Ayda L; Yunis, Emilio J; Yunis, Juan J

    2012-05-01

    The quality and efficiency of a standard organic DNA isolation method and a silica-based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real-time PCR, and STR profiles were obtained using the AmpFlSTR(®) Identifiler(®) PCR amplification kit. Overall, the silica-based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica-based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica-based method does not improve neither the quality nor the quantity of DNA for STR profiling.

  8. An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees.

    PubMed

    Novaes, R M L; Rodrigues, J G; Lovato, M B

    2009-02-03

    Traditionally, molecular studies of plant species have used leaves as the source of DNA. However, sampling leaves from tall tree species can be quite difficult and expensive. We developed a sequence of procedures for using stem bark as a source of DNA from Leguminosae trees of the Atlantic Forest and the Cerrado. Leguminosae is an important species-rich family in these two highly diverse and endangered biomes. A modified CTAB protocol for DNA isolation is described, and details of the procedures for sampling and storage of the bark are given. The procedures were initially developed for three species, and then their applicability for 15 other species was evaluated. DNA of satisfactory quality was obtained from the bark of all species. The amounts of DNA obtained from leaves were slightly higher than from bark samples, while its purity was the same. Storing the bark frozen or by drying in silica gel yielded similar results. Polymerase chain reaction amplification worked for both plastid and nuclear genomes. This alternative for isolating DNA from bark samples of trees facilitates field work with these tree species.

  9. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

    PubMed

    Olsen, B S; Johansen, I E

    2001-01-01

    The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

  10. Isolation of DNA-methyltransferase genes from strawberry (Fragaria x ananassa Duch.) and their expression in relation to micropropagation.

    PubMed

    Chang, Linlin; Zhang, Zhihong; Han, Baiming; Li, He; Dai, Hongyan; He, Ping; Tian, Hongzhe

    2009-09-01

    DNA methylation can control gene expression and may also play a role in plant development. Methylation of cytosine residues in DNA is enzymatically catalyzed by DNA methyltransferases. In this study, full-length genomic genes and cDNAs of methyltransferase (MET1) and domain-rearranged methyltransferase (DRM) were isolated from strawberry (Fragaria x ananassa Duch.). Two genomic clones (FaMET1a and FaMET1b) encoding MET1 had open-reading frame of 4,695 and 4,671 nucleotides with two introns, respectively. Amino acid sequence comparison indicated high similarity (98.72% identity) of strawberry MET1 protein to other plant MET1 sequences. The full-length cDNA of strawberry DRM genes (FaDRMa, FaDRMb and FaDRMc) were 2,273, 2,282 and 2,288 bp, respectively. Ten introns with different sizes were dispersed in FaDRM genes. Similarly, FaDRMa, FaDRMb and FaDRMc had high-sequence similarity overall. Expressions of strawberry MET1 and DRM genes were compared among in vitro-micropropagated plants, generations of micropropagated plants and conventionally propagated plants. The transcriptional expressions of both FaMET1 and FaDRM genes were downregulated in micropropagated plants, and they were recovered in the first and second runner generations of micropropagated plants. However, there was a slighter difference in global DNA methylation rates between micropropagated plants and conventionally propagated plants. Therefore, there was no positive relation between global DNA methylation rates and the expression levels of MET1 and DRM genes.

  11. Enhanced production of poly-γ-glutamic acid by a newly-isolated Bacillus subtilis.

    PubMed

    Ju, Wan-Taek; Song, Yong-Su; Jung, Woo-Jin; Park, Ro-Dong

    2014-11-01

    Application of poly-gamma-glutamic acid (γ-PGA), an unusual macromolecular anionic polypeptide, is limited due to the high cost associated with its low productivity. Screening bacterial strains to find a more efficient producer is one approach to overcome this limitation. Strain MJ80 was isolated as a γ-PGA producer among 1,500 bacterial colonies obtained from soil samples. It was identified as Bacillus subtilis, based on the biochemical and morphological properties and 16S rDNA gene sequencing. It produced γ-PGA from both glutamic acid and soybean powder, identifying it as a facultative glutamic acid-metabolizing bacterium. After optimization of its culture conditions, B. subtilis MJ80 showed γ-PGA productivity of 75.5 and 68.7 g/l in 3 and 300 l jar fermenters for 3 days cultivation, respectively, the highest productivity reported to date, suggesting MJ80 to be a promising strain for γ-PGA production.

  12. Biodegradation of Picolinic Acid by a Newly Isolated Bacterium Alcaligenes faecalis Strain JQ135.

    PubMed

    Qiu, Jiguo; Zhang, Junjie; Zhang, Yanting; Wang, Yuhong; Tong, Lu; Hong, Qing; He, Jian

    2017-04-01

    We isolated a bacterial strain JQ135 from municipal wastewater, which was capable of efficiently degrading picolinic acid (PA). Based on the physico-biochemical characteristics and 16S rDNA analysis, strain JQ135 was identified as Alcaligenes faecalis. In addition, strain JQ135 produced an orange pigment when cultured in the Luria-Bertani medium, which is different from the previously reported strains of A. faecalis. During the degradation of PA by the resting strain JQ135 cells, only one intermediate, 6-hydroxypicolinic acid (6HPA), was detected by ultraviolet spectrophotometry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry. A random transposon mutagenesis library of strain JQ135 was constructed. One mutant, Mut-G31, could convert PA into 6HPA without further degradation. The disrupted gene (orf2) was amplified from Mut-G31, and its product showed 32% identity to the 3-deoxy-D-manno-octulosonic acid kinase (KdkA) from Haemophilus influenzae. Results from complementation analysis confirmed that GTG was the initiation codon of the kdkA-like orf2, and that it was essential for PA biodegradation by strain JQ135. This study provides the first genetic evidence for the bacterial degradation of PA.

  13. Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

    PubMed Central

    Loureiro, João; Rodriguez, Eleazar; Doležel, Jaroslav; Santos, Conceição

    2007-01-01

    Background and Aims After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. Methods GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high

  14. Protective Effect of Folic Acid on Oxidative DNA Damage

    PubMed Central

    Guo, Xiaojuan; Cui, Huan; Zhang, Haiyang; Guan, Xiaoju; Zhang, Zheng; Jia, Chaonan; Wu, Jia; Yang, Hui; Qiu, Wenting; Zhang, Chuanwu; Yang, Zuopeng; Chen, Zhu; Mao, Guangyun

    2015-01-01

    Abstract Although previous reports have linked DNA damage with both transmissions across generations as well as our own survival, it is unknown how to reverse the lesion. Based on the data from a Randomized, Double-blind, Placebo Controlled Clinical Trial, this study aimed to assess the efficacy of folic acid supplementation (FAS) on DNA oxidative damage reversal. In this randomized clinical trial (RCT), a total of 450 participants were enrolled and randomly assigned to 3 groups to receive folic acid (FA) 0.4 mg/day (low-FA), 0.8 mg/day (high-FA), or placebo (control) for 8 weeks. The urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) and creatinine (Cr) concentration at pre- and post-FAS were measured with modified enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. A multivariate general linear model was applied to assess the individual effects of FAS and the joint effects between FAS and hypercholesterolemia on oxidative DNA damage improvement. This clinical trial was registered with ClinicalTrials.gov, number NCT02235948. Of the 438 subjects that received FA fortification or placebo, the median (first quartile, third quartile) of urinary 8-OHdG/Cr for placebo, low-FA, and high-FA groups were 58.19 (43.90, 82.26), 53.51 (38.97, 72.74), 54.73 (39.58, 76.63) ng/mg at baseline and 57.77 (44.35, 81.33), 51.73 (38.20, 71.30), and 50.65 (37.64, 76.17) ng/mg at the 56th day, respectively. A significant decrease of urinary 8-OHdG was observed after 56 days FA fortification (P < 0.001). Compared with the placebo, after adjusting for some potential confounding factors, including the baseline urinary 8-OHdG/Cr, the urinary 8-OHdG/Cr concentration significantly decreased after 56 days FAS [β (95% confidence interval) = −0.88 (−1.62, −0.14) and P = 0.020 for low-FA; and β (95% confidence interval) = −2.68 (−3.42, −1.94) and P < 0.001 for high-FA] in a dose-response fashion (Ptrend

  15. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

    SciTech Connect

    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-05-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. (/sup 3/H)Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

  16. Paenibacillus aceti sp. nov., isolated from the traditional solid-state acetic acid fermentation culture of Chinese cereal vinegar.

    PubMed

    Li, Pan; Lin, Weifeng; Liu, Xiong; Li, Sha; Luo, Lixin; Lin, Wei-Tie

    2016-09-01

    A Gram-stain-negative, rod-shaped, motile, endospore-forming, facultatively anaerobic bacterium, designated strain L14T, was isolated from the traditional acetic acid fermentation culture of Chinese cereal vinegars. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain L14T was affiliated to the genus Paenibacillus, most closely related to Paenibacillus motobuensis MC10T with 97.8 % similarity. Chemotaxonomic characterization supported the allocation of the strain to the genus Paenibacillus. The polar lipid profile of strain L14T contained the major compounds diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The predominant menaquinone was MK-7, and the major fatty acid components were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. The DNA G+C content of strain L14T was 49.9 mol%. The DNA-DNA relatedness value between strain L14T and P. motobuensis MC10T was 51.2 %. The results of physiological and biochemical tests allowed phenotypic differentiation of strain L14T from closely related species. On the basis of phenotypic and chemotaxonomic analyses, phylogenetic analysis and DNA-DNA relatedness values, strain L14T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus aceti sp. nov. is proposed. The type strain is L14T (=CGMCC 1.15420T=JCM 31170T).

  17. Actinocrinis puniceicyclus gen. nov., sp. nov., an actinobacterium isolated from an acidic spring.

    PubMed

    Kim, Joong-Jae; Marjerrison, Colbran E; Cornish Shartau, Sabrina L; Brady, Allyson L; Sharp, Christine E; Rijpstra, W Irene C; Sinninghe Damsté, Jaap S; Schumann, Peter; Grasby, Stephen E; Dunfield, Peter F

    2017-03-01

    An aerobic, mildly acidophilic actinobacterium was isolated from the Ochre Beds bog in Kootenay National Park, Canada. Cells of isolate OB1T were Gram-stain-positive, non-motile, pink- to purple-pigmented filaments. The pH range for growth was pH 3.5-6.5 (optimum pH 5.5), and the temperature range was 13-30°C. The major cellular fatty acids were i-C16 : 0 (28.5 %), i-C15 : 0 (14.6 %) and ai-C15 : 0 (14.3 %), and the major polar lipid was phosphohexose. The major quinone was menaquinone-11 (MK-11), and the peptidoglycan type was A1γ. The DNA G+C content was 70.2 %. Along with growth on complex media including yeast extract, proteose peptone, casamino acids and tryptic soy broth, growth occured on mono- and disaccharides (glucose, sucrose, galactose and xylose) and polysaccharides (starch, gellan, pectin, xylan and alginate). Anaerobic growth was not observed. The cells did not fix atmospheric nitrogen. On the basis of comparative 16S rRNA gene sequence analysis, this isolate belonged to the family Actinospicaceae, in the suborder Catenulisporineae of the order Actinomycetales. The most closely related species was Actinospica robiniae. However, the 16S rRNA gene sequence identity to this bacterium was low (92.8 %) and there were several chemotaxonomic differences from this species. We therefore propose a novel genus and species, Actinocrinis puniceicyclus gen. nov., sp. nov., with strain OB1T (=DSM 45618T=ATCC BAA-2771T) as the type strain.

  18. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    ABSTRACT

    Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  19. Direct DNA isolation from solid biological sources without pretreatments with proteinase-K and/or homogenization through automated DNA extraction.

    PubMed

    Ki, Jang-Seu; Chang, Ki Byum; Roh, Hee June; Lee, Bong Youb; Yoon, Joon Yong; Jang, Gi Young

    2007-03-01

    Genomic DNA from solid biomaterials was directly isolated with an automated DNA extractor, which was based on magnetic bead technology with a bore-mediated grinding (BMG) system. The movement of the bore broke down the solid biomaterials, mixed crude lysates thoroughly with reagents to isolate the DNA, and carried the beads to the next step. The BMG system was suitable for the mechanical homogenization of the solid biomaterials and valid as an automated system for purifying the DNA from the solid biomaterials without the need for pretreatment or disruption procedures prior to the application of the solid biomaterials.

  20. Isolation of PCR ready-human DNA using copper nanoparticles from skeletal remains.

    PubMed

    Lodha, Anand; Ansari, Niha; Shah, Shahil; Rao, M V; Menon, Shobhana K

    2017-01-01

    Present study represents a novel approach of PCR ready-human DNA extraction method from skeletal remains using copper nanoparticles (CuNPs) for personnel identification. To achieve rapid, cost effective, sensitive and non-hazardous method for DNA extraction we utilized CuNPs synthesized using microwave. The applicability of this approach was first tested in blood samples and afterwards, this system was extended to skeletal remains' samples also. This method yields good quality DNA that are ready for PCR reactions from small quantities of blood and skeletal remains. Consequently, even small quantities of nanoparticles could be potentially utilized for a highly efficient isolation of DNA from skeletal remains as well as from ancient archaeological samples. The present method has the advantages that it is quick with high yield, inexpensive, robust, environment friendly and does not require use of hazardous organic solvents.

  1. Isolation of acetic, propionic and butyric acid-forming bacteria from biogas plants.

    PubMed

    Cibis, Katharina Gabriela; Gneipel, Armin; König, Helmut

    2016-02-20

    In this study, acetic, propionic and butyric acid-forming bacteria were isolated from thermophilic and mesophilic biogas plants (BGP) located in Germany. The fermenters were fed with maize silage and cattle or swine manure. Furthermore, pressurized laboratory fermenters digesting maize silage were sampled. Enrichment cultures for the isolation of acid-forming bacteria were grown in minimal medium supplemented with one of the following carbon sources: Na(+)-dl-lactate, succinate, ethanol, glycerol, glucose or a mixture of amino acids. These substrates could be converted by the isolates to acetic, propionic or butyric acid. In total, 49 isolates were obtained, which belonged to the phyla Firmicutes, Tenericutes or Thermotogae. According to 16S rRNA gene sequences, most isolates were related to Clostridium sporosphaeroides, Defluviitoga tunisiensis and Dendrosporobacter quercicolus. Acetic, propionic or butyric acid were produced in cultures of isolates affiliated to Bacillus thermoamylovorans, Clostridium aminovalericum, Clostridium cochlearium/Clostridium tetani, C. sporosphaeroides, D. quercicolus, Proteiniborus ethanoligenes, Selenomonas bovis and Tepidanaerobacter sp. Isolates related to Thermoanaerobacterium thermosaccharolyticum produced acetic, butyric and lactic acid, and isolates related to D. tunisiensis formed acetic acid. Specific primer sets targeting 16S rRNA gene sequences were designed and used for real-time quantitative PCR (qPCR). The isolates were physiologically characterized and their role in BGP discussed.

  2. Phage type and DNA plasmid profile of Salmonella typhimurium isolates in the area of Isernia, Italy.

    PubMed Central

    Fantasia, M.; Ricci, N.; Manuppella, A.; Martini, A.; Filetici, E.; Laurelli, T.

    1990-01-01

    Thirty-eight Salmonella typhimurium strains isolated from December 1987 to March 1988 in Isernia, Central Italy, were characterized on the basis of their phage type, resistance to antimicrobials and plasmid profiles. According to their phage types, the isolates could be assigned to one of six groups, the prevalent one being PT 195 which accounted for 73.6% of isolates. On the basis of their plasmid content, the isolates could be assigned to one of ten groups. The prevalent plasmid profile (60.0; 6.0; 4.3; 4.0; 3.2 megadaltons) was found in 60.4% of isolates. All the isolates from a particular food (salsicce), and as most of isolates from humans who had consumed this food belonged to phage type 195 and were of the same plasmid profile. The combined use of phage typing and DNA plasmid analysis proved to be a useful tool in identifying epidemiologically related isolates in this investigation. Images Fig. 2 PMID:2209736

  3. Investigation of irradiated rats DNA in the presence of Cu(II) chelates of amino acids Schiff bases.

    PubMed

    Karapetyan, N H; Torosyan, A L; Malakyan, M; Bajinyan, S A; Haroutiunian, S G

    2016-01-01

    The new synthesized Cu(II) chelates of amino acids Schiff bases were studied as a potential radioprotectors. Male albino rats of Wistar strain were exposed to X-ray whole-body irradiation at 4.8 Gy. This dose caused 30% mortality of the animals (LD30). The survival of animals exposed to radiation after preliminary administration of 10 mg/kg Cu(II)(Nicotinyl-L-Tyrosinate)2 or Cu(II)(Nicotinyl-L-Tryptophanate)2 prior to irradiation was registered about 80 and 100% correspondingly. Using spectrophotometric melting and agarose gel electrophoresis methods, the differences between the DNA isolated from irradiated rats and rats pretreated with Cu(II) chelates were studied. The fragments of DNA with different breaks were revealed in DNA samples isolated from irradiated animals. While, the repair of the DNA structure was observed for animals pretreated with the Cu(II) chelates. The results suggested that pretreatment of the irradiated rats with Cu(II)(Nicotinyl-L-Tyrosinate)2 and Cu(II)(Nicotinyl-L-Tryptophanate)2 compounds improves the liver DNA characteristics.

  4. DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates

    PubMed Central

    Sater, Mohamad R. Abdul; Lamelas, Araceli; Wang, Guilin; Clark, Tyson A.; Röltgen, Katharina; Mane, Shrikant; Korlach, Jonas; Pluschke, Gerd; Schmid, Christoph D.

    2015-01-01

    The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of

  5. Physiological Characteristics and Production of Folic Acid of Lactobacillus plantarum JA71 Isolated from Jeotgal, a Traditional Korean Fermented Seafood

    PubMed Central

    Lim, Sang-Dong

    2014-01-01

    Folic acid, one of the B group of vitamins, is an essential substance for maintaining the functions of the nervous system, and is also known to decrease the level of homocysteine in plasma. Homocysteine influences the lowering of the cognitive function in humans, and especially in elderly people. In order to determine the strains with a strong capacity to produce folic acid, 190 bacteria were isolated from various kinds of jeotgal and chungkuk-jang. In our test experiment, JA71 was found to contain 9.03μg/mL of folic acid after 24 h of incubation in an MRS broth. This showed that JA71 has the highest folic acid production ability compared to the other lactic acid bacteria that were isolated. JA71 was identified as Lactobacillus plantarum by the result of API carbohydrate fermentation pattern and 16s rDNA sequence. JA71 was investigated for its physiological characteristics. The optimum growth temperature of JA71 was 37℃, and the cultures took 12 h to reach pH 4.4. JA71 proved more sensitive to bacitracin when compared with fifteen different antibiotics, and showed most resistance to neomycin and vancomycin. Moreover, it was comparatively tolerant of bile juice and acid, and displayed resistance to Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus with restraint rates of 60.4%, 96.7%, and 76.2%, respectively. These results demonstrate that JA71 could be an excellent strain for application to functional products. PMID:26760752

  6. Increased Amoxicillin–Clavulanic Acid Resistance in Escherichia coli Blood Isolates, Spain

    PubMed Central

    Oteo, Jesús; Lázaro, Edurne; Cuevas, Óscar; García-Cobos, Silvia; Pérez-Vázquez, María; de Abajo, F. J.

    2008-01-01

    To determine the evolution and trends of amoxicillin–clavulanic acid resistance among Escherichia coli isolates in Spain, we tested 9,090 blood isolates from 42 Spanish hospitals and compared resistance with trends in outpatient consumption. These isolates were collected by Spanish hospitals that participated in the European Antimicrobial Resistance Surveillance System network from April 2003 through December 2006. PMID:18680650

  7. Increased amoxicillin-clavulanic acid resistance in Escherichia coli blood isolates, Spain.

    PubMed

    Oteo, Jesús; Campos, José; Lázaro, Edurne; Cuevas, Oscar; García-Cobos, Silvia; Pérez-Vázquez, María; de Abajo, F J

    2008-08-01

    To determine the evolution and trends of amoxicillin-clavulanic acid resistance among Escherichia coli isolates in Spain, we tested 9,090 blood isolates from 42 Spanish hospitals and compared resistance with trends in outpatient consumption. These isolates were collected by Spanish hospitals that participated in the European Antimicrobial Resistance Surveillance System network from April 2003 through December 2006.

  8. Rhizobium acidisoli sp. nov., isolated from root nodules of Phaseolus vulgaris in acid soils.

    PubMed

    Román-Ponce, Brenda; Jing Zhang, Yu; Soledad Vásquez-Murrieta, María; Hua Sui, Xin; Feng Chen, Wen; Carlos Alberto Padilla, Juan; Wu Guo, Xian; Lian Gao, Jun; Yan, Jun; Hong Wei, Ge; Tao Wang, En

    2016-01-01

    Two Gram-negative, aerobic, non-motile, rod-shaped bacterial strains, FH13T and FH23, representing a novel group of Rhizobium isolated from root nodules of Phaseolus vulgaris in Mexico, were studied by a polyphasic analysis. Phylogeny of 16S rRNA gene sequences revealed them to be members of the genus Rhizobium related most closely to 'Rhizobium anhuiense' CCBAU 23252 (99.7 % similarity), Rhizobium leguminosarum USDA 2370T (98.6 %), and Rhizobium sophorae CCBAU 03386T and others ( ≤ 98.3 %). In sequence analyses of the housekeeping genes recA, glnII and atpD, both strains formed a subclade distinct from all defined species of the genus Rhizobium at sequence similarities of 82.3-94.0 %, demonstrating that they represented a novel genomic species in the genus Rhizobium. Mean levels of DNA-DNA relatedness between the reference strain FH13T and the type strains of related species varied between 13.0 ± 2.0 and 52.1 ± 1.2 %. The DNA G+C content of strain FH13T was 63.5 mol% (Tm). The major cellular fatty acids were 16 : 0, 17 : 0 anteiso, 18 : 0, summed feature 2 (12 : 0 aldehyde/unknown 10.928) and summed feature 8 (18 : 1ω7c). The fatty acid 17 : 1ω5c was unique for this strain. Some phenotypic features, such as failure to utilize adonitol, l-arabinose, d-fructose and d-fucose, and ability to utilize d-galacturonic acid and itaconic acid as carbon source, could also be used to distinguish strain FH13T from the type strains of related species. Based upon these results, a novel species, Rhizobium acidisoli sp. nov., is proposed, with FH13T ( = CCBAU 101094T = HAMBI 3626T = LMG 28672T) as the type strain.

  9. [FATTY ACID COMPOSITION ALTEROMONAS-LIKE BACTERIA ISOLATED FROM THE BLACK SEA WATER].

    PubMed

    Klochko, V V; Avdeeva, L V

    2015-01-01

    Alteromonas macleodii strains isolated from the Black sea water were similar in their fatty acids composition with the type strain of this species. Analysis of lipid composition of 10 A. macleodii strains isolated from the deep and surface water layers in different World ocean regions including the Black sea water has shown that the deep and surface isolates of this species formed two groups different in their fatty acids profiles. The Black sea isolates of Pseudoalteromonas haloplanktis, P. citrea, P. flavipulchra conformed to these species type strains in their fatty acids composition. On the basis of the fatty acids spectra similarity of three Pseudoalteromonas species strains with Plipolytica described in 2010 has been established. Presence of three isomers C16:1ψ7, C 16:1ψ9 and C16:1ψ6--components of hexadecenic acid in the Black sea isolates of Shewanella baltica has been shown.

  10. Isolation and characterization of new highly polymorphic DNA markers from the Huntington disease region

    SciTech Connect

    Weber, B.; Hedrick, A.; Andrew, S.; Riess, O.; Collins, C.; Kowbel, D.; Hayden, M.R. )

    1992-02-01

    The defect causing Huntington disease (HD) has been mapped to 4p16.3, distal to the DNA marker D4S10. Subsequently, additional polymorphic markers closer to the HD gene have been isolated, which has led to the establishment of predictive testing programs for individuals at risk for HD. Approximately 17% of persons presenting to the Canadian collaborative study for predictive testing for HD have not received any modification of risk, in part because of limited informativeness of currently available DNA markers. Therefore, more highly polymorphic DNA markers are needed, which well further increase the accuracy and availability of predictive testing, specifically for families with complex or incomplete pedigree structures. In addition, new markers are urgently needed in order to refine the breakpoints in the few known recombinant HD chromosomes, which could allow a more accurate localization of the HD gene within 4p16.3 and, therefore, accelerate the cloning of the disease gene. In this study, the authors present the identification and characterization of nine new polymorphic DNA markers, including three markers which detect highly informative multiallelic VNTR-like polymorphisms with PIC values of up to .84. These markers have been isolated from a cloned region of DNA which has been previously mapped approximately 1,000 kb from the 4p telomere.

  11. Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott).

    PubMed Central

    Rhoads, D M; McIntosh, L

    1991-01-01

    Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUC119 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybrid-selected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of 42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein. Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane alpha-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aox1, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum. Images PMID:1706518

  12. Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage.

    PubMed

    Tang, M; Subbiah, M T

    1996-01-19

    The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu2+ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled OX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17 beta significantly decreased the formation of single and double strand breaks in DNA induced by H2O2 alone or with Cu2+. Equilin (an equine estrogen) was more effective than estradiol-17 beta at the doses tested. Arachidonic acid in the presence of Cu2+ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.

  13. Chromatin Isolation and DNA Sequence Analysis in Large Undergraduate Laboratory Sections

    NASA Astrophysics Data System (ADS)

    Hagerman, Ann E.

    1999-10-01

    A pair of exercises that introduce undergraduate students to basic techniques and concepts of molecular biology and that are appropriate for classes with large enrollments are described. One exercise is a simple laboratory experiment in which chromatin is isolated from chicken liver and is resolved into histone proteins and DNA by ion-exchange chromatography. The other is a series of computer simulations that introduce DNA sequencing, mapping, and sequence analysis to the students. The final step of the simulation is submission of a sequence to a database on the World Wide Web for identification of the protein product of the gene.

  14. Microsatellite genotyping of DNA isolated from claws left on tanned carnivore hides.

    PubMed

    Hedmark, Eva; Ellegren, Hans

    2005-11-01

    Tanned hides, a common form of preservation of mammalian specimens, are usually resistant to DNA analysis. However, we show that DNA isolated from the pulp of claws of tanned hides amplifies well for microsatellite markers. For eight wolverine and eight lynx hides tanned 5-20 years ago, 93-98% of replicate amplifications gave distinct PCR products. Genotypes obtained in analysis of tissue samples of the same individuals were in all cases in agreement with those obtained by analysis of claws. We thus conclude that the use of claws from tanned hides offers new possibilities to genetic studies of preserved mammalian specimens, for instance, in the monitoring of illegal trade.

  15. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis.

    PubMed

    Liu, Ying; Xu, Qiao-Xian; Xi, Pei-Yu; Chen, Hong-Hao; Liu, Chun-Sheng

    2013-05-01

    The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

  16. Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase.

    PubMed

    Naggert, J; Witkowski, A; Mikkelsen, J; Smith, S

    1988-01-25

    A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.

  17. Isolation of cDNA clones for the catalytic gamma subunit of mouse muscle phosphorylase kinase: expression of mRNA in normal and mutant Phk mice.

    PubMed Central

    Chamberlain, J S; VanTuinen, P; Reeves, A A; Philip, B A; Caskey, C T

    1987-01-01

    We have isolated and characterized cDNA clones for the gamma subunit of mouse muscle phosphorylase kinase (gamma-Phk). These clones were isolated from a lambda gt11 mouse muscle cDNA library via screening with a synthetic oligonucleotide probe corresponding to a portion of the rabbit gamma-Phk amino acid sequence. The gamma-Phk cDNA clones code for a 387-amino acid protein that shares 93% amino acid sequence identity with the corresponding rabbit amino acid sequence. RNA gel blot analysis reveals that the muscle gamma-Phk probe hybridizes to two mRNA species (2.4 and 1.6 kilobases) in skeletal muscle, cardiac muscle, and brain, but does not hybridize to liver RNA. Phk-deficient I-strain (Phk) mouse muscle contains reduced levels of gamma-Phk mRNA as compared with control mice. Although the Phk defect is an X-linked recessive trait, hybridization to a human-rodent somatic cell hybrid mapping panel shows that the gamma-Phk gene is not located on the X chromosome. Rather, the gamma-Phk cross-hybridizing human restriction fragments map to human chromosomes 7 (multiple) and 11 (single). Reduced gamma-Phk mRNA in I-strain mice, therefore, appears to be a consequence of the Phk-mutant trait and does not stem from a mutant gamma-subunit gene. Images PMID:3472241

  18. Role of Amino Acid Insertions on Intermolecular Forces between Arginine Peptide Condensed DNA Helices

    PubMed Central

    DeRouchey, Jason E.; Rau, Donald C.

    2011-01-01

    In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads. PMID:21994948

  19. Spherical Nucleic Acids: A New Form of DNA

    NASA Astrophysics Data System (ADS)

    Cutler, Joshua Isaac

    Spherical Nucleic Acids (SNAs) are a new class of nucleic acid-based nanomaterials that exhibit unique properties currently being explored in the contexts of gene-based cancer therapies and in the design of programmable nanoparticle-based materials. The properties of SNAs differ from canonical, linear nucleic acids by virtue of their dense packing into an oriented 3-dimensional array. SNAs can be synthesized from a number of useful nanoparticle templates, such as plasmonic gold and silver, magnetic oxides, luminescent semi-conductor quantum dots, and silica. In addition, by crosslinking the oligonucleotides and dissolving the core, they can be made in a hollow form as well. This dissertation describes the evolution of SNAs from initial studies of inorganic nanoparticle-based materials densely functionalized with oligonucleotides to the proving of a hypothesis that their unique properties can be observed in a core-less structure if the nucleic acids are densely packed and highly oriented. Chapter two describes the synthesis of densely functionalized polyvalent oligonucleotide superparamagnetic iron oxide nanoparticles using the copper-catalyzed azide-alkyne cycloaddition reaction. These particles are shown to exhibit cooperative binding in a density- and salt concentration-dependent fashion, with nearly identical behaviors to those of SNA-functionalized gold nanoparticles. Importantly, these particles are the first non-gold particles shown to be capable of entering cells in high numbers via the SNA-mediated cellular uptake pathway, and provided the first evidence that SNA-mediated cellular uptake is core-independent. In the third chapter, a gold nanoparticle catalyzed alkyne cross-linking reaction is described that is capable of forming hollow organic nanoparticles using polymers with alkyne-functionalized backbones. With this method, the alkyne-modified polymers adsorb to the particle surfaces, cross-link on the surface, allowing the gold nanoparticle to be

  20. Analysis of the Genome of a Korean Isolate of the Pieris rapae Granulovirus Enabled by Its Separation from Total Host Genomic DNA by Pulse-Field Electrophoresis

    PubMed Central

    Jo, Yong Hun; Patnaik, Bharat Bhusan; Kang, Se Won; Chae, Sung-Hwa; Oh, Seunghan; Kim, Dong Hyun; Noh, Mi Young; Seo, Gi Won; Jeong, Heon Cheon; Noh, Ju Young; Jeong, Ji Eun; Hwang, Hee Ju; Ko, Kisung; Han, Yeon Soo; Lee, Yong Seok

    2013-01-01

    Background Most traditional genome sequencing projects involving viruses include the culture and purification of the virus particles. However, purification of virions may yield insufficient material for traditional sequencing. The electrophoretic method described here provides a strategy whereby the genomic DNA of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) could be recovered in sufficient amounts for sequencing by purifying it directly from total host DNA by pulse-field gel electrophoresis (PFGE). Methodology/Principal Findings The total genomic DNA of infected P. rapae was embedded in agarose plugs, treated with restriction nuclease and methylase, and then PFGE was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the purified viral DNA. The double-stranded circular genome of PiraGV-K was found to encode 120 open reading frames (ORFs), which covered 92% of the sequence. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (∼99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF 11), involved in the liquefaction of the host, were found in the genome. Conclusions/Significance The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the electrophoretic method. The method appears to be generally applicable to the analysis of genomes of large viruses. PMID:24391907

  1. Isolation of lactic acid bacteria with potential protective culture characteristics from fruits

    NASA Astrophysics Data System (ADS)

    Hashim, Nurul Huda; Sani, Norrakiah Abdullah

    2015-09-01

    Lactic acid bacteria are also known as beneficial microorganisms abundantly found in fermented food products. In this study, lactic acid bacteria were isolated from fresh cut fruits obtained from local markets. Throughout the isolation process from 11 samples of fruits, 225 presumptive lactic acid bacteria were isolated on MRS agar medium. After catalase and oxidase tests, 149 resulted to fit the characteristics of lactic acid bacteria. Further identification using Gram staining was conducted to identify the Gram positive bacteria. After this confirmation, the fermentation characteristics of these isolates were identified. It was found that 87 (58.4%) isolates were heterofermentative, while the rest of 62 (41.6%) are homofermentative lactic acid bacteria. Later, all these isolates were investigated for the ability to inhibit growth of Staphylococcus aureus using agar spot assay method. Seven (4.7%) isolates showed strong antagonistic capacity, while 127 (85.2%) and 8 (5.4%) isolates have medium and weak antagonistic capacity, respectively. The other 7 (4.7%) isolates indicated to have no antagonistic effect on S. aureus. Results support the potential of LAB isolated in this study which showed strong antagonistic activity against S. aureus may be manipulated to become protective cultures in food products. While the homofermentative or heterofermentative LAB can be utilized in fermentation of food and non-food products depending on the by-products required during the fermentation.

  2. A fluorescence-based analysis of aristolochic acid-derived DNA adducts.

    PubMed

    Romanov, Victor; Sidorenko, Victoria; Rosenquist, Thomas A; Whyard, Terry; Grollman, Arthur P

    2012-08-01

    Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.

  3. Comparison of ribosomal DNA length and restriction site polymorphisms in Gremmeniella and Ascocalyx isolates.

    PubMed Central

    Bernier, L; Hamelin, R C; Ouellette, G B

    1994-01-01

    The small subunit (SSU) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA genes from 27 specimens of the fungal genera Gremmeniella and Ascocalyx were amplified by PCR. Length polymorphisms were observed in the SSU and allowed the differentiation of four groups among the isolates tested: (i) Ascocalyx abietis; (ii) Gremmeniella isolates from Picea spp.; (iii) Gremmeniella isolates from Abies balsamea; and (iv) Gremmeniella isolates from Abies sacchalinensis, Larix spp., and Pinus spp. The amplified ITS was the same length for all Gremmeniella specimens and was 60 bp longer in A. abietis. Phylogenetic analysis of length polymorphisms and of 24 restriction sites in the SSU and ITS showed that Gremmeniella isolates were more related to each other than to the Ascocalyx isolate. Furthermore, seven groups were evident within the genus Gremmeniella. Our results confirm that Gremmeniella and Ascocalyx should be kept as different taxa and suggest that the taxonomy of the former could be revised to consider isolates from Abies balsamea and from Picea spp. to be two different varieties while incorporating Gremmeniella laricina into G. abietina, as a new variety. Images PMID:7912501

  4. Human genomic DNA isolation from whole blood using a simple microfluidic system with silica- and polymer-based stationary phases.

    PubMed

    Günal, Gülçin; Kip, Çiğdem; Öğüt, Sevim Eda; Usta, Duygu Deniz; Şenlik, Erhan; Kibar, Güneş; Tuncel, Ali

    2017-05-01

    Monodisperse-porous silica microspheres 5.1μm in size with a bimodal pore-size distribution (including both mesoporous and macroporous compartments) were obtained using a newly developed staged-shape templated hydrolysis and condensation protocol. Synthesized silica microspheres and monodisperse-porous polymer-based microspheres with different functionalities, synthesized by staged-shape template polymerization, were comparatively tested as sorbents for human genomic DNA (hgDNA) isolation in a microfluidic system. Microcolumns with a permeability range of 1.8-8.5×10(-13)m(2) were fabricated by the slurry-packing of silica- or polymer-based microspheres. The monodisperse-porous silica microspheres showed the best performance in hgDNA isolation in an aqueous buffer medium; >2500ng of hgDNA was recovered with an isolation yield of about 50%, using an hgDNA feed concentration of 100ng/μL. Monodisperse-porous silica microspheres were also evaluated as a sorbent for genomic DNA isolation from human whole blood in the microfluidic system; 14ng of hgDNA was obtained from 10μL of whole blood lysate with an isolation yield of 64%. Based on these results, we conclude that monodisperse-porous silica microspheres with a bimodal pore size distribution are a promising sorbent for the isolation of hgDNA in larger amounts and with higher yields compared to the sorbents previously tried in similar microfluidic systems.

  5. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.

    PubMed

    Yoo, Dae-goon; Floyd, Madison; Winn, Matthew; Moskowitz, Samuel M; Rada, Balázs

    2014-08-01

    Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO-DNA complexes and established a new HNE-DNA ELISA. We show that these methods reliably quantify MPO-DNA and HNE-DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired "early" and "late" bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways.

  6. Pasteurella multocida isolated from wild birds of North America: a serotype and DNA fingerprint study of isolates from 1978 to 1993

    USGS Publications Warehouse

    Wilson, M.A.; Duncan, R.M.; Nordholm, G.E.; Berlowski, B.M.

    1995-01-01

    Serotype and DNA fingerprint methods were used to study Pasteurella multocida isolated from 320 wild birds of North America. Isolates were collected during 1978-93. The HhaI profiles of 314 isolates matched the HhaI profile of somatic reference type 1, strain X-73; somatic type 1 antigen was expressed by 310 isolates, and the serotype of four isolates was undetected. Differentiation of the 314 isolates was observed by digestion of DNA with HpaII. None of the HpaII profiles matched the HpaII profile of X-73 (designated HhaI 001/HpaII 001). Three HpaII profiles were recognized among the somatic type 1 isolates: HpaII 002 (n = 18), HpaII 003 (n = 122), and HpaII 004 (n = 174). Profile HpaII 002 was found among isolates collected during 1979-83. Profile HpaII 003 was identified from isolates collected during 1979-89, with the exception of two isolates in 1992. The HpaII 004 profile was identified from isolates collected during 1983-93. Of the six remaining isolates, four expressed somatic type 4 and had HhaI profiles identical to the somatic type 4 reference strain P-1662 profile (designated HhaI 004); these isolates were differentiated by digestion of DNA with HpaII. One isolate was identified as serotype F:11, and another was serotype A:3,4. In the present study, 314 of 316 (99.4%) isolates from wild birds in the Central, Mississippi, and Pacific flyways during 1978-93, were P. multocida somatic type 1.

  7. 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates

    PubMed Central

    Drancourt, Michel; Bollet, Claude; Carlioz, Antoine; Martelin, Rolland; Gayral, Jean-Pierre; Raoult, Didier

    2000-01-01

    Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides

  8. Overexpression of ubiquitin and amino acid permease genes in association with antimony resistance in Leishmania tropica field isolates.

    PubMed

    Kazemi-Rad, Elham; Mohebali, Mehdi; Khadem-Erfan, Mohammad Bagher; Hajjaran, Homa; Hadighi, Ramtin; Khamesipour, Ali; Rezaie, Sassan; Saffari, Mojtaba; Raoofian, Reza; Heidari, Mansour

    2013-08-01

    The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime®) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

  9. Enhancing DNA electro-transformation efficiency on a clinical Staphylococcus capitis isolate.

    PubMed

    Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

    2015-02-01

    Clinical staphylococcus isolates possess a stronger restriction-modification (RM) barrier than laboratory strains. Clinical isolates are therefore more resistant to acceptance of foreign genetic material than laboratory strains, as their restriction systems more readily recognize and destroy foreign DNA. This stronger barrier consequently restricts genetic studies to a small number of domestic strains that are capable of accepting foreign DNA. In this study, an isolate of Staphylococcus capitis, obtained from the blood of a very low birth-weight baby, was transformed with a shuttle vector, pBT2. Optimal conditions for electro-transformation were as follows: cells were harvested at mid-log phase, electro-competent cells were prepared; cells were pre-treated at 55°C for 1min; 3μg of plasmid DNA was mixed with 70-80μL of competent cells (3-4×10(10)cells/mL) at 20°C in 0.5M sucrose, 10% glycerol; and electroporation was conducted using 2.1kV/cm field strength with a 0.1cm gap. Compared to the conventional method, which involves DNA electroporation of Staphylococcus aureus RN4220 as an intermediate strain to overcome the restriction barrier, our proposed approach exhibits a higher level (3 log10 units) of transformation efficiency. Heat treatment was used to temporarily inactivate the recipient RM barrier. Other important parameters contributing to improved electro-transformation efficiency were growth stage for cell harvesting, the quantity of DNA, the transformation temperature and field strength. The approach described here may facilitate genetic manipulations of this opportunistic pathogen.

  10. DNA polymorphism of Mycobacterium tuberculosis PE_PGRS33 gene among clinical isolates of pediatric TB patients and its associations with clinical presentation.

    PubMed

    Wang, Jun; Huang, Yanfeng; Zhang, Aihua; Zhu, Chaomin; Yang, Zhenhua; Xu, Hongmei

    2011-07-01

    In vitro and in animal studies have suggested an important role for the Mycobacterium tuberculosis PE_PGRS33 protein in the pathogenesis of TB. A significant level of PE_PGRS33 gene DNA polymorphism among clinical isolates from adult tuberculosis (TB) patients and its association with clinical and epidemiological phenotypes of the disease has been found. To better understand the role of PE_PGRS33 protein in the pathogenesis pediatric TB, we investigated DNA polymorphism of the PE_PGRS33 gene among 101 of pediatric TB patients' isolates and assessed the relationship between the PE_PGRS33 sequence variation and clinical characteristics of TB. Twelve different PE_PGRS33 sequence variations representing 12 different alleles were observed among the 101 M. tuberculosis clinical isolates investigated. Of these 101 isolates, 62(59.41%) had PE_PGRS33 alleles that would result in a change in the amino acid sequence of the PE_PGRS33 protein. The degree of DNA polymorphism within individual M. tuberculosis isolates from pediatric TB patients was remarkably lower than that previously found in M. tuberculosis isolates from adults TB patients. The frequency distribution of isolates having PE_PGRS33 gene sequence variations was similar between Beijing and non-Beijing families of the pathogen. Patients having TB meningitis and negative PPD skin test results appeared to be more likely to be infected by isolates having a mutant type of the PE_PGRS33 gene than patients who had no TB meningitis (OR 2.54, 95% CI [1.11-5.84]) and patients who had positive PPD-skin test results (OR 4.26, 95% CI [1.14-12.86]), respectively. This study provides new insight into the molecular pathogenesis of pediatric TB.

  11. Detection and isolation of single tumor cells containing mutated DNA sequences

    NASA Astrophysics Data System (ADS)

    Leary, James F.; He, Feng; Reece, Lisa M.

    1999-04-01

    One of the problems in treating breast cancer patients is discovering the gene rearrangements that are occurring while the patient is in apparent remission. Spontaneous mutations in DNA sequences, particularly in tumor suppressor genes, can lead to the evolution of new clones of tumor cells that may be able to evade both clinical treatments and the patient's immune surveillance system. Isolation of these tumor clones is extremely difficult. Rare-event analysis and single-cell sorting techniques must be used to successfully detect and isolate these tumor clones. PCR amplification of selected gene sequences followed by TA cloning techniques can then be used to perform single-cell DNA sequencing in those gene regions. In this paper we present preliminary data showing successful detection and single-cell sorting of rare tumor clones from defined cell mixtures. Using TA cloning techniques and PCR we have been able to detect a single base-pair mutation in the PTEN tumor suppressor gene in single cells from a breast cancer cell line. Thus, while extremely difficult, it should in the future be possible to isolate tumor clones form a patient for subsequent molecular analyses of DNA mutations in critical gene regions.

  12. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography.

    PubMed

    Hilbrig, Frank; Freitag, Ruth

    2012-01-01

    Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.

  13. Typing and subtyping of 83 clinical isolates purified from surgically implanted silicone feeding tubes by random amplified polymorphic DNA amplification.

    PubMed

    Dautle, Melanie P; Ulrich, Ricky L; Hughes, Thomas A

    2002-02-01

    In this study, 83 clinical isolates purified from biofilms colonizing 18 silicone gastrostomy devices (12 "buttons" and six tubes converted to skin level devices) were selected for subtype characterization utilizing genetic analysis. The tubes, previously used for feeding, remained in place for 3 to 47 months (mean, 20.0 months) in children ranging in age from 6 months to 17 years. Classification of specific microbes using random amplified polymorphic DNA (RAPD) analysis revealed genetic similarities and differences among isolates belonging to the same genus. Both gram-positive and -negative bacteria were investigated, including 2 isolates of Bacillus brevis, 4 isolates of Bacillus licheniformis, 2 isolates of Bacillus pumilus, 3 isolates of Enterococcus durans, 19 isolates of Enterococcus faecalis, 8 isolates of Enterococcus faecium, 2 isolates of Enterococcus hirae, 7 isolates of Escherichia coli, 8 isolates of Lactobacillus plantarum, 19 isolates of Staphylococcus aureus, 2 isolates of Staphylococcus epidermidis, and 7 isolates of Staphylococcus saprophyticus. Amplified DNA fragments (amplicons) provided species-specific fingerprints for comparison by agarose gel electrophoresis. A total of 62 distinct RAPD types were categorized from the five genera studied. Typing analysis suggested cross acquisition of E. coli, E. faecalis, and S. aureus in three patient pairs. Genomic polymorphism detection proved efficient and reliable for classifying bacterial subtypes isolated from biofilms adhering to various portions of commonly employed enteral access tubes.

  14. Isolation of Proteins on Nascent DNA in Hypoxia and Reoxygenation Conditions.

    PubMed

    Olcina, Monica M; Giaccia, Amato J; Hammond, Ester M

    2016-01-01

    During S-phase both DNA replication and histone deposition must be co-ordinated at and around the replication fork. Replication stress can interfere with the fidelity of this process and can result in genomic instability. The study of proteins associated with DNA replication forks is important for a detailed understanding of DNA replication and chromatin assembly both under basal as well as replication stress conditions. iPOND (isolation of Proteins on Nascent DNA) allows the temporal study of proteins and protein modifications associated with replication forks in a variety of conditions, allowing the 'tracing' of protein association and histone deposition and maturation at active, stalled and damaged replication forks. Importantly, low oxygen (hypoxic) conditions, found in tumours, can result in replication stress. Here we describe the adaptation of the iPOND technique allowing the isolation of proteins and protein modifications specifically with replication forks undergoing hypoxia-induced replication stress. Furthermore, we describe the adaptation of this method for the study of factors associated with replication forks recovering from hypoxia-induced replication stress following periods of reoxygenation. These adaptations are important in order to study proteins associated with replication forks undergoing replication stress in physiologically relevant conditions.

  15. Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

    PubMed Central

    Darrasse, A; Kotoujansky, A; Bertheau, Y

    1994-01-01

    Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed. Images PMID:8117082

  16. Isolation and mapping of human chromosome 21 cDNA: Progress in constructing a chromosome 21 expression map

    SciTech Connect

    Jan-Fang Cheng; Boyartchuk, V.; Zhu Y.

    1994-09-01

    We have isolated 175 cDNA clones from a fetal brain library by direct cDNA selection using genomic DNA isolated from pools of human chromosome 21 (HC21) cosmids. DNA sequences have revealed that 16 of these cDNA clones contain overlapping sequences. Of the other 159 cDNA sequences, 10 match previously identified HC21 genes, and 9 match previously determined cDNA sequences, including the Wilms tumor related transcript (QM), the human testican cDNA, the mammalian calponin cDNA, and 6 anonymous expressed sequence tags. All isolated cDNAs were hybridized to their corresponding cosmids, which suggests that they originated from HC21. We have localized 92 cDNA clones to previously reported HC21q YACs. The remaining unmapped cDNAs contain either sequences not included in the isolated HC21q YACs or sequences that hybridize to yeast DNA. The cDNAs not included in the YACs should be useful in isolating new YACs to bridge the gaps. PCR primers were derived from 4 novel cDNA sequences that had been mapped to the YACs in the suspected Down syndrome region and used in RT-PCR analysis. All 4 primer sequences amplified RNA fragments with the expected sizes, suggesting that these sequences could be used for expression analysis. The construction of a chromosome 21 cDNA map not only is important in the refinement of physical maps, but also will identify a set of genes in the disease regions for detailed characterization. 30 refs., 2 figs., 2 tabs.

  17. Inhibition of N-methyl-N-nitrosourea-induced mutagenicity and DNA methylation by ellagic acid.

    PubMed Central

    Dixit, R; Gold, B

    1986-01-01

    Ellagic acid, a naturally occurring plant phenol, inhibits the activity of the direct-acting mutagen N-methyl-N-nitrosourea (MeNU) in Salmonella typhimurium TA100. Ellagic acid at 0.10, 0.25, 0.50, and 1.00 mM inhibited the mutagenicity of MeNU (0.40 mM) by 3%, 13%, 45%, and 60%, respectively. Ellagic acid (3 mM) also inhibited the mutagenic activity of N,N-dimethylnitrosamine (25-200 mM) in the presence of pyrazole-induced rat liver fraction S-9. The effect of ellagic acid on DNA methylation was studied by incubating 0, 0.72, 1.32, 2.64, and 6.60 mM ellagic acid with DNA (0.9 mM nucleotide) and [3H]MeNU (0.66 mM). HPLC analysis of DNA hydrolysates showed that ellagic acid caused a dose-dependent 36-84% decrease in O6-methylguanine but only a 20% decrease in the 7-methylguanine adduct. Under conditions where methylation at the O6 position of guanine in double-stranded DNA was inhibited 65% by ellagic acid, no significant inhibition of either O6- or 7-methylguanine formation was detected in single-stranded DNA. Affinity-binding studies revealed that [3H]ellagic acid binds equally to double-stranded or single-stranded DNA but that poly(dA X dT) binds 1.5 times as much ellagic acid as does poly(dG X dC). The binding of ellagic acid to DNA is dependent on the concentration of both ellagic acid and DNA. The specific inhibition of O6-methylguanine formation only in double-stranded DNA and the relatively low inhibition of 7-methylguanine formation rule out the possibility that ellagic acid prevents DNA alkylation by scavenging the electrophilic intermediate generated in the hydrolysis of MeNU. The results suggest that ellagic acid inhibition of MeNU-induced mutagenicity is due to specific inhibition of methylation at the O6 position of guanine through an ellagic acid-duplex DNA affinity-binding mechanism. PMID:3464940

  18. Influence of amino acids Shiff bases on irradiated DNA stability in vivo.

    PubMed

    Karapetyan, N H; Malakyan, M H; Bajinyan, S A; Torosyan, A L; Grigoryan, I E; Haroutiunian, S G

    2013-01-01

    To reveal protective role of the new Mn(II) complexes with Nicotinyl-L-Tyrosinate and Nicotinyl-L-Tryptophanate Schiff Bases against ionizing radiation. The DNA of the rats liver was isolated on 7, 14, and 30 days after X-ray irradiation. The differences between the DNA of irradiated rats and rats pre-treated with Mn(II) complexes were studied using the melting, microcalorimetry, and electrophoresis methods. The melting parameters and the melting enthalpy of rats livers DNA were changed after the X-ray irradiation: melting temperature and melting enthalpy were decreased and melting interval was increased. These results can be explained by destruction of DNA molecules. It was shown that pre-treatment of rats with Mn(II) complexes approximates the melting parameters to norm. Agarose gel electrophoresis data confirmed the results of melting studies. The separate DNA fragments were revealed in DNA samples isolated from irradiated animals. The DNA isolated from animals pre-treated with the Mn(II) chelates had better electrophoretic characteristics, which correspond to healthy DNA. Pre-treatment of the irradiated rats with Mn(II)(Nicotinil-L-Tyrosinate) and Mn(II)(Nicotinil-L-Tryptophanate)2 improves the DNA characteristics.

  19. Genotypic heterogeneity within Giardia lamblia isolates demonstrated by M13 DNA fingerprinting.

    PubMed Central

    Carnaby, S; Katelaris, P H; Naeem, A; Farthing, M J

    1994-01-01

    There has been considerable speculation regarding the possible relationship between the phenotypic and genotypic heterogeneity seen among human isolates of Giardia lamblia and the wide clinical spectrum of human giardiasis. Several workers have suggested that human giardiasis may be a mixed infection consisting of variant strains or subgroups which are present in the same infection and which are selectable, but it is not clear whether these apparent variant strains represent a truly heterogeneous infection or whether the genotypic heterogeneity observed is due to the susceptibility of the Giardia genome to a high rate of structural genetic rearrangement. We have therefore studied variation in Giardia intestinalis genotypes in 19 isolates in vitro and in vivo by using the technique of M13 DNA fingerprinting. Genotypes of isolates changed with time when cultured under standard conditions and when pressured with bile. Sequential isolates and their clones taken from a patient with chronic giardiasis both before and after several treatments with metronidazole had different genotypes. Finally, clones of isolate WB had different initial genotypes, which changed after 4 months in culture. These findings suggest that the apparent genotypic heterogeneity at least in these G. intestinalis isolates is more likely to be due to the plasticity of the Giardia genome than to the presence of a truly mixed population of strains within the same infection. Images PMID:8168952

  20. Modified salting-out method for DNA isolation from newborn cord blood nucleated cells.

    PubMed

    Noguera, N I; Tallano, C E; Bragós, I M; Milani, A C

    2000-01-01

    The present work describes modification of a widely used salting-out procedure to rapidly extract DNA suitable for PCR, using the ARMS method to amplify a target sequence in the beta-globin gene. The salting-out DNA extraction procedure did not completely remove or decrease the presence of inhibitors to PCR in a considerable number of cord blood samples. By introducing a simple phenol/chloroform step, before ethanol precipitation of the nucleic acid, to certain samples, we were able to eliminate or substantially reduce the presence of inhibitors to PCR without having to re-extract the samples.

  1. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  2. Identification and genetic homogeneity of Trichophyton tonsurans isolated from several regions by random amplified polymorphic DNA.

    PubMed

    Kim, J A; Takizawa, K; Fukushima, K; Nishimura, K; Miyaji, M

    1999-01-01

    Trichophyton tonsurans is an anthropophilic dermatophyte mostly causing tinea capitis and tinea corporis. This study was carried out to identify T. tonsurans and to clarify whether it has any genetic differences depending on the phenotype or region of isolation by random amplified polymorphic DNA (RAPD) analysis with three random primers. The assay was performed in 11 Korean, 2 Japanese, 2 Taiwanese, 5 Brazilian and 1 American isolates of T. tonsurans together with the other 10 anamorphic species of dermatophytes and 3 Arthroderma spp. All tested species of dermatophytes showed distinct bands and T. tonsurans was differentiated from other dermatophytes. It was most clearly differentiated from T. mentagrophytes by using primer 5'-GAAGGCTCCC-3' (OPAO-15). No difference was found in RAPD band patterns in all strains of T. tonsurans with these random primers. It was considered that T. tonsurans is a genetically homogeneous species regardless of its isolation regions, morphologic or physiologic characteristics.

  3. Genetic diversity of Clavispora lusitaniae isolated from Agave fourcroydes Lem, as revealed by DNA fingerprinting.

    PubMed

    Pérez-Brito, Daisy; Magaña-Alvarez, Anuar; Lappe-Oliveras, Patricia; Cortes-Velazquez, Alberto; Torres-Calzada, Claudia; Herrera-Suarez, Teófilo; Larqué-Saavedra, Alfonso; Tapia-Tussell, Raul

    2015-01-01

    This study characterized Clavispora lusitaniae strains isolated from different stages of the processing and early fermentation of a henequen (Agave fourcroydes) spirit produced in Yucatan, Mexico using a molecular technique. Sixteen strains identified based on morphological features, obtained from different substrates, were typed molecularly. Nine different versions of the divergent D1/D2 domain of the large-subunit ribosomal DNA sequence were identified among the C. lusitaniae strains. The greatest degree of polymorphism was found in the 90-bp structural motif of the D2 domain. The MSP-PCR technique was able to differentiate 100% of the isolates. This study provides significant insight into the genetic diversity of the mycobiota present during the henequen fermentation process, especially that of C. lusitaniae, for which only a few studies in plants have been published. The applied MSP-PCR markers were very efficient in revealing olymorphisms between isolates of this species.

  4. Low copy number DNA profiling from isolated sperm using the aureka®-micromanipulation system.

    PubMed

    Schneider, C; Müller, U; Kilper, R; Siebertz, B

    2012-07-01

    A new cell isolation technique linked to the aureka® micromanipulation system (aureka®) was used to pick sperm from mixed samples containing sperm and epithelial cells. Both cell types were stained using the HY-LITER™ high-resolution, fluorescent staining kit. To isolate a single sperm of interest under a fluorescent microscope, a specific microsphere picking technique was used. This sensitive and reliable cell identification and isolation technique enables low-copy-number (LCN) DNA profiling, as few as 20 sperm are sufficient for obtaining a full short tandem repeat (STR) profile without any allelic drop out. The presented protocol covers the whole workflow, from sample staining and cell pick up to STR analysis.

  5. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    DOEpatents

    Croteau, Rodney Bruce; Crock, John E.

    2005-01-25

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  6. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

    PubMed Central

    Devault, A; Lazure, C; Nault, C; Le Moual, H; Seidah, N G; Chrétien, M; Kahn, P; Powell, J; Mallet, J; Beaumont, A

    1987-01-01

    Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase. Images Fig. 1. Fig. 3. PMID:2440677

  7. Nucleic acid chemistry in the organic phase: from functionalized oligonucleotides to DNA side chain polymers.

    PubMed

    Liu, Kai; Zheng, Lifei; Liu, Qing; de Vries, Jan Willem; Gerasimov, Jennifer Y; Herrmann, Andreas

    2014-10-08

    DNA-incorporating hydrophobic moieties can be synthesized by either solid-phase or solution-phase coupling. On a solid support the DNA is protected, and hydrophobic units are usually attached employing phosphoramidite chemistry involving a DNA synthesizer. On the other hand, solution coupling in aqueous medium results in low yields due to the solvent incompatibility of DNA and hydrophobic compounds. Hence, the development of a general coupling method for producing amphiphilic DNA conjugates with high yield in solution remains a major challenge. Here, we report an organic-phase coupling strategy for nucleic acid modification and polymerization by introducing a hydrophobic DNA-surfactant complex as a reactive scaffold. A remarkable range of amphiphile-DNA structures (DNA-pyrene, DNA-triphenylphosphine, DNA-hydrocarbon, and DNA block copolymers) and a series of new brush-type DNA side-chain homopolymers with high DNA grafting density are produced efficiently. We believe that this method is an important breakthrough in developing a generalized approach to synthesizing functional DNA molecules for self-assembly and related technological applications.

  8. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  9. Isolation of fatty acids and aromatics from cell suspension cultures of Lavandula angustifolia.

    PubMed

    Topçu, Gülaçti; Herrmann, Gabriele; Kolak, Ufuk; Gören, C; Porzel, Andrea; Kutchan, Toni M

    2007-02-01

    Cell suspension cultures of Lavandula angustifolia Mill. ssp. angustifolia (syn.: L. officinalis Chaix.) afforded a fatty acid composition, cis and trans p-coumaric acids (=p-hydroxy cinnamic acids), and beta-sitosterol. The fatty acid composition was analyzed by GC-MS, and the structures of the isolated three compounds were determined by 1H- and 13C-NMR, and MS spectroscopic techniques.

  10. Insights from computational analysis of full-length β-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms

    PubMed Central

    Bhore, Subhash J.; Cha, Thye S.; Amelia, Kassim; Shah, Farida H.

    2014-01-01

    Background: Palm oil derived from fruits (mesocarp) of African oil palm (Elaeis guineensis Jacq. Tenera) and American oil palm (E. oleifera) is important for food industry. Due to high yield, Elaeis guineensis (Tenera) is cultivated on commercial scale, though its oil contains high (~54%) level of saturated fatty acids. The rate-limiting activity of beta-ketoacyl-[ACP] synthase-II (KAS-II) is considered mainly responsible for the high (44%) level of palmitic acid (C16:0) in the oil obtained from E. guineensis. Objective: The objective of this study was to annotate KAS-II cDNA isolated from American and African oil palms. Materials and Methods: The full-length E. oleifera KAS-II (EoKAS-II) cDNA clone was isolated using random method of gene isolation. Whereas, the E. guineensis KAS-II (EgTKAS-II) cDNA was isolated using reverse transcriptase polymerase chain reaction (RT-PCR) technique; and missing ends were obtained by employing 5’and 3’ RACE technique. Results: The results show that EoKAS-II and EgTKAS-II open reading frames (ORFs) are of 1689 and 1721 bp in length, respectively. Further analysis of the both EoKAS-II and EgTKAS-II predicted protein illustrates that they contains conserved domains for ‘KAS-I and II’, ‘elongating’ condensing enzymes, ‘condensing enzymes super-family’, and ‘3-oxoacyl-[ACP] synthase II’. The predicted protein sequences shows 95% similarity with each other. Consecutively, the three active sites (Cys, His, and His) were identified in both proteins. However, difference in positions of two active Histidine (His) residues was noticed. Conclusion: These insights may serve as the foundation in understanding the variable activity of KAS-II in American and African oil palms; and cDNA clones could be useful in the genetic engineering of oil palms. PMID:24678202

  11. Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation.

    PubMed

    Yang, J; Cao, Y; Cai, Y; Terada, F

    2010-07-01

    Natural populations of lactic acid bacteria (LAB) and silage fermentation of vegetable residues were studied. Fifty-two strains of LAB isolated from cabbage, Chinese cabbage, and lettuce residues were identified and characterized. The LAB strains were gram-positive and catalase-negative bacteria, which were divided into 6 groups (A to F) according to morphological and biochemical characteristics. The strains in group A were rods that did not produce gas from glucose and formed the d and l isomers of lactate. Groups B and C were homofermentative cocci that formed l-lactic acid. Groups D, E, and F were heterofermentative cocci that formed d-lactic acid. Based on 16S rDNA gene sequence analysis, group A to F strains were identified as Lactobacillus plantarum, Lactococcus piscium, Lactococcus lactis, Leuconostoc citreum, Weissella soli and Leuconostoc gelidum, respectively. The prevalent LAB, predominantly homofermentative lactobacilli, consisted of Lactobacillus plantarum (34.6%), Weissella soli (19.2%), Leuconostoc gelidum (15.4%), Leuconostoc citreum (13.5%), Lactococcus lactis (9.6%), and Lactococcus piscium (7.7%). Lactobacillus plantarum was the dominant member of the LAB population in 3 types of vegetable residues. These vegetable residues contained a high level of crude protein (20.2 to 28.4% of dry matter). These silages prepared by using a small-scale fermentation system were well preserved, with low pH and a relatively high content of lactate. This study suggests that the vegetable residues contain abundant LAB species and nutrients, and that they could be well preserved by making silage, which is a potentially good vegetable protein source for livestock diets.

  12. Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

    PubMed Central

    Prat, S; Cortadas, J; Puigdomènech, P; Palau, J

    1985-01-01

    The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins. Images PMID:3839076

  13. Genotyping and DNA microarray based characterization of Staphylococcus aureus isolates from rabbit carcasses.

    PubMed

    Merz, Axel; Stephan, Roger; Johler, Sophia

    2016-02-01

    Staphylococcus aureus can cause staphylococcal food poisoning. Although the organism is frequently detected on rabbit carcasses, little is known about the characteristics of S. aureus strains contaminating rabbit meat. In this study, 137 S. aureus isolates originating from 137 rabbit carcasses were spa typed and characterized by DNA microarray. The isolates were assigned to CC5, CC7, CC8, CC15, CC96, CC101, CC121, and ST890, and to 13 spa types (t056, t085, t091, t160, t179, t681, t741, t745, t1190, t1773, t4770, t8456, t14871). Enterotoxin genes detected included sea, sed, sej, and ser. In addition, the egc operon, encoding the newly described staphylococcal enterotoxins SEG/SEI/SElM/SElN/SElO/SElU, was found in all isolates except those of t091. While none of the examined isolates presented genes conferring methicillin, vancomycin, or aminoglycoside resistance, we frequently detected blaZ/I/R conferring resistance to penicillin. The isolates represented a heterogeneous group assigned to clonal lineages detected among humans and animals, with two spa types exclusively associated with rabbit meat (t4770, t8456).

  14. Molecular profiles of Venezuelan isolates of Trypanosoma sp. by random amplified polymorphic DNA method.

    PubMed

    Perrone, T M; Gonzatti, M I; Villamizar, G; Escalante, A; Aso, P M

    2009-05-12

    Nine Trypanosoma sp. Venezuelan isolates, initially presumed to be T. evansi, were collected from three different hosts, capybara (Apure state), horse (Apure state) and donkey (Guarico state) and compared by the random amplification polymorphic DNA technique (RAPD). Thirty-one to 46 reproducible fragments were obtained with 12 of the 40 primers that were used. Most of the primers detected molecular profiles with few polymorphisms between the seven horse, capybara and donkey isolates. Quantitative analyses of the RAPD profiles of these isolates revealed a high degree of genetic conservation with similarity coefficients between 85.7% and 98.5%. Ten of the primers generated polymorphic RAPD profiles with two of the three Trypanosoma sp. horse isolates, namely TeAp-N/D1 and TeGu-N/D1. The similarity coefficient between these two isolates and the rest, ranged from 57.9% to 68.4% and the corresponding dendrogram clustered TeAp-N/D1 and Te Gu-N/D1 in a genetically distinct group.

  15. Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India.

    PubMed

    Sutar, Sasmita Kumari Das; Gupta, Bhavna; Ranjit, Manoranjan; Kar, Shantanu Kumar; Das, Aparup

    2011-02-01

    The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

  16. Transcriptional analysis of the DNA polymerase gene of Bombyx mori parvo-like virus (China isolate).

    PubMed

    Wang, Yong-Jie; Chen, Ke-Ping; Yao, Qin; Han, Xu

    2007-04-01

    The Bombyx mori parvo-like virus (China isolate) DNA polymerase (BmDNV-3 dnapol) gene has been tentatively identified based on the presence of conserved motifs. In the present study, we perform a transcriptional analysis of the BmDNV-3 dnapol gene using the total RNA isolated from BmDNV-3 infected silkworm at different times. Northern blot analysis with a BmDNV-3 dnapol-specific riboprobe showed a major transcript of 3.3 kb. 5'-RACE revealed that the major transcription start point was located 20 nucleotides downstream of the TATA box. In a temporal expression analysis using differential RT-PCR, BmDNV-3 dnapol transcript was detected at low levels at 6 h.p.i., increased from 6 to 36 h.p.i., and remained fairly constant thereafter. Analysis of the predicted DNA polymerase sequence using neighborjoining and protein parsimony algorithms indicated that the predicted 1115-residue polypeptide contained five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The molecular phylogenetic analysis of this gene supported the placement of Bombyx mori parvo-like virus in a separate virus family.

  17. Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

    PubMed

    O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

    2015-08-03

    The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms.

  18. Nucleic acid isolation from ecological samples--fungal associations, mycorrhizae.

    PubMed

    Koide, Roger T

    2005-01-01

    Mycorrhizal fungi are among the most common symbioses found in terrestrial ecosystems, both natural and managed. They are important for many reasons, but most notably because of their positive effects on plant growth, which are mediated by their uptake of nutrients from the soil and transport of these to the roots. Moreover, many edible fungi are mycorrhizal. The study of mycorrhizal fungi has been hampered by the inability to identify species and individuals in the soil. This has been greatly aided by DNA-based methods, which first require the extraction of DNA. Herein, I discuss some general concerns that must be considered when extracting and purifying DNA from ecological samples and offer specific methods for soil, mycorrhizal roots, and fruiting bodies. These methods are rapid, safe, effective, relatively inexpensive, and convenient because they are based on commercially available kits.

  19. Infectivity and complete nucleotide sequence of cucumber fruit mottle mosaic virus isolate Cm cDNA.

    PubMed

    Rhee, Sun-Ju; Hong, Jin-Sung; Lee, Gung Pyo

    2014-07-01

    Three isolates of cucumber fruit mottle mosaic virus (CFMMV) were collected from melon, cucumber, and pumpkin plants in Korea. A full-length cDNA clone of CFMMV-Cm (melon isolate) was produced and evaluated for infectivity after T7 transcription in vitro (pT7CF-Cmflc). The complete CFMMV genome sequence of the infectious clone pT7CF-Cmflc was determined. The genome of CFMMV-Cm consisted of 6,571 nucleotides and shared high nucleotide sequence identity (98.8 %) with the Israel isolate of CFMMV. Based on the infectious clone pT7CF-Cmflc, a CaMV 35S-promoter driven cDNA clone (p35SCF-Cmflc) was subsequently constructed and sequenced. Mechanical inoculation with RNA transcripts of pT7CF-Cmflc and agro-inoculation with p35SCF-Cmflc resulted in systemic infection of cucumber and melon, producing symptoms similar to those produced by CFMMV-Cm. Progeny virus in infected plants was detected by RT-PCR, western blot assay, and transmission electron microscopy.

  20. Bacillus acidicola sp. nov., a novel mesophilic, acidophilic species isolated from acidic Sphagnum peat bogs in Wisconsin.

    PubMed

    Albert, Richard A; Archambault, Julieta; Rosselló-Mora, Ramón; Tindall, Brian J; Matheny, Mike

    2005-09-01

    A mesophilic, acidophilic, spore-forming bacterium, strain 105-2(T), was isolated from an acidic Sphagnum peat bog in Wisconsin, USA. Strain 105-2(T) has 16S rRNA gene sequence similarity to Bacillus sporothermodurans DSM 10599(T) and Bacillus oleronius DSM 9356(T) of 97.4 and 97.8%, respectively. The primary lipoquinone is MK-7 and the major fatty acids are 15:0 iso, 15:0 anteiso and 17:0 anteiso. The predominant polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and a glycolipid. The DNA G+C content was found to be 43.2 mol%. The phenotypic, chemotaxonomic and molecular analyses identified strain 105-2(T) as a novel Bacillus species, for which the name Bacillus acidicola is proposed. The type strain is 105-2(T) (=DSM 14745(T)=ATCC BAA-366(T)=NRRL B-23453(T)).

  1. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

    SciTech Connect

    Travis, G.H.; Sutcliffe, J.G.

    1988-03-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.

  2. [Preparative isolation of tri-, tetra-, penta- and hexapyrimidine nucleotides from hydrolysates of depurinated herring sperm DNA (author's transl)].

    PubMed

    Schott, H

    1979-04-21

    The pyrimidine nucleotides p(dC)3p, p(dT)3p and p(dT)4p and mixtures of the sequence isomers p(dC3, dT), (dC3, dT)p; p(dC3, dT)p; p(dC2, dT2)p; p(dC, dT3)p; p(dC3, dT2)p; p(dC2, dT3); p(dC2, dT3)p; p(dC, dT4)p; p(dC4, dT2); p(dC3, dT3); p(dC3, dT3)p and p(dC2, dT4)p have been isolated on a preparative scale from hydrolysates of depurinated herring sperm DNA. The DNA hydrolysate is first separated into a high- and a low-molecular weight pyrimidine nucleotide mixture by column chromatography at pH 7.5 on DEAE-cellulose. The high-molecular-weight pyrimidine nucleotide mixture is further separated into four peaks on QAE-Sephadex at pH 7.5. The second peak is re-chromatographed on QAE-Sephadex at pH 3.5. Pyrimidine nucleotides containing predominantly cytidylic acid units may thus be separated from these with predominantly thymidylic acid units. Subsequent separation according to number of phosphate groups at pH 7.5 on QAE-Sephadex yields products of 70--93% purity. In a final separation step, the pyrimidine nucleotides and mixtures of sequence isomers are once again chromatographed on QAE-Sephadex with 7 M urea at pH 7.5. The products thus obtained are generally chromatographically pure. Impurities which are not fully removed by column chromatography are separated by paper chromatography. The structure of the isolated DNA fragments and the composition of the mixtures of sequence isomers are determined from the chromatographic data, absorption characteristics and by enzymatic degradation.

  3. Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa.

    PubMed

    Meerak, Jomkhwan; Yukphan, Pattaraporn; Miyashita, Mika; Sato, Hajime; Nakagawa, Yasuyoshi; Tahara, Yasutaka

    2008-06-01

    Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.

  4. DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from cecal samples of chickens in Grenada

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To speciate Campylobacter strains from the ceca of chickens in Grenada by PCR and to evaluate DNA-based typing methods for the characterization of these isolates. Isolates were speciated with two multiplex PCR assays and were typed with flaA-RFLP, PFGE and MLST. Results confirmed that C. coli strain...

  5. Exopolysaccharides Isolated from Milk Fermented with Lactic Acid Bacteria Prevent Ultraviolet-Induced Skin Damage in Hairless Mice

    PubMed Central

    Morifuji, Masashi; Kitade, Masami; Fukasawa, Tomoyuki; Yamaji, Taketo; Ichihashi, Masamitsu

    2017-01-01

    Background: We studied the mechanism by which fermented milk ameliorates UV-B-induced skin damage and determined the active components in milk fermented with lactic acid bacteria by evaluating erythema formation, dryness, epidermal proliferation, DNA damage and cytokine mRNA levels in hairless mice exposed to acute UV-B irradiation. Methods: Nine week-old hairless mice were given fermented milk (1.3 g/kg BW/day) or exopolysaccharide (EPS) concentrate (70 mg/kg BW/day) orally for ten days. Seven days after fermented milk or EPS administration began, the dorsal skin of the mice was exposed to a single dose of UV-B (20 mJ/cm2). Results: Ingestion of either fermented milk or EPS significantly attenuated UV-B-induced erythema formation, dryness and epidermal proliferation in mouse skin. Both fermented milk and EPS were associated with a significant decrease in cyclobutane pyrimidine dimers and upregulated mRNA levels of xeroderma pigmentosum complementation group A (XPA), which is involved in DNA repair. Furthermore, administration of either fermented milk or EPS significantly suppressed increases in the ratio of interleukin (IL)-10/IL-12a and IL-10/interferon-gamma mRNA levels. Conclusion: Together, these results indicate that EPS isolated from milk fermented with lactic acid bacteria enhanced DNA repair mechanisms and modulated skin immunity to protect skin against UV damage. PMID:28098755

  6. Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

    PubMed Central

    Kasarda, D D; Okita, T W; Bernardin, J E; Baecker, P A; Nimmo, C C; Lew, E J; Dietler, M D; Greene, F C

    1984-01-01

    The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently. Images PMID:6589619

  7. Antileukemia component, dehydroeburicoic acid from Antrodia camphorata induces DNA damage and apoptosis in vitro and in vivo models.

    PubMed

    Du, Ying-Chi; Chang, Fang-Rong; Wu, Tung-Ying; Hsu, Yu-Ming; El-Shazly, Mohamed; Chen, Chieh-Fu; Sung, Ping-Jyun; Lin, Yan-Yu; Lin, Yi-Hsin; Wu, Yang-Chang; Lu, Mei-Chin

    2012-06-15

    Antrodia camphorata (AC) is a native Taiwanese mushroom which is used in Asian folk medicine as a chemopreventive agent. The triterpenoid-rich fraction (FEA) was obtained from the ethanolic extract of AC and characterized by high performance liquid chromatography (HPLC). FEA caused DNA damage in leukemia HL 60 cells which was characterized by phosphorylation of H2A.X and Chk2. It also exhibited apoptotic effect which was correlated to the enhancement of PARP cleavage and to the activation of caspase 3. Five major triterpenoids, antcin K (1), antcin C (2), zhankuic acid C (3), zhankuic acid A (4), and dehydroeburicoic acid (5) were isolated from FEA. The cytotoxicity of FEA major components (1-5) was investigated showing that dehydroeburicoic acid (DeEA) was the most potent cytotoxic component. DeEA activated DNA damage and apoptosis biomarkers similar to FEA and also inhibited topoisomerase II. In HL 60 cells xenograft animal model, DeEA treatment resulted in a marked decrease of tumor weight and size without any significant decrease in mice body weights. Taken together, our results provided the first evidence that pure AC component inhibited tumor growth in vivo model backing the traditional anticancer use of AC in Asian countries.

  8. Characterization of a DNA uptake reaction through the nuclear membrane of isolated yeast nuclei. [Saccharomyces cerevisiae

    SciTech Connect

    Tsuchiya, E.; Shakuto, S.; Miyakawa, T.; Fukui, S.

    1988-02-01

    Isolated yeast nuclei were able to incorporate /sup 3/H-labeled pJDB219 DNA in vitro in the presence of ATP and Mg/sup 2 +/. The number of plasmid molecules incorporated into each nucleus was calculated to be 60 under the conditions we used. Enzyme-histochemical staining of the incorporated biotinylated pJDB219 with streptavidin-biotinylated-peroxidase complex indicated a uniform distribution of the incorporated plasmids within each nucleus. After intranuclear incorporation, substrate pJDB219 DNAs (open and closed circular forms) were changed to the linear form and were weakly digested over the longer incubation period (over 60 min). Facile release of the once-incorporated plasmid DNA was never observable; discharge of the incorporated (/sup 3/H)pJDB219 during a 60-min incubation was less than 5%. The addition of adenylyl-imidodiphosphate, N,N'-dicyclohexylcarbodiimide (DCCD), or quercetin inhibited in vitro DNA uptake reaction. DCCD and quercetin inhibited the nuclear ATPase and apparent protein kinase, respectively; hence, the involvement of these enzymes in the nuclear DNA transport system was suggested.

  9. Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

    PubMed Central

    Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

    1986-01-01

    A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

  10. A new small temperate DNA phage BcP15 isolated from Burkholderia cepacia DR11.

    PubMed

    Hens, D K; Ghosh, A N; Kumar, R

    2005-12-01

    A Burkholderia cepacia DR11 strain was isolated during the survey of microorganisms from coastal water of deltaic Sunderbans. This strain always released temperate phage BcP15 into culture supernatant. UV irradiation of the strain also induced phage induction. The phage titer was 2.3 x 10(8). New temperate phage BcP15 has unusual structure. It has a hexagonal head, 65 nm in diameter and a tail 200 nm long, attached with single thick wavy tail fiber (424-705 nm). Phage DNA is double stranded 11.9 kb long. Southern hybridization result indicated that the phage DNA was in lysogenic state into the B. cepacia DR11 genome. SDS-PAGE of phage protein showed two major bands of molecular weight 20 kDa and 40 kDa.

  11. Ultrafast capillary electrophoresis isolation of DNA aptamer for the PCR amplification-based small analyte sensing.

    PubMed

    Fiore, Emmanuelle; Dausse, Eric; Dubouchaud, Hervé; Peyrin, Eric; Ravelet, Corinne

    2015-01-01

    Here, we report a new homogeneous DNA amplification-based aptamer assay for small analyte sensing. The aptamer of adenosine chosen as the model analyte was split into two fragments able to assemble in the presence of target. Primers were introduced at extremities of one fragment in order to generate the amplifiable DNA component. The amount of amplifiable fragment was quantifiable by Real-Time Polymerase Chain Reaction (RT-PCR) amplification and directly reliable on adenosine concentration. This approach combines the very high separation efficiency and the homogeneous format (without immobilization) of capillary electrophoresis (CE) and the sensitivity of real time PCR amplification. An ultrafast isolation of target-bound split aptamer (60 s) was developed by designing a CE input/ouput scheme. Such method was successfully applied to the determination of adenosine with a LOD of 1 μM.

  12. Ultrafast Capillary Electrophoresis Isolation of DNA Aptamer for the PCR Amplification-Based Small Analyte Sensing

    NASA Astrophysics Data System (ADS)

    Fiore, Emmanuelle; Dausse, Eric; Dubouchaud, Hervé; Peyrin, Eric; Ravelet, Corinne

    2015-08-01

    Here, we report a new homogeneous DNA amplification-based aptamer assay for small analyte sensing. The aptamer of adenosine chosen as the model analyte was split into two fragments able to assemble in the presence of target. Primers were introduced at extremities of one fragment in order to generate the amplifiable DNA component. The amount of amplifiable fragment was quantifiable by Real-Time Polymerase Chain Reaction (RT-PCR) amplification and directly reliable on adenosine concentration. This approach combines the very high separation efficiency and the homogeneous format (without immobilization) of capillary electrophoresis and the sensitivity of real time PCR amplification. An ultrafast isolation of target-bound split aptamer (60 s) was developed by designing a capillary electrophoresis input/ouput scheme. Such method was successfully applied to the determination of adenosine with a LOD of 1 µM.

  13. First isolation of tandemly repeated DNA sequences in New World vultures and phylogenetic implications.

    PubMed

    Keyser, C; Montagnon, D; Schlee, M; Ludes, B; Pfitzinger, H; Mangin, P

    1996-02-01

    A highly repeated DNA sequence composed of closely related subunits that ranged from 171 to 176 base pairs has been cloned and characterized in the king vulture (Sarcoramphus papa). Related sequences were also isolated in the black vulture (Coragyps atratus). This new family of avian repetitive DNA elements is here termed the "HaeIII family." Genomic DNAs from a number of avian species were probed with one of the king vulture restriction fragments. In the cathartids, the hybridization patterns showed no individual or sexual variations. A strong HaeIII ladder was present in the two aforementioned species as well as in the Andean condor (Vultur gryphus), but in the black vulture the bands of the ladder alternated in intensity. Weaker hybridization signals were obtained in two ciconids, the jabiru stork (Jabiru mycteria) and the white stork (Ciconia ciconia). The HaeIII repeat was not detected in accipitrid birds of prey, a Polyborinae falconid, pelecanids, and psittacids.

  14. Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

    PubMed

    Baldini, A; Archidiacono, N; Carbone, R; Bolino, A; Shridhar, V; Miller, O J; Miller, D A; Ward, D C; Rocchi, M

    1992-01-01

    We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

  15. Isolation and identification of a cDNA clone coding for an HLA-DR transplantation antigen alpha-chain.

    PubMed

    Gustafsson, K; Bill, P; Larhammar, D; Wiman, K; Claesson, L; Schenning, L; Servenius, B; Sundelin, J; Rask, L; Peterson, P A

    1982-10-01

    Membrane-bound mRNA was isolated from Raji cells and enriched for message coding for the HLA-DR transplantation antigen alpha-chain by sucrose gradient centrifugation. Double-stranded cDNA was constructed from this mRNA fraction, ligated to plasmid pBR322, and cloned into Escherichia coli. By hybrid selection, a plasmid, pDR-alpha-1, able to hybridize with mRNA coding for the HLA-DR alpha-chain was identified. From the nucleotide sequence of one end of the insert an amino acid sequence was predicted which is identical to part of the amino-terminal sequence of an HLA-DR alpha-chain preparation isolated from Raji cells. This clearly shows that pDR-alpha-1 carries almost the complete message for an HLD-DR alpha-chain. From the nucleotide sequence of this plasmid it will be possible to predict the primary structure of an HLA-DR alpha-chain.

  16. Isolation of novel cDNA encompassing the ADU balanced translocation break point in the DiGeorge critical region.

    PubMed

    Kim, M H; Hur, H; Park, J; Kim, Y J

    2001-03-01

    DiGeorge syndrome (DGS) is a developmental field defect of the third and fourth pharyngeal pouches that are associated with congenital heart defects, hypoparathyroidism, cell-mediated immunodeficiency, velopharyngeal insufficiency, and craniofacial anomalities. Approximately 90% of patients exhibit monosomy in the 22q11 region. In order to isolate the critical gene responsible for DGS, the cDNA libraries were screened with a probe containing the ADU balanced translocation break point, that is a locus reported in one patient (ADU) caused by a balanced translocation between chromosomes 22 and 2. Out of 10(6) clones, three independent overlapping clones were isolated, which were assumed to have originated from a single transcript, DGCR7. This transcript contained a 175-aa long open reading frame (ORF), encoding an acidic (pI = 5.81) and a proline-rich peptide, which are often found in the activation domain of several transcription factors. Also, it was predicted to be a nuclear protein. Northern hybridization detected an approx 1.9 kb transcript in all fetal and adult tissues tested, with strong expression in the fetal liver and kidney. In the case of adult tissues, strong expression was also detected in areas such as the heart, skeletal muscle, liver, and kidney.

  17. DNA Diagnostics: Nanotechnology-enhanced Electrochemical Detection of Nucleic Acids

    PubMed Central

    Wei, Fang; Lillehoj, Peter B.; Ho, Chih-Ming

    2010-01-01

    The detection of mismatched base pairs in DNA plays a crucial role in the diagnosis of genetic-related diseases and conditions, especially for early stage treatment. Among the various biosensors that have been employed for DNA detection, electrochemical sensors show great promise since they are capable of precise DNA recognition and efficient signal transduction. Advancements in micro- and nanotechnologies, specifically fabrication techniques and new nanomaterials, have enabled for the development of highly sensitive, highly specific sensors making them attractive for the detection of small sequence variations. Furthermore, the integration of sensors with sample preparation and fluidic processes enables for rapid, multiplexed DNA detection for point-of-care (POC) clinical diagnostics. PMID:20075759

  18. Anti-inflammatory and apoptotic activities of pomolic acid isolated from Cecropia pachystachya.

    PubMed

    Schinella, Guillermo; Aquila, Silvia; Dade, Martín; Giner, Rosa; Recio, María del Carmen; Spegazzini, Etile; de Buschiazzo, Perla; Tournier, Horacio; Ríos, José Luis

    2008-02-01

    The dichloromethane extract and pomolic acid ( 5) obtained from leaves of Cecropia pachystachya both reduced carrageenan-induced paw oedema in mice. Interestingly, while the triterpenoid inhibited the in vivo production of interleukin-1beta by 39 %, it had no effect on tumour necrosis factor-alpha production. We also demonstrated that both the dichloromethane extract and 5 inhibited the viability of human polymorphonuclear (PMN) cells in a time- and dose-dependent fashion. The PMN membrane integrity was determined with the aid of flow cytometry by means of the exclusion of propidium iodide as assay. Although the cell membrane integrity was altered, neither the extract nor 5 produced cellular necrosis. Moreover, the development of hypodiploid nuclei and DNA fragmentation in the PMN cells were both dependent on dose and time. Finally, in the annexin V-FITC binding assay, compound 5 increased the total of apoptotic cells by 42 % at 100 microM and by 71 % at 200 microM with respect to the control group. In conclusion, both the dichloromethane extract of ambay and isolated compound 5 inhibit the viability of PMN cells through apoptosis. Since they can regulate human neutrophil functions in this way, it is likely that these substances can also limit inflammation.

  19. Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds.

    PubMed

    Games, Patrícia D; Dos Santos, Izabela S; Mello, Erica O; Diz, Mariângela S S; Carvalho, André O; de Souza-Filho, Gonçalo A; Da Cunha, Maura; Vasconcelos, Ilka M; Ferreira, Beatriz Dos S; Gomes, Valdirene M

    2008-12-01

    The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.

  20. Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells

    SciTech Connect

    Ishibe, M.; Rosier, R.N.; Puzas, J.E. )

    1991-10-01

    Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.

  1. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    PubMed Central

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  2. Isolation and characterization of pyrimidine-psoralen-pyrimidine photodiadducts from DNA. [Ultraviolet radiation

    SciTech Connect

    Kanne, D.; Straub, K.; Hearst, J.E.; Rapoport, H.

    1982-12-01

    The isolation and characterization of pyrimidine-psoralen-pyrimidine photodiadducts from DNA are reported for the first time. For each of the four psoralens studied, a single pair of diastereomeric thymidine-psoralen-thymidine photodiadducts, each with cis-syn stereochemistry, was found to account for > 90% of the diadducts formed. Additionally, pulse-chase experiments that establish that these photo cross-links are formed by cycloaddition of a second thymidine residue to the 3,4 double bond (pyrone side) of an initially formed 4',5' (furan-side) psoralen-thymidine photomonoadduct have been carried out.

  3. Genotyping Clostridium botulinum toxinotype A isolates from patients using amplified rDNA restriction analysis.

    PubMed

    Pourshafie, M; Vahdani, P; Popoff, M

    2005-10-01

    In this study, the application of amplified rDNA restriction analysis (ARDRA) for characterizing Clostridium botulinum toxinotype A strains isolated from individuals with botulism was evaluated. Ten restriction enzymes were tested for their suitability in ARDRA as a typing method and HhaI was selected for the best outcome. Analysis of HhaI restriction profiles of the amplified products divided C. botulinum isolates into three clusters. Non-toxigenic Clostridium sporogenes strains showed an ARDRA restriction pattern that was distinct from those observed for C. botulinum. The successful use of ARDRA for subdivision of C. botulinum in this study confirmed that this technique is a powerful method for typing of C. botulinum toxinotype A clonal diversity. In addition, it is rapid, sensitive and simple.

  4. Novel lactic acid bacteria isolated from the bumble bee gut: Convivina intestini gen. nov., sp. nov., Lactobacillus bombicola sp. nov., and Weissella bombi sp. nov.

    PubMed

    Praet, Jessy; Meeus, Ivan; Cnockaert, Margo; Houf, Kurt; Smagghe, Guy; Vandamme, Peter

    2015-05-01

    Twelve isolates of lactic acid bacteria (LAB) were obtained in the course of a bumble bee gut microbiota study and grouped into four matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry clusters. Comparative 16S rRNA gene sequence analysis revealed that cluster 1 isolates, represented by strain LMG 28288(T), are most closely related to Lactobacillus apis (97.0% sequence similarity to that of L. apis LMG 26964(T)). Cluster 2 isolates represented by strain LMG 28290(T) are most closely related to Weissella hellenica (99.6% sequence similarity to that of W. hellenica LMG 15125(T)). The single cluster 3 and 4 isolates had identical 16S rRNA gene sequences which were 94.8% similar to that of Leuconostoc mesenteroides subsp. mesenteroides LMG 6893(T), their nearest phylogenetic neighbour. A polyphasic taxonomic study additionally including comparative pheS sequence analysis, DNA-DNA hybridization experiments, DNA G+C content analysis, (GTG)5-PCR fingerprinting and a biochemical characterization, demonstrated that cluster 1 isolates represent a novel Lactobacillus species for which we propose the name Lactobacillus bombicola sp. nov. with LMG 28288(T) (= DSM 28793(T)) as the type strain; and that cluster 2 isolates represent a novel Weissella species for which we propose the name Weissella bombi sp. nov. with LMG 28290(T) (= DSM 28794(T)) as the type strain. Cluster 3 and 4 isolates, in contrast, represented a very distinct, novel taxon that could be distinguished from members of the genera Leuconostoc and Fructobacillus, its nearest phylogenetic neighbours, by its cellular morphology, non-fructophilic metabolism and DNA G+C content. We therefore classify both isolates into a novel species representing a novel LAB genus for which the name Convivina intestini gen. nov., sp. nov. is proposed with LMG 28291(T) (= DSM 28795(T)) as the type strain.

  5. [Studies on the interaction of the metal complex of hydrazide of podophyllic acid with DNA].

    PubMed

    Wang, Ping-Hong; Zhang, Qi; Wang, Liu-Fang; Song, Yu-Min; Qu, Jian-Qiang; Liu, Ying-Qian

    2006-05-01

    The interaction between the metal complex of hydrazide of podophyllic acid and calf thymus (CT) DNA was studied by using absorption spectra, fluorescence spectra and DNA heat denaturation. It was found that the intensity of the maximal absorption peaks from absorption spectra is weakened in the presence of the metal complex of hydrazide of podophyllic acid compared with that in the absence of the metal complex. All the experimental results show that the intercalation mode was proved to exist between HDPP-Ni complexes and CT DNA.

  6. Synthesis of bile acid monosulphates by the isolated perfused rat kidney.

    PubMed Central

    Summerfield, J A; Gollan, J L; Billing, B H

    1976-01-01

    Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3% being converted into polar metabolities in 1h. These metabolities were neither glycine nor taurine conjugates, nor bile acid glucuronides, and on solovolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14C]chenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]sulphate was incorporated in chenodeoxycholic acid by the kidney, resulting in a similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man. PMID:942413

  7. Molecular characterization of clinical isolates of Moraxella catarrhalis by randomly amplified polymorphic DNA fingerprinting.

    PubMed

    Theoga Raj, Christol James; Shankar, Esaki Muthu; Rothan, Hussin A; Rao, Usha Anand

    2014-01-01

    Moraxella catarrhalis, a less virulent microorganism that colonizes the upper respiratory tract, has recently been associated with lower respiratory disease, especially in HIV-positive immunocompromised individuals and children. Here, we correlated the DNA clustering pattern of 24 clinical isolates of M. catarrhalis for β-lactamase production and drug resistance, from different disease groups using three different arbitrarily selected primers, P1 (5'-TCACGATGCA-3'), P14 (5'-GATCAAGTCC-3') and P17 (5'-GATCTGACAC-3'). M. catarrhalis revealed three distinct banding patterns with primer P1, four with P14 and P17. 71% (n = 17) of the isolates revealed pattern 2 with primer P1, which discriminated majority (12/21) of the isolates grouped under the major branch of the dendrogram. The minor branch had only three isolates. Separation of M. catarrhalis into two subpopulations (major and minor clusters) with primer P1 is suggestive of diverse genetic lineage. A high level of concordance between RAPD and antibiotic profile was observed. Clustering of M. catarrhalis recovered from different disease groups reflect the identical clinical background or the common geographical/temporal factors. The presence or absence of β-lactamase in a cluster confirmed their single source of origin.

  8. Surface water-borne multidrug and heavy metal-resistant Staphylococcus isolates characterized by 16S rDNA sequencing.

    PubMed

    Yilmaz, Fadime; Orman, Nazlı; Serim, Gamze; Kochan, Ceren; Ergene, Aysun; Icgen, Bulent

    2013-12-01

    Four Staphylococcus isolates having both multidrug- and multimetal-resistant ability were isolated from surface water. Further identification of the isolates was obtained through biochemical tests and 16S rDNA gene sequencing. One methicillin-resistant and two methicillin-sensitive isolates were determined as Staphylococcus aureus. The other isolate was identified as Staphylococcus warneri. The antibiotic and heavy metal resistance profiles of the Staphylococcus isolates were determined by using 26 antibiotics and 17 heavy metals. S. aureus isolates displayed resistance to most of the β-lactam antibiotics tested. All Staphylococcus isolates were resistant to heavy metals including silver, lithium, and barium. Due to a possible health risk of these pathogenic bacteria, a need exists for an accurate assessment of their acquired resistance to multiple drugs and metals.

  9. Isolation of SMA candidate genes from a YAC contig by direct selection of cDNA clones from normalized cDNA libraries

    SciTech Connect

    Ross, B.M.; Bonaldo, M.F.; Vitale, E.

    1994-09-01

    A YAC contig has been constructed across the spinal muscular atrophy (SMA) region of chromosome 5 (5q11-13). Further definition by pedigree analysis has yielded a minimal genetic region of 400 kb. For isolation of candidate genes in this region, the following cDNA selection method was hybridized to directionally cloned normalized (Cot 1 DNA-preannealed) cDNA libraries in the form of single-stranded circles. The libraries used were constructed from SMA infant brain and normal fetal liver+spleen. Hybridizing circles were eluted off the filter, partially converted into duplexes and electroporated into bacteria. The selected clones were then sequentially hybridized with a human Cot 1 DNA probe (BRL), and a probe made from the corresponding YAC DNA. Clones that hybridized only to the YAC DNA probe were verified to map to the critical region by genomic Southern analyses. Ten different cDNA clones have been isolated by this procedure so far. Three of them have been definitively mapped back to the region. Four of the ten clones are now completely sequenced. One clone shows sequence homology to a transcriptional initiation factor; another has homology to a prokaryotic attachment site sequence for the lipid moiety of membrane lipoproteins. Two clones show no homology to sequences represented in the public databases. We are continuing the full characterization of the cDNA clones as candidates for the SMA gene.

  10. A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes.

    PubMed Central

    Sturrock, S S; Dryden, D T

    1997-01-01

    The S subunits of type I DNA restriction/modification enzymes are responsible for recognising the DNA target sequence for the enzyme. They contain two domains of approximately 150 amino acids, each of which is responsible for recognising one half of the bipartite asymmetric target. In the absence of any known tertiary structure for type I enzymes or recognisable DNA recognition motifs in the highly variable amino acid sequences of the S subunits, it has previously not been possible to predict which amino acids are responsible for sequence recognition. Using a combination of sequence alignment and secondary structure prediction methods to analyse the sequences of S subunits, we predict that all of the 51 known target recognition domains (TRDs) have the same tertiary structure. Furthermore, this structure is similar to the structure of the TRD of the C5-cytosine methyltransferase, Hha I, which recognises its DNA target via interactions with two short polypeptide loops and a beta strand. Our results predict the location of these sequence recognition structures within the TRDs of all type I S subunits. PMID:9254696

  11. Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

    PubMed Central

    Ferreira, M A; Tooley, P W; Hatziloukas, E; Castro, C; Schaad, N W

    1996-01-01

    Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds. PMID:8572716

  12. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    SciTech Connect

    Cool, D.E.; Tonks, N.K.; Charbonneau, H.; Walsh, K.A.; Fischer, E.H.; Krebs, E.G. )

    1989-07-01

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3{prime} untranslated end, although a poly(A){sup +} tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a M{sub r} 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low M{sub r} PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.

  13. TECHNICAL BRIEF: Isolation of total DNA from postmortem human eye tissues and quality comparison between iris and retina

    PubMed Central

    Wang, Jay Ching Chieh; Wang, Aikun; Gao, Jiangyuan; Cao, Sijia; Samad, Idris; Zhang, Dean; Ritland, Carol; Cui, Jing Z.

    2012-01-01

    Background Recent genomic technologies have propelled our understanding of the mechanisms underlying complex eye diseases such as age-related macular degeneration (AMD). Genotyping postmortem eye tissues for known single nucleotide polymorphisms (SNPs) associated with AMD may prove valuable, especially when combined with information obtained through other methods such as immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA), and proteomics. Initially intending to genotype postmortem eye tissues for AMD-related SNPs, our group became interested in isolating and comparing the quality of DNA from the iris and retina of postmortem donor eyes. Since there is no previously published protocol in the literature on this topic, we present a protocol suitable for isolating high-quality DNA from postmortem eye tissues for genomic studies. Methods DNA from 33 retinal samples and 35 iris samples was extracted using the phenol-chloroform-isoamyl method from postmortem donor eye tissues. The quantity of DNA was measured with a spectrophotometer while the quality was checked using gel electrophoresis. The DNA samples were then amplified with PCR for the complement factor H (CFH) gene. The purified amplified products were then genotyped for the SNPs in the CFH gene. Results Regarding concentration, the retina yielded 936 ng/μl of DNA, while the iris yielded 78 ng/μl of DNA. Retinal DNA was also purer than iris DNA (260/280=1.78 vs. 1.46, respectively), and produced superior PCR results. Retinal tissue yielded significantly more DNA than the iris tissue per mg of sample (21.7 ng/μl/mg vs. 7.42 ng/μl/mg). Retinal DNA can be readily amplified with PCR, while iris DNA can also be amplified by adding bovine serum albumin. Overall, retinal tissues yielded DNA of superior quality, quantity, and suitability for genotyping and genomic studies. Conclusions The protocol presented here provides a clear and reliable method for isolating total DNA from postmortem eye

  14. Isolation of organic acids from large volumes of water by adsorption chromatography

    USGS Publications Warehouse

    Aiken, George R.

    1984-01-01

    The concentrations of dissolved organic carbon from most natural waters ranges from 1 to 20 milligrams carbon per liter, of which approximately 75 percent are organic acids. These acids can be chromatographically fractionated into hydrophobic organic acids, such as humic substances, and hydrophilic organic acids. To effectively study any of these organic acids, they must be isolated from other organic and inorganic species, and concentrated. Usually, large volumes of water must be processed to obtain sufficient quantities of material, and adsorption chromatography on synthetic, macroporous resins has proven to be a particularly effective method for this purpose. The use of the nonionic Amberlite XAD-8 and Amberlite XAD-4 resins and the anion exchange resin Duolite A-7 for isolating and concentrating organic acids from water is presented.

  15. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA.

    PubMed

    Sirbu, Bianca M; Couch, Frank B; Cortez, David

    2012-03-01

    Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol.

  16. Biosorption of acidic textile dyestuffs from aqueous solution by Paecilomyces sp. isolated from acidic mine drainage.

    PubMed

    Çabuk, Ahmet; Aytar, Pınar; Gedikli, Serap; Özel, Yasemin Kevser; Kocabıyık, Erçin

    2013-07-01

    Removal of textile dyestuffs from aqueous solution by biosorption onto a dead fungal biomass isolated from acidic mine drainage in the Çanakkale Region of Turkey was investigated. The fungus was found to be a promising biosorbent and identified as Paecilomyces sp. The optimal conditions for bioremediation were as follows: pH, 2.0; initial dyestuff concentration, 50 mg l(-1) for Reactive Yellow 85 and Reactive Orange 12, and 75 mg l(-1) for Reactive Black 8; biomass dosage, 2 g l(-1) for Reactive Yellow 85, 3 g l(-1) for Reactive Orange 12, 4 g l(-1) for Reactive Black 8; temperature, 25 °C; and agitation rate, 100 rpm. Zeta potential measurements indicated an electrostatic interaction between the binding sites and dye anions. Fourier transform infrared spectroscopy showed that amine, hydroxyl, carbonyl, and amide bonds were involved in the dyestuff biosorption. A toxicity investigation was also carried out before and after the biosorption process. These results showed that the toxicities for the reactive dyestuffs in aqueous solutions after biosorption studies decreased. The Freundlich and Langmuir adsorption models were used for the mathematical description of the biosorption equilibrium, and isotherm constants were evaluated for each dyestuff. Equilibrium data of biosorption of RY85 and RO12 dyestuffs fitted well to both models at the studied concentration and temperature.

  17. Senescence in isolated carnation petals : effects of indoleacetic Acid and inhibitors of protein synthesis.

    PubMed

    Wulster, G; Sacalis, J; Janes, H W

    1982-10-01

    Indoleacetic acid induces senescence in isolated carnation (Dianthus caryophyllus, cv. White Sim) petals, increasing the duration and amount of ethylene production. This effect is inhibited by Actinomycin D, an inhibitor of RNA synthesis, and cycloheximide, a translational inhibitor of protein synthesis. The ability of petals to respond to indoleacetic acid appears to be a function of physiological age. Indoleacetic acid is capable of enhancing ethylene evolution and senescence only in specific portions of the petal.

  18. Ent-kaurene synthase from the fungus Phaeosphaeria sp. L487. cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase in fungal gibberellin biosynthesis.

    PubMed

    Kawaide, H; Imai, R; Sassa, T; Kamiya, Y

    1997-08-29

    ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi. In plants, ent-kaurene is synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase and ent-kaurene synthase. A cell-free system of the fungus Phaeosphaeria sp. L487 converted labeled geranylgeranyl diphosphate to ent-kaurene. A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes. Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide. The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively. A fusion protein produced by expression of the cDNA in Escherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene. Amo-1618 completely inhibited the copalyl diphosphate synthase activity of the enzyme at 10(-6) M, whereas it did not inhibit the ent-kaurene synthase activity even at 10(-4) M. These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate catalytic sites for the two cyclization reactions.

  19. Isolation and characterization of halophilic lactic acid bacteria isolated from "terasi" shrimp paste: a traditional fermented seafood product in Indonesia.

    PubMed

    Kobayashi, Takeshi; Kajiwara, Michika; Wahyuni, Mita; Kitakado, Toshihide; Hamada-Sato, Naoko; Imada, Chiaki; Watanabe, Etsuo

    2003-10-01

    Lactic acid bacteria from "terasi" shrimp paste, a highly popular fermented seafood in Indonesia were isolated and characterized. Viable cell counts were 10(4) to 10(6) cfu/g on MRS medium. All the isolates were catalase-negative, gram-positive cocci and were able to grow at 15% NaCl. Numerical phenotypic analysis showed that the isolates clustered into one group. However, they could be classified into two types: the Tetragenococcus halophilus group and the T. muriaticus group as revealed by a restriction fragment length polymorphism (RFLP) analysis and sequencing of the 16S rRNA gene. This study is the first to show that both species of Tetragenococcus are distributed in Indonesian fermented foods.

  20. Amino acid racemization in amber-entombed insects: implications for DNA preservation

    NASA Technical Reports Server (NTRS)

    Bada, J. L.; Wang, X. S.; Poinar, H. N.; Paabo, S.; Poinar, G. O.

    1994-01-01

    DNA depurination and amino acid racemization take place at similar rates in aqueous solution at neutral pH. This relationship suggests that amino acid racemization may be useful in accessing the extent of DNA chain breakage in ancient biological remains. To test this suggestion, we have investigated the amino acids in insects entombed in fossilized tree resins ranging in age from <100 years to 130 million years. The amino acids present in 40 to 130 million year old amber-entombed insects resemble those in a modern fly and are probably the most ancient, unaltered amino acids found so far on Earth. In comparison to other geochemical environments on the surface of the Earth, the amino acid racemization rate in amber insect inclusions is retarded by a factor of >10(4). These results suggest that in amber insect inclusions DNA depurination rates would also likely be retarded in comparison to aqueous solution measurements, and thus DNA fragments containing many hundreds of base pairs should be preserved. This conclusion is consistent with the reported successful retrieval of DNA sequences from amber-entombed organisms.

  1. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

    PubMed Central

    2010-01-01

    Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should

  2. Isolation and characterization of cDNA for a plant mitochondrial phosphate translocator (Mpt1): ozone stress induces Mpt1 mRNA accumulation in birch (Betula pendula Roth).

    PubMed

    Kiiskinen, M; Korhonen, M; Kangasjärvi, J

    1997-10-01

    We have isolated by DDRT-PCR (differential-display reverse-transcription polymerase chain reaction) and cDNA library screening a 1.3 kb cDNA corresponding to a strongly ozone-inducible transcript from birch (Betula pendula Roth). Nucleotide sequence analysis suggests that it encodes a mitochondrial phosphate translocator protein (Pic), the first one isolated from plants. The isolated birch mitochondrial phosphate translocator cDNA (designated Mpt1) contains an open reading frame of 1092 bases encoding a 364 amino acid polypeptide. The deduced protein is 66% similar to bovine Pic isoform B. Comparison of the N-terminal amino acid sequence to known mammalian Pic proteins and the existence of an in-frame stop codon upstream of the initiation codon suggest that the isolated cDNA is full-length. Southern hybridization analysis of birch genomic DNA shows that Mpt1 is a single-copy gene. Accumulation of Mpt1 mRNA during oxidative stress imposed by ozone is detectable already at 2 h and it is at maximum ca. 12 h after the beginning of an 8 h ozone exposure (150 ppb). A second O3 peak at 48-56 h did not increase transcript levels further. O3 exposure for 2 h was sufficient for Mpt1 induction. Birch Mpt1 transcript levels remain at moderately low level during leaf development and is lower in roots and leaves when compared to young shoots undergoing wood formation and lignification.

  3. Comparative genomics profiling of clinical isolates of Aeromonas salmonicida using DNA microarrays

    PubMed Central

    Nash, John HE; Findlay, Wendy A; Luebbert, Christian C; Mykytczuk, Oksana L; Foote, Simon J; Taboada, Eduardo N; Carrillo, Catherine D; Boyd, Jessica M; Colquhoun, Duncan J; Reith, Michael E; Brown, Laura L

    2006-01-01

    Background Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations. Results Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested. Conclusion We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research. PMID:16522207

  4. Evaluation of three methods for DNA fingerprinting of Corynebacterium pseudotuberculosis strains isolated from goats in Poland.

    PubMed

    Stefańska, Ilona; Rzewuska, Magdalena; Binek, Marian

    2008-01-01

    Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.

  5. Isolation of anacardic acid from natural cashew nut shell liquid (CNSL) using supercritical carbon dioxide.

    PubMed

    Philip, Joseph Y N; Da Cruz Francisco, José; Dey, Estera S; Buchweishaija, Joseph; Mkayula, Lupituko L; Ye, Lei

    2008-10-22

    Solvent extracted cashew nut shell liquid (CNSL), conventionally known as natural CNSL, is a mixture of several alkenyl phenols. One of these alkenyl phenols is anacardic acid, which is present at the highest concentration. In view of anticipated industrial applications of anacardic acid, the objective of this work was to isolate anacardic acid from natural CNSL by supercritical carbon dioxide (scCO 2). In this study, the solubility data for natural CNSL in scCO 2 under a range of operating conditions of pressure (100, 200, and 300 bar), temperature (40 and 50 degrees C), and CO 2 flow rate (5, 10, and 15 g min (-1)) were established. The best scCO 2 working conditions were found to be 50 degrees C and 300 bar at a flow rate of 5 g min (-1) CO 2. Using 3 g of sample (CNSL/solid adsorbent = 1/2) under these scCO 2 conditions, it was possible to quantitatively isolate high purity anacardic acid from crude natural CNSL (82% of total anacardic acid) within 150 min. The anacardic acid isolated by scCO 2 was analyzed by different spectroscopic techniques (UV-vis, FT-IR, and (1)H NMR) and HPLC analysis, indicating that the anacardic acid isolated by scCO 2 has better quality than that obtained through a conventional method involving several chemical conversion steps.

  6. Analysis of 16S rRNA gene lactic acid bacteria (LAB) isolate from Markisa fruit (Passiflora sp.) as a producer of protease enzyme and probiotics

    NASA Astrophysics Data System (ADS)

    Hidayat, Habibi

    2017-03-01

    16S rRNA gene analysis of bacteria lactic acid (LAB) isolate from Markisa Kuning Fruit (Passiflora edulis var. flavicarpa) as a producer of protease enzyme and probiotics has been done. The aim of the study is to determine the protease enzyme activity and 16S rRNA gene amplification using PCR. The calculation procedure was done to M4 isolate bacteria lactic acid (LAB) Isolate which has been resistant to acids with pH 2.0 in the manner of screening protease enzyme activity test result 6.5 to clear zone is 13 mm againts colony diametre is 2 mm. The results of study enzyme activity used spectrophotometer UV-Vis obtainable the regression equation Y=0.02983+0.001312X, with levels of protein M4 isolate is 0.6594 mg/mL and enzyme activity of obtainable is 0.8626 unit/ml while the spesific enzyme activity produced is 1.308 unit/mg. Then, 16S rRNA gene amplificatiom and DNA sequencing has been done. The results of study showed that the bacteria species contained from M4 bacteria lactic acid (LAB) isolate is Weisella cibiria strain II-I-59. Weisella cibiria strain II-I-59 is one of bacteria could be utilized in the digestive tract.

  7. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus

    SciTech Connect

    Kwon, B.S.; Haq, A.K.; Pomerantz, S.H.; Halaban, R.

    1987-11-01

    Screening of a lambdagt11 human melanocyte cDNA library with antibodies against hamster tyrosinase resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were form related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.

  8. Absorption by DNA single strands of adenine isolated in vacuo: The role of multiple chromophores

    NASA Astrophysics Data System (ADS)

    Nielsen, Lisbeth Munksgaard; Pedersen, Sara Øvad; Kirketerp, Maj-Britt Suhr; Nielsen, Steen Brøndsted

    2012-02-01

    The degree of electronic coupling between DNA bases is a topic being up for much debate. Here we report on the intrinsic electronic properties of isolated DNA strands in vacuo free of solvent, which is a good starting point for high-level excited states calculations. Action spectra of DNA single strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (˜3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar to that for the adenine molecule and the dAMP mononucleotide. Desolvation has little effect on the bandwidth, which implies that inhomogenous broadening of the absorption bands in aqueous solution is of minor importance compared to, e.g., conformational disorder. Finally, at high photon energies, internal conversion competes with electron detachment since dissociation of the bare photoexcited ions on the microsecond time scale is measured.

  9. Expression and isolation of antimicrobial small molecules from soil DNA libraries.

    PubMed

    MacNeil, I A; Tiong, C L; Minor, C; August, P R; Grossman, T H; Loiacono, K A; Lynch, B A; Phillips, T; Narula, S; Sundaramoorthi, R; Tyler, A; Aldredge, T; Long, H; Gilman, M; Holt, D; Osburne, M S

    2001-04-01

    Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

  10. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  11. Method Optimization of Deoxyribonucleic Acid (DNA) Thin Films for Biotronics

    DTIC Science & Technology

    2011-09-01

    Added to the Spin-coater ......................................................................4 3.3 Comparison of Spin - Coating Speed and Sample...precipitate after centrifugation. ..............................3 Figure 3. Diagram of spin - coating method. First, the DNA-CTMA solution was pipetted onto... spin - coating speeds. ...................................................................................................................6 Figure 5

  12. Uracil misincorporation into DNA and folic acid supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE: We evaluated the relation between UrMis in different ...

  13. Switchable reconfiguration of nucleic acid nanostructures by stimuli-responsive DNA machines.

    PubMed

    Liu, Xiaoqing; Lu, Chun-Hua; Willner, Itamar

    2014-06-17

    CONSPECTUS: The base sequence in DNA dictates structural and reactivity features of the biopolymer. These properties are implemented to use DNA as a unique material for developing the area of DNA nanotechnology. The design of DNA machines represents a rapidly developing research field in the area of DNA nanotechnology. The present Account discusses the switchable reconfiguration of nucleic acid nanostructures by stimuli-responsive DNA machines, and it highlights potential applications and future perspectives of the area. Programmed switchable DNA machines driven by various fuels and antifuels, such as pH, Hg(2+) ions/cysteine, or nucleic acid strands/antistrands, are described. These include the assembly of DNA tweezers, walkers, a rotor, a pendulum, and more. Using a pH-oscillatory system, the oscillatory mechanical operation of a DNA pendulum is presented. Specifically, the synthesis and "mechanical" properties of interlocked DNA rings are described. This is exemplified with the preparation of interlocked DNA catenanes and a DNA rotaxane. The dynamic fuel-driven reconfiguration of the catenane/rotaxane structures is followed by fluorescence spectroscopy. The use of DNA machines as functional scaffolds to reconfigurate Au nanoparticle assemblies and to switch the fluorescence features within fluorophore/Au nanoparticle conjugates between quenching and surface-enhanced fluorescence states are addressed. Specifically, the fluorescence features of the different DNA machines are characterized as a function of the spatial separation between the fluorophore and Au nanoparticles. The experimental results are supported by theoretical calculations. The future development of reconfigurable stimuli-responsive DNA machines involves fundamental challenges, such as the synthesis of molecular devices exhibiting enhanced complexities, the introduction of new fuels and antifuels, and the integration of new payloads being reconfigured by the molecular devices, such as enzymes or

  14. Phytate degrading activities of lactic acid bacteria isolated from traditional fermented food

    NASA Astrophysics Data System (ADS)

    Damayanti, Ema; Ratisiwi, Febiyani Ndaru; Istiqomah, Lusty; Sembiring, Langkah; Febrisiantosa, Andi

    2017-03-01

    The objective of this study was to determine the potential of LAB with phytate degrading activity from fermented traditional food grain-based and legume-based. Lactic acid bacteria were isolated from different sources of traditional fermented food from Gunungkidul Yogyakarta Indonesia such as gembus tempeh (tofu waste), soybean tempeh, lamtoro tempeh (Leucaena bean) and kara tempeh. Isolation of LAB was performed using Total Plate Count (TPC) on de Man Rogosa Sharpe Agar (MRSA) medium supplemented with CaCO3. They were screened for their ability to degrade myo-inositol hexaphosphate or IP6 by using qualitative streak platemethod with modified de Man Rogosa-MorpholinoPropanesulfonic Acid Sharpe (MRS-MOPS) medium contained sodium salt of phytic acid as substrate and cobalt chloride staining (plate assay) method. The selected isolates were further assayed for phytase activities using quantitative method with spectrophotometer and the two selected isolates growth were optimized. Furthermore, thhe isolates that shown the highest phytase activity was characterized and identified using API 50 CH kitand 16S rRNA gene sequencing. The results showed that there were 18 LAB isolates obtained from samplesand 13 isolates were able to degrade sodium phytate based on qualitative screening. According to quantitative assay, the highest phytate degrading activities were found in TG-2(23.562 U/mL) and TG-1 (19.641 U/mL) isolated from gembus tempeh. The phytate activity of TG-2 was optimum at 37 °C with agitation, while the phytate activity of TG-1 was optimum at 45 °C without agitation. Characterization and identification of TG-2 isolate with the highest phytate degrading activity using API 50 CH and 16S rRNA showed that TG-2had homology with Lactobacillus fermentum. It could be concluded that LAB from from fermented traditional food grain-based and legume-based produced the extracellular phytase. Keywords: lactic acid bacteria, tempeh, phytatedegrading activity

  15. Isolation of a cDNA clone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway

    SciTech Connect

    Bernhard, W.R.; Thoma, S.; Botella, J.; Somerville, C.R. )

    1991-01-01

    A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.

  16. RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

    PubMed Central

    Sirois, Cherilyn M.; Jin, Tengchuan; Miller, Allison L.; Bertheloot, Damien; Nakamura, Hirotaka; Horvath, Gabor L.; Mian, Abubakar; Jiang, Jiansheng; Schrum, Jacob; Bossaller, Lukas; Pelka, Karin; Garbi, Natalio; Brewah, Yambasu; Tian, Jane; Chang, ChewShun; Chowdhury, Partha S.; Sims, Gary P.; Kolbeck, Roland; Coyle, Anthony J.; Humbles, Alison A.

    2013-01-01

    Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo. PMID:24081950

  17. Low-Temperature Production of Urocanic Acid by Spoilage Bacteria Isolated from Mahimahi (Coryphaena hippurus).

    PubMed

    Baranowski, J D

    1985-08-01

    Urocanic acid production was studied in 166 bacterial cultures isolated from mahimahi which had been stored for 14 days on ice in seawater. After 4 days of incubation at 10 degrees C in histidine-supplemented Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.), urocanic acid was measured by thin-layer chromatography. Fifty-eight of the cultures were positive for urocanic acid, with the eight most active isolates producing ca. 10 mg/100 ml. These observations, coupled with a report that urocanic acid may be the predominant histidine metabolite in fish held at low temperatures, all suggest that urocanic acid may be a useful alternative to histamine as a spoilage index in scombroid and other fish that are rich in endogenous histidine.

  18. [The effect of spermine on acid-base equilibrium in DNA molecule].

    PubMed

    Slonitskiĭ, S V; Kuptsov, V Iu

    1990-01-01

    The influence of spermine (Sp) on the acid-induced predenaturational and denaturational transitions in the DNA molecule structure has been studied by means of circular dichroism, spectrophotometric and viscometric titration at supporting electrolyte concentration 10 mM NaCl. The data available indicate that at [N]/[P] less than or equal to 0.60 (here [N] and [P] are molar concentrations of Sp nitrogen and DNA phosphours, respectively) the cooperative structural B----B(+)----S transitions are accompanied by the DNA double-helice winding. No competition for proton acceptor sites in the DNA molecule between H+ and Sp4+ cations has been observed when binding to neutral macromolecule. At 0.60 less than or equal to [N]/[P] less than or equal to 0.75 the displacement of the B----B(+)----S transitions midpoints to acidic pH region has been established. This is accompanied by DNA condensation and the appearance of differential scattering of circularly polarized light. The calculations carried out in the framework of the two-variable Manning theory have shown that the acid-induced reduction of the effective polyion charge density facilitates the Sp-induced DNA condensation. It has been shown that the acid-base equilibrium in the DNA molecule is determined by local [H+] in the 2-3 A hydrated monolayer of the macromolecule. An adequate estimation of [H+] can be obtained on the basis of the Poisson-Boltzman approach. The data obtained are consistent with recently proposed hypothesis of polyelectrolyte invariance of the acid-base equilibrium in the DNA molecule.

  19. Isolation of hydrophilic organic acids from water using nonionic macroporous resins

    USGS Publications Warehouse

    Aiken, G.R.; McKnight, Diane M.; Thorn, K.A.; Thurman, E.M.

    1992-01-01

    A method has been developed for the isolation of hydrophilic organic acids from aquatic environments using Amberlite* * Use of trade names in this report is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey. XAD-4 resin. The method uses a two column array of XAD-8 and XAD-4 resins in series. The hydrophobic organic acids, composed primarily of aquatic fulvic acid, are removed from the sample on XAD-8, followed by the isolation of the more hydrophilic organic acids on XAD-4. For samples from a number of diverse environments, more of the dissolved organic carbon was isolated on the XAD-8 resin (23-58%) than on the XAD-4 resin (7-25%). For these samples, the hydrophilic acids have lower carbon and hydrogen contents, higher oxygen and nitrogen contents, and are lower in molecular weight than the corresponding fulvic acids. 13C NMR analyses indicate that the hydrophilic acids have a lower concentration of aromatic carbon and greater heteroaliphatic, ketone and carboxyl content than the fulvic acid. ?? 1992.

  20. Genomic analyses of DNA transformation and penicillin resistance in Streptococcus pneumoniae clinical isolates.

    PubMed

    Fani, Fereshteh; Leprohon, Philippe; Zhanel, George G; Bergeron, Michel G; Ouellette, Marc

    2014-01-01

    Alterations in penicillin-binding proteins, the target enzymes for β-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing of S. pneumoniae R6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7 kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

  1. Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

    PubMed Central

    2012-01-01

    Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. Results The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. Conclusions The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. PMID:22703293

  2. The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size

    PubMed Central

    Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

    2016-01-01

    Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of −0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process. PMID:27104527

  3. The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.

    PubMed

    Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

    2016-04-20

    Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of -0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process.

  4. The storage period of the formalin-fixed paraffin-embedded tumor blocks does not influence the concentration and purity of the isolated DNA in a series of 83 renal and thyroid carcinomas.

    PubMed

    Nechifor-Boilă, Adela Corina; Loghin, Andrada; Vacariu, Victor; Halaţiu, Vasile Bogdan; Borda, Angela

    2015-01-01

    Optimal recovery of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues is highly dependent on a series of pre-extraction steps, mainly related (but not limited) to fixation. The aim of our study was to investigate if the storage period of the FFPE blocks had a significant effect on the isolated DNA. We examined the quantity and purity of the isolated DNA from 83 FFPE blocks, corresponding to malignant thyroid (n=28) and renal (n=55) carcinomas that had been stored in our department for up to eight years. The DNA extraction protocol was based on a precipitation method (MasterPure™ DNA Purification Kit, Epicentre), in accordance to the manufacturer instructions, optimized in our laboratory. A spectrophotometer was used to determine the yield (A260) and purity (A260/A280 ratio) of the isolated DNA. We successfully isolated good DNA quantity and purity from all our study cases (mean concentration: 223.4 ± 104.16 ng/μL; mean A260/A280 ratio: 1.68 ± 0.09). Moreover, no statistically significant differences were observed between tumor blocks stored for 2-3 years and 7-8 years, respectively, both in terms of DNA quantity (p=0.196) and purity (p=0.663). In conclusion, we successfully validated an efficient, reproducible DNA extraction technique that provided a good range of DNA concentrations and purity, regardless the type of tissue (thyroid or kidney). Moreover, we demonstrated that the storage period of the FFPE blocks does not have a significant influence on the DNA quantity and purity.

  5. Poly(N-vinylimidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids.

    PubMed

    Schemeth, Dieter; Noël, Jean-Christophe; Jakschitz, Thomas; Rainer, Matthias; Tessadri, Richard; Huck, Christian W; Bonn, Günther K

    2015-07-23

    In this study we report the novel polymeric resin poly(N-vinyl imidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids. The monomer to crosslinker ratio and the porogen composition were optimized for isolating phenolic acids diluted in acetonitrile at normal phase chromatography conditions, first. Acetonitrile serves as polar, aprotic solvent, dissolving phenolic acids but not interrupting interactions with the stationary phase due to the approved Hansen solubility parameters. The optimized resin demonstrated high loading capacities and adsorption abilities particularly for phenolic acids in both, acetonitrile and aqueous solutions. The adsorption behavior of aqueous standards can be attributed to ion exchange effects due to electrostatic interactions between protonated imidazole residues and deprotonated phenolic acids. Furthermore, adsorption experiments and subsequent curve fittings provide information of maximum loading capacities of single standards according to the Langmuir adsorption model. Recovery studies of the optimized polymer in the normal-phase and ion-exchange mode illustrate the powerful isolation properties for phenolic acids and are comparable or even better than typical, commercially available solid phase extraction materials. In order to prove the applicability, a highly complex extract of rosemary leaves was purified by poly(N-vinyl imidazole/ethylene glycol dimethacrylate) and the isolated compounds were identified using UHPLC-qTOF-MS.

  6. The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

    PubMed Central

    Senthong, Pattama; Millington, Christopher L.; Wilkinson, Oliver J.; Marriott, Andrew S.; Watson, Amanda J.; Reamtong, Onrapak; Eyers, Claire E.; Williams, David M.; Margison, Geoffrey P.; Povey, Andrew C.

    2013-01-01

    The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O6-carboxymethylguanine (O6-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O6-CMG is not a substrate for the human version of the DNA damage reversal protein O6-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O6-alkylguanine lesions by removing alkyl groups from the O6-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O6-methylguanine (O6-MeG) or O6-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O6-alkyl group and its transfer to the active-site cysteine residue of MGMT. O6-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O6-CMG is a potential causative agent. PMID:23335782

  7. Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni.

    PubMed Central

    Davis, A H; Blanton, R; Rottman, F; Maurer, R; Mahmoud, A

    1986-01-01

    Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni. To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals. We report here on the properties of three recombinant clones that derive from developmentally regulated genes. Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli. Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins. The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle. Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs. A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs. Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms. Clone 8-2 contained tandem cDNA inserts. One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms. Images PMID:3461448

  8. Antroquinonol D, isolated from Antrodia camphorata, with DNA demethylation and anticancer potential.

    PubMed

    Wang, Sheng-Chao; Lee, Tzong-Huei; Hsu, Chun-Hua; Chang, Yu-Jia; Chang, Man-Shan; Wang, Yi-Ching; Ho, Yuan-Soon; Wen, Wu-Che; Lin, Ruo-Kai

    2014-06-18

    DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in various human diseases, including cancer. A rational approach to preventing tumorigenesis involves the use of pharmacologic inhibitors of DNA methylation; these inhibitors should reactivate tumor suppressor genes (TSGs) in tumor cells and restore tumor suppressor pathways. Antroquinonol D (3-demethoxyl antroquinonol), a new DNMT1 inhibitor, was isolated from Antrodia camphorata and identified using nuclear magnetic resonance. Antroquinonol D inhibited the growth of MCF7, T47D, and MDA-MB-231 breast cancer cells without harming normal MCF10A and IMR-90 cells. The SRB assay showed that the 50% growth inhibition (GI50) in MCF7, T47D, and MDA-MB-231 breast cancer cells following treatment with antroquinonol D was 8.01, 3.57, and 25.08 μM, respectively. d-Antroquinonol also inhibited the migratory ability of MDA-MB-231 breast cancer cells in wound healing and Transwell assays. In addition, antroquinonol D inhibited DNMT1 activity, as assessed by the DNMT1 methyltransferase activity assay. As the cofactor SAM level increased, the inhibitory effects of d-antroquinonol on DNMT1 gradually decreased. An enzyme activity assay and molecular modeling revealed that antroquinonol D is bound to the catalytic domain of DNMT1 and competes for the same binding pocket in the DNMT1 enzyme as the cofactor SAM, but does not compete for the binding pocket in the DNMT3B enzyme. An Illumina Methylation 450 K array-based assay and real-time PCR assay revealed that antroquinonol D decreased the methylation status and reactivated the expression of multiple TSGs in MDA-MB-231 breast cancer cells. In conclusion, we showed that antroquinonol D induces DNA demethylation and the recovery of multiple tumor suppressor genes, while inhibiting breast cancer growth and migration potential.

  9. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes.

    PubMed

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2'-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of -22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL(-1). In the presence of divalent Ca(2+), the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca(2+)/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca(2+) and the synthesized copolymer on DNA transfection was observed. The use of Ca(2+) or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca(2+)/DNA polyplex could serve as a promising candidate for gene delivery.

  10. Deoxyribonucleic acid (DNA)-Ni-nanostrands composites for EMI shielding

    NASA Astrophysics Data System (ADS)

    Ouchen, Fahima; Wilson, Benjamin G.; Yaney, Perry P.; Salour, Michael M.; Grote, James G.

    2016-09-01

    In this study, we demonstrated the use of DNA-CTMA (DC) in combination with Nickel Nanostrands (NiNs) for application in Electromagnetic Interference (EMI) shielding. The addition of NiNs fillers to DC led to films with higher shielding effectiveness (SE) than when Silver nanoparticles were used. An enhanced EMI shielding effectiveness (SE) was also achieved by the fabrication of the DC-NiNs shielding film structure in a layered architecture. Very thin layer of Guanine ( 60 nm) were inserted between layers of DNA-NiNs ( 100um each) to total a thickness of 500um of the shielding film. An increase of the SE by 6-8 dB for the layered structure as compared to the bulk thick film with NiNs loadings up to 10 wt%. At higher loadings (>10 wt. %), a significant physical degradation of the films was observed for all films regardless of the thickness or the process of fabrication.

  11. Role of amino acid insertions on intermolecular forces between arginine peptide condensed DNA helices: implications for protamine-DNA packaging in sperm.

    PubMed

    DeRouchey, Jason E; Rau, Donald C

    2011-12-09

    In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.

  12. Evaluation of the probiotic characteristics of newly isolated lactic acid bacteria.

    PubMed

    Aswathy, Ravindran Girija; Ismail, Bindhumol; John, Rojan Pappy; Nampoothiri, Kesavan Madhavan

    2008-12-01

    Lactic acid bacteria were isolated from fermented vegetables, sour dough, milk products, sheep and human excreta. The newly isolated cultures were evaluated for a number of probiotic characteristics like bile salt resistance, salt tolerance in general, survival in low pH, hydrophobicity of the cell surface, resistance to low phenol concentration, antimicrobial activity and susceptibility pattern against vancomycin and erythromycin. The selected cultures were further screened for their ability to produce the nutraceticals such as folic acid and exopolysaccharide (EPS). Two potent isolates, CB2 (from cabbage) and SD2 (from sour dough) were found to produce both extracellular and intracellular folate. One of the isolates from yogurt (MC-1) and the one from whey (W3) produced significant amount of EPS with a maximum production of 8.79 +/- 0.05 g/l by MC-1.

  13. Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries.

    PubMed

    Suárez, V B; Quiberoni, A; Binetti, A G; Reinheimer, J A

    2002-10-01

    Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.

  14. Bioprocess development for docosahexaenoic acid from novel fungal isolate of Fusarium solani.

    PubMed

    Jini, Sugatha; Hridya, Azhikodan; Pandey, Ashok; Binod, Parameswaran

    2015-06-01

    Fungal cultures were isolated from soil samples collected from the Western Ghats regions of Kerala. Primary screening of isolated strains were done by Sudan black staining method and 15 lipid producing cultures were isolated. The fatty acid profiling of the positive strains were analyzed for docosahexaenoic acid (DHA) production. Selected oleaginous cultures were grown in submerged culture condition to study the biomass yield and poly unsaturated fatty acid, DHA production. The optimization of production process under submerged conditions was carried out using statistical experimental design and confirmation of DHA was done by GC analysis. Maximum DHA production of 150 mg/l was achieved on 4 days of incubation at submerged condition in the presence of glucose as carbon source.

  15. A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP.

    PubMed Central

    Kim, C S; Lee, C H; Shin, J S; Chung, Y S; Hyung, N I

    1997-01-01

    Because DNA degradation is mediated by secondary plant products such as phenolic terpenoids, the isolation of high quality DNA from plants containing a high content of polyphenolics has been a difficult problem. We demonstrate an easy extraction process by modifying several existing ones. Using this process we have found it possible to isolate DNAs from four fruit trees, grape (Vitis spp.), apple (Malus spp.), pear (Pyrus spp.) and persimmon (Diospyros spp.) and four species of conifer, Pinus densiflora, Pinus koraiensis,Taxus cuspidata and Juniperus chinensis within a few hours. Compared with the existing method, we have isolated high quality intact DNAs (260/280 = 1.8-2.0) routinely yielding 250-500 ng/microl (total 7.5-15 microg DNA from four to five tissue discs). PMID:9023124

  16. [First successful DNA isolation and profiling from bone using an approach that is non-destructive toward bone surface].

    PubMed

    Pluta, Dominika; Lebioda, Arleta; Jonkisz, Anna; Dobosz, Tadeusz

    Commonly used destructive DNA isolation techniques consist of pulverization which leads to the complete destruction of smaller bones or irreversible damage to larger bones through the cutting of extensive fragments. The procedure is totally unacceptable when handling bones which are valuable for anthropological or religious reasons, as museum exhibits or due to emotional factors. Since the Department's team has already resolved this problem in the case of human teeth (A. Pilecka), efforts have been undertaken to develop a similar non-destructive technique for the isolation of DNA from human bones. To the best knowledge of the authors of the report, the study has yielded the world's first STR profile derived from DNA isolated from human bones by a technique that is non-destructive toward the bone surface.

  17. Switchable mechanical DNA ``arms'' operating on nucleic acid scaffolds associated with electrodes or semiconductor quantum dots

    NASA Astrophysics Data System (ADS)

    Pelossof, Gilad; Tel-Vered, Ran; Liu, Xiaoqing; Willner, Itamar

    2013-09-01

    Functional footholds linked to DNA scaffolds associated with surfaces provide nano-engineered assemblies acting as switching devices. By the assembly of a β-cyclodextrin receptor on one foothold, and a ferrocene-modified nucleic acid on a second foothold, the switchable and reversible, fuel-driven activation of ``molecular arms'' proceeds, transduced by electrochemical or optical signals.Functional footholds linked to DNA scaffolds associated with surfaces provide nano-engineered assemblies acting as switching devices. By the assembly of a β-cyclodextrin receptor on one foothold, and a ferrocene-modified nucleic acid on a second foothold, the switchable and reversible, fuel-driven activation of ``molecular arms'' proceeds, transduced by electrochemical or optical signals. Electronic supplementary information (ESI) available: Experimental procedures, time-dependent deactivation of a DNA ``arm'' using a DNA anti-fuel, and control experiments, excluding β-cyclodextrin from the systems. See DOI: 10.1039/c3nr02653a

  18. Isolation, characterization, and ecology of sulfur-respiring crenarchaea inhabiting acid-sulfate-chloride-containing geothermal springs in Yellowstone National Park.

    PubMed

    Boyd, Eric S; Jackson, Robert A; Encarnacion, Gem; Zahn, James A; Beard, Trevor; Leavitt, William D; Pi, Yundan; Zhang, Chuanlun L; Pearson, Ann; Geesey, Gill G

    2007-10-01

    Elemental sulfur (S(0)) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S(0)-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72 degrees C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81 degrees C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S(0), and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S(0) flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.

  19. Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park▿ †

    PubMed Central

    Boyd, Eric S.; Jackson, Robert A.; Encarnacion, Gem; Zahn, James A.; Beard, Trevor; Leavitt, William D.; Pi, Yundan; Zhang, Chuanlun L.; Pearson, Ann; Geesey, Gill G.

    2007-01-01

    Elemental sulfur (S0) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S0-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S0, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S0 flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively. PMID:17720836

  20. A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

    PubMed Central

    Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

    2014-01-01

    The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

  1. Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones.

    PubMed

    Ozdemir-Kaynak, Elif; Yesil-Celiktas, Ozlem

    2015-10-01

    A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol-chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2min instead of 24h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8±0.2°C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40ng/μl and proved to be a superior alternative to the phenol-chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.

  2. A rapid and efficient method for isolating high quality DNA from leaves of carnivorous plants from the Drosera genus.

    PubMed

    Biteau, Flore; Nisse, Estelle; Hehn, Alain; Miguel, Sissi; Hannewald, Paul; Bourgaud, Frédéric

    2012-07-01

    Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 μg/g of fresh weight to 35.29 μg/g depending of the species of Drosera, with a A₂₆₀/A₂₈₀ ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h.

  3. Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L. epidermis.

    PubMed

    Guerrero, Consuelo; Martín-Rufián, M; Reina, José J; Heredia, Antonio

    2006-01-01

    A cDNA encoding an acyl-CoA binding protein (ACBP) homologue has been cloned from a cDNA library made from mRNA isolated from epidermis of young leaves of Agave americana L. The derived amino acid sequence reveals a protein corresponding to the membrane-associated form of ACBPs only previously described in Arabidopsis and rice. Northern blot analysis showed that the A. americana ACBP gene is mainly expressed in the epidermis of mature zone of the leaves. The epidermis of A. americana leaves have a well developed cuticle with the highest amounts of the cuticular components waxes, cutin and cutan suggesting a potential role of the protein in cuticle formation.

  4. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

    SciTech Connect

    Vieira, P.; De Waal-Malefyt, R.; Dang, M.N.; Johnson, K.E.; Kastelein, R.; Fiorentino, D.F.; DeVries, J.E.; Roncarolo, M.G.; Mosmann, T.R.; Moore, K.W. )

    1991-02-15

    The authors demonstrated the existence of human cytokine synthesis inhibitory factor (DSIF) (interleukin 10 (IL-10)). cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BDRFL. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this way, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.

  5. Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology

    PubMed Central

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

  6. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli.

  7. Superimposed Code Theoretic Analysis of Deoxyribonucleic Acid (DNA) Codes and DNA Computing

    DTIC Science & Technology

    2010-01-01

    hybridization that occurs between a DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research addresses how the...are 5′→3′ and strands with strikethrough are 3′→5′. A dsDNA duplex formed between a strand and its reverse complement is called a Watson - Crick (WC...3’ 5’ 3’ 5’TACGCGACTTTC3’ 5’GAAAGTCGCGTA3’ ATCAAACGATGC GCATCGTTTGAT Watson Crick (WC) Duplexes TACGCGACTTTC

  8. A trimethoxyellagic acid glucuronide from Conocarpus erectus leaves: isolation, characterization and assay of antioxidant capacity.

    PubMed

    Ayoub, Nahla A

    2010-03-01

    The new trimethoxy-ellagic glycoside, 3,3',4'-tri-O-methylellagic acid 4-O-beta-glucupyranuronide and twelve known phenolics were isolated from the leaves of Conocarpus erectus L. (Combretaceae). Structures of all compounds were determined on the basis of spectroscopic methods and chemical degradation. The new compound, together with four of the isolated known constituents and the plant extract itself, showed potent inhibitory effect against reactive oxygen species attack on salicylic acid in a dose-dependent manner adopting xanthine/hypoxanthine oxidase assay.

  9. Bacteriocin-Producing Lactic Acid Bacteria Isolated from Traditional Fermented Food

    PubMed Central

    Kormin, Salasiah; Rusul, Gulam; Radu, Son; Ling, Foo Hooi

    2001-01-01

    Lactic Acid Bacteria (LAB) isolated from several traditional fermented foods such as “tempeh”, “tempoyak” and “tapai” were screened for the production of bacteriocin. One strain isolated from “tempeh” gives an inhibitory activity against several LAB. The strain was later identified as Lactobacillus plantarum BS2. Study shows that the inhibitory activity was not caused by hydrogen peroxide, organic acids or bacteriophage. The bacteriocin production was maximum after 10 hours of incubation with an activity of 200 AU/ml. The bacteriocin was found to be sensitive towards trypsin, α-chymotrypsin, β-chymotrypsin, α-amylase and lysozyme. PMID:22973159

  10. Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

    2011-01-01

    Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

  11. DnaJ sequences of Bacillus cereus strains isolated from outbreaks of hospital infection are highly similar to Bacillus anthracis.

    PubMed

    Zhang, Jiwei; van Hung, Pham; Hayashi, Masahiro; Yoshida, Shigeru; Ohkusu, Kiyofumi; Ezaki, Takayuki

    2011-07-01

    Bacillus cereus is becoming an important nomosomial pathogen because of frequent isolation from blood cultures and from severe systemic infections. To differentiate highly pathogenic outbreak strain of B. cereus from other sources of the Bacillus cereus, we attempted to analyze their dnaJ sequences. Assays indicated that dnaJ sequence similarity of all of 52 blood culture isolates of B. cereus ranged from 92.8% to 100%. The distance between B. anthracis and B. cereus except six outbreak isolates ranged from 3.8% to 6.4%. The dnaJ sequences of six outbreak strains of B. cereus (GTC 02891, GTC 02896, GTC 02916, GTC 02917, GTC 03221, and GTC 03222) were closely related to those of B. anthracis (99.2%-99.5% sequence similarity). Ba813 sequences were only found in the six outbreak strains of B. cereus. The other pathogenic factors of B. anthracis were not found in these six outbreak strains, with the exception of GTC 02891 (cap-positive). The six outbreak strains formed clear β-hemolytic colonies on a sheep blood agar plate. Our findings suggest that outbreak strains of B. cereus isolated from blood cultures are likely to have the risk of causing serious infection, and dnaJ and Ba813 are important markers to identify such strains. Phylogenetic analysis of dnaJ and MLST revealed that the six outbreak strains of B. cereus are closely related to B. anthracis.

  12. Associations between whole peripheral blood fatty acids and DNA methylation in humans

    PubMed Central

    de la Rocha, Carmen; Pérez-Mojica, J. Eduardo; León, Silvia Zenteno-De; Cervantes-Paz, Braulio; Tristán-Flores, Fabiola E.; Rodríguez-Ríos, Dalia; Molina-Torres, Jorge; Ramírez-Chávez, Enrique; Alvarado-Caudillo, Yolanda; Carmona, F. Javier; Esteller, Manel; Hernández-Rivas, Rosaura; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio; Lund, Gertrud

    2016-01-01

    Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5’UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage. PMID:27181711

  13. Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

    PubMed Central

    Fojta, M; Vetterl, V; Tomschik, M; Jelen, F; Nielsen, P; Wang, J; Palecek, E

    1997-01-01

    Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. PMID:9129832

  14. Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

    PubMed

    Yao, Dongbao; Song, Tingjie; Sun, Xianbao; Xiao, Shiyan; Huang, Fujian; Liang, Haojun

    2015-11-11

    Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures.

  15. Binding of retinoic acid receptor heterodimers to DNA. A role for histones NH2 termini.

    PubMed

    Lefebvre, P; Mouchon, A; Lefebvre, B; Formstecher, P

    1998-05-15

    The retinoic acid signaling pathway is controlled essentially through two types of nuclear receptors, RARs and RXRs. Ligand dependent activation or repression of retinoid-regulated genes is dependent on the binding of retinoic acid receptor (RAR)/9-cis-retinoic acid receptor (RXR) heterodimers to retinoic acid response element (RARE). Although unliganded RXR/RAR heterodimers bind constitutively to DNA in vitro, a clear in vivo ligand-dependent occupancy of the RARE present in the RARbeta2 gene promoter has been reported (Dey, A., Minucci, S., and Ozato, K. (1994) Mol. Cell. Biol. 14, 8191-8201). Nucleosomes are viewed as general repressors of the transcriptional machinery, in part by preventing the access of transcription factors to DNA. The ability of hRXRalpha/hRARalpha heterodimers to bind to a nucleosomal template in vitro has therefore been examined. The assembly of a fragment from the RARbeta2 gene promoter, which contains a canonical DR5 RARE, into a nucleosome core prevented hRXRalpha/hRARalpha binding to this DNA, in conditions where a strong interaction is observed with a linear DNA template. However, histone tails removal by limited proteolysis and histone hyperacetylation yielded nucleosomal RAREs able to bind to hRXRalpha/hRARalpha heterodimers. These data establish therefore the role of histones NH2 termini as a major impediment to retinoid receptors access to DNA, and identify histone hyperacetylation as a potential physiological regulator of retinoid-induced transcription.

  16. Analysis of chromosome-sized DNA and genome typing of isolated strains of Taylorella equigenitalis.

    PubMed

    Matsuda, M; Asami, Y; Miyazawa, T; Samata, T; Isayama, Y; Honda, M; Ide, Y

    1994-01-01

    Analysis of chromosome-sized DNA and genome typing of Taylorella equigenitalis NCTC11184, Kentucky 188, and five strains of T. equigenitalis isolated in Japan were carried out. The three restriction enzymes used, ApaI, NaeI and NotI, cleaved the genomic DNAs of five Japanese strains of T. equigenitalis into relatively limited numbers of restriction fragments, which were well resolved on crossed-field gel electrophoresis (CFGE). The respective profiles after CFGE of the restriction fragments from all five strains were essentially identical to each other after digestion by ApaI, NaeI or NotI. Hence it appears that these strains have a common genome type with respect to these three restriction enzymes. It was also shown that the respective profiles from these strains were essentially different from those of T. equigenitalis NCTC11184 and those of Kentucky 188 after digestion with ApaI, NaeI or NotI.

  17. Isolation and characterization of two homologous cDNA clones from Torpedo electromotor neurons.

    PubMed

    Ngsee, J K; Scheller, R H

    1989-10-01

    Two homologous cDNA clones were isolated from a Torpedo california electric lobe lambda gt11 expression library using a polyclonal antiserum directed against proteins associated with synaptic vesicles. Northern blotting reveals an 8- to 9-kb transcript in the electric lobe and the spinal cord, but not in the brain or other non-neuronal tissues. Antibodies generated against a fusion protein synthesized in Escherichia coli reacted with a 85- to 90-kD species in the neurons of the electric lobe. The immunoreactivity is associated with microsomal membranes and can be extracted readily with high salt. Immunohistochemical studies demonstrated a sparse punctate staining pattern in the cell body which colocalized with a subpopulation of post-Golgi vesicles.

  18. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification.

    PubMed

    Mauk, Michael G; Liu, Changchun; Song, Jinzhao; Bau, Haim H

    2015-10-20

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of "lab on a chip" NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase ("membrane") to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 10³ virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  19. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    PubMed Central

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed. PMID:27600235

  20. Isolation and characterization of hyaluronic acid from the liver of marine stingray Aetobatus narinari.

    PubMed

    Sadhasivam, Giji; Muthuvel, Arumugam; Pachaiyappan, Abirami; Thangavel, Balasubramanian

    2013-03-01

    Although hyaluronic acid research pursuits ahead in exploring its biomedical perspective, very limited investigations were carried out in their isolation shape view point, furthermore, most of the investigations were targeted towards the terrestrial source. To swerve from that, the present study was projected through the marine superstore, where in high molecular weight hyaluronic acid of 13, 65,863 Da was isolated from the liver of stingray Aetobatus narinari. The purified HA was confirmed at the preliminary level by their stains all dye binding nature. Their analytical composition including carbon, hydrogen, nitrogen, N-acetyl glucosamine, glucuronic acid contents was analysed. The HA was characterized by agarose-gel electrophoresis, FTIR, HPTLC, and (1)H NMR. The DPPH radical scavenging activity of HA and its reducing power was evident to all the tested concentrations, but lower than that of ascorbic acid. HA showed significant inhibition against the proliferation of cells, substantiating its influence in regulation of cell functions.

  1. Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA.

    PubMed Central

    Evans, C T; Ledesma, D B; Schulz, T Z; Simpson, E R; Mendelson, C R

    1986-01-01

    A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene. Images PMID:3018730

  2. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  3. Molecular dynamics simulations of G-DNA and perspectives on the simulation of nucleic acid structures

    PubMed Central

    šponer, Jiří; Cang, Xiaohui; Cheatham, Thomas E.

    2013-01-01

    The article reviews the application of biomolecular simulation methods to understand the structure, dynamics and interactions of nucleic acids with a focus on explicit solvent molecular dynamics simulations of guanine quadruplex (G-DNA and G-RNA) molecules. While primarily dealing with these exciting and highly relevant four-stranded systems, where recent and past simulations have provided several interesting results and novel insight into G-DNA structure, the review provides some general perspectives on the applicability of the simulation techniques to nucleic acids. PMID:22525788

  4. The aflatoxin B1 isolating potential of two lactic acid bacteria

    PubMed Central

    Hamidi, Adel; Mirnejad, Reza; Yahaghi, Emad; Behnod, Vahid; Mirhosseini, Ali; Amani, Sajad; Sattari, Sara; Darian, Ebrahim Khodaverdi

    2013-01-01

    Objective To determine lactic acid bacteria's capability to enhance the process of binding and isolating aflatoxin B1 and to utilize such lactic acid bacteria as a food supplement or probiotic products for preventing absorption of aflatoxin B1 in human and animal bodies. Methods In the present research, the bacteria were isolated from five different sources. For surveying the capability of the bacteria in isolating aflatoxin B1, ELISA method was implemented, and for identifying the resultant strains through 16S rRNA sequencing method, universal primers were applied. Results Among the strains which were isolated, two strains of Lactobacillus pentosus and Lactobacillus beveris exhibited the capability of absorbing and isolating aflatoxin B1 by respectively absorbing and discharging 17.4% and 34.7% of the aforementioned toxin existing in the experiment solution. Conclusions Strains of Lactobacillus pentosus and Lactobacillus beveris were isolated from human feces and local milk samples, respectively. And both strains has the ability to isolate or bind with aflatoxin B1. PMID:23998015

  5. Zinc complexes of the antibacterial drug oxolinic acid: structure and DNA-binding properties.

    PubMed

    Tarushi, Alketa; Psomas, George; Raptopoulou, Catherine P; Kessissoglou, Dimitris P

    2009-06-01

    The neutral mononuclear zinc complexes with the quinolone antibacterial drug oxolinic acid in the absence or presence of a nitrogen donor heterocyclic ligand 2,2'-bipyridine or 1,10-phenanthroline have been synthesized and characterized. The experimental data suggest that oxolinic acid is on deprotonated mode acting as a bidentate ligand coordinated to the metal ion through the ketone and one carboxylato oxygen atoms. The crystal structures of (chloro)(oxolinato)(2,2'-bipyridine)zinc(II), 2, and bis(oxolinato)(1,10-phenanthroline)zinc(II), 3, have been determined with X-ray crystallography. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the DNA-binding constants have been calculated. Competitive studies with ethidium bromide (EB) have shown that complex 3 exhibits the ability to displace the DNA-bound EB indicating that it binds to DNA in strong competition with EB.

  6. Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures.

    PubMed

    Majoros, L; Kardos, G; Belák, A; Maráz, A; Asztalos, L; Csánky, E; Barta, Z; Szabó, B

    2003-11-01

    We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua. Although C. norvegensis is considered a pseudohypha-producing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua.

  7. Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2000-01-01

    A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

  8. Unambiguous typing of canine adenovirus isolates by deoxyribonucleic acid restriction-endonuclease analysis.

    PubMed Central

    Assaf, R; Marsolais, G; Yelle, J; Hamelin, C

    1983-01-01

    Viral deoxyribonucleic acid extracted from a limited number of cells infected with canine adenovirus type 1 or type 2 was cleaved with several restriction endonucleases. Agarose gel electrophoresis of the limit digests showed stable differences between the canine adenovirus type 1 and type 2 cleavage patterns. Rapid and accurate typing of large numbers of clinical isolates may thus be done by deoxyribonucleic acid restriction-endonuclease analysis. Images Fig. 1. Fig. 2. PMID:6321002

  9. Isolation and cDNA cloning of a potassium channel peptide toxin from the sea anemone Anemonia erythraea.

    PubMed

    Hasegawa, Yuichi; Honma, Tomohiro; Nagai, Hiroshi; Ishida, Masami; Nagashima, Yuji; Shiomi, Kazuo

    2006-10-01

    A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.

  10. Characterization of Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss) feed and larvae: safety, DNA fingerprinting, and bacteriocinogenicity.

    PubMed

    Araújo, Carlos; Muñoz-Atienza, Estefanía; Poeta, Patrícia; Igrejas, Gilberto; Hernández, Pablo E; Herranz, Carmen; Cintas, Luis M

    2016-05-03

    The use of lactic acid bacteria (LAB) as probiotics constitutes an alternative or complementary strategy to chemotherapy and vaccination for disease control in aquaculture. The objectives of this work were (1) the in vitro safety assessment of 8 Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) feed and larvae; (2) the evaluation of their genetic relatedness; (3) the study of their antimicrobial/bacteriocin activity against fish pathogens; and (4) the biochemical and genetic characterization of the bacteriocin produced by the strain displaying the greatest antimicrobial activity. Concerning the safety assessment, none of the pediococci showed antibiotic resistance nor produced hemolysin or gelatinase, degraded gastric mucin, or deconjugated bile salts. Four strains (50%) produced tyramine or putrescine, but the corresponding genes were not amplified by PCR. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting allowed clustering of the pediococci into 2 well-defined groups (68% similarity). From the 8 pediococci displaying direct antimicrobial activity against at least 3 out of 9 fish pathogens, 6 strains (75%) were identified as bacteriocin producers. The bacteriocin produced by P. acidilactici L-14 was purified, and mass spectrometry and DNA sequencing revealed its identity to pediocin PA-1 (PedPA-1). Altogether, our results allowed the identification of 4 (50%) putatively safe pediococci, including 2 bacteriocinogenic strains. ERIC-PCR fingerprinting was a valuable tool for genetic profiling of P. acidilactici strains. This work reports for the first time the characterization of a PedPA-1-producing P. acidilactici strain isolated from an aquatic environment (rainbow trout larvae), which shows interesting properties related to its potential use as a probiotic in aquaculture.

  11. Human synaptonemal complex protein 1 (SCP1): Isolation and characterization of the cDNA and chromosomal localization of the gene

    SciTech Connect

    Meuwissen, R.L.J.; Meerts, I.; Heyting, C.

    1997-02-01

    Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes (homologs) during meiotic prophase. They consist of two proteinaceous axes, one along each homolog, that are connected along their length by numerous transverse filaments (TFs). The cDNA encoding one major component of TFs of SCs of the rat, rnSCP1, has recently been isolated and characterized. In this paper we describe the isolation and characterization of the cDNA encoding the human protein homologous to rnSCP1, hsSCP1. hsSCP1 and rnSCP1 have 75% amino acid identity. The most prominent structural features and amino acid sequence motifs of rnSCP1 have been conserved in hsSCP1. Most probably, hsSCP1 is functionally homologous to rnSCP1. The hsSCP1 gene was assigned to human chromosome 1p12-p13 by fluorescence in situ hybridization. 44 refs., 4 figs.

  12. Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles

    PubMed Central

    Gott, Jonatha M.; Naegele, Gregory M.; Howell, Scott J.

    2016-01-01

    Mitochondrial RNAs in the acellular slime mold Physarum polycephalum contain nucleotides that are not encoded in the mitochondrial genes from which they are transcribed. These site-specific changes are quite extensive, comprising ~4% of the residues within mRNAs and ~2% of rRNAs and tRNAs. These “extra” nucleotides are added co-transcriptionally, but the means by which this is accomplished have not been elucidated. The cox1 mRNA also contains four sites of C to U changes, which occur post-transcriptionally, most likely via targeted deamination. The currently available in vitro systems for studying P. polycephalum editing are limited in that the template is the entire ~63,000 bp mitochondrial genome. This presents a significant challenge when trying to define the signals that specify editing sites. In an attempt to overcome this issue, a method for introducing DNA into isolated P. polycephalum mitochondria via electroporation has been developed. Exogenous DNA is expressed, but the transcripts synthesized from these templates are not edited under the conditions tested. However, transcripts derived from the mitochondrial genome are accurately edited after electroporation, indicating that the editing machinery is still functional. These findings suggest that this method may ultimately provide a feasible approach to elucidating editing signals. PMID:27983641

  13. Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles.

    PubMed

    Sheline, C; Melendy, T; Ray, D S

    1989-01-01

    We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.

  14. Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

    PubMed Central

    Elhag, G A; Thomas, F J; McCreery, T P; Bourque, D P

    1992-01-01

    Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco. Images PMID:1542565

  15. Isolation, Solubility, and Characterization of D-Mannitol Esters of 4-Methoxybenzeneboronic Acid.

    PubMed

    Lopalco, Antonio; Marinaro, William A; Day, Victor W; Stella, Valentino J

    2017-02-01

    The purpose of this study was to determine the aqueous solubility of a model phenyl boronic acid, 4-methoxybenzeneboronic acid, as a function of pH both in the absence and in the presence of varying D-mannitol concentration. Solid isolated D-mannitol esters were characterized by differential scanning calorimetry, thermogravimetric analysis, powder X-ray diffraction, and single-crystal X-ray studies, and the boronic acid-to-D-mannitol ratio was quantified by HPLC. Hydrolysis of the monoester was studied using UV spectral differences between the monoester and the parent boronic acid. Two D-mannitol esters of 4-methoxybenzeneboronic acid were isolated. The triboronate ester was very insoluble whereas a symmetrical monoboronate monohydrate was also less soluble than the parent. Both esters were crystalline. The monoboronate monohydrate was, however, more soluble than the parent at alkaline pH values due to its lower pKa value (6.53) compared to the parent acid (9.41). Hydrolysis of the monoboronate was extremely fast when even small amount of water was added to dry acetonitrile solutions of the ester. The hydrolysis was buffer concentration dependent and apparent pH sensitive with hydrolysis accelerated by acid. Implications affecting the formulation of future boronic acid drugs are discussed.

  16. Cloning of the. gamma. -aminobutyric acid (GABA). rho. sub 1 cDNA: A GABA receptor subunit highly expressed in the retina

    SciTech Connect

    Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr. ); O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi ); Uhl, G.R. Johns Hopkins Univ. School of Medicine, Baltimore, MD )

    1991-04-01

    Type A {gamma}-aminobutyric acid (GABA{sub A}) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA{sub A} subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA {rho}{sub 1}, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

  17. NtPolI-like1 and NtPolI-like2, bacterial DNA polymerase I homologs isolated from BY-2 cultured tobacco cells, encode DNA polymerases engaged in DNA replication in both plastids and mitochondria.

    PubMed

    Ono, Yuriko; Sakai, Atsushi; Takechi, Katsuaki; Takio, Susumu; Takusagawa, Mari; Takano, Hiroyoshi

    2007-12-01

    Two cDNAs encoding homologs of bacterial DNA polymerase I were isolated from cultured tobacco (Nicotiana tabacum) BY-2 cells, and the corresponding genes were named NtPolI-like1 and NtPolI-like2. High sequence similarity suggested that they are orthologous genes each derived from respective parental species of N. tabacum, an allotetraploid plant. Each of the NtPolI-like1/2 gene products had a putative transit peptide for plastid localization at the N-terminus, followed by a 3'-5' exonuclease domain in the internal region, and a DNA polymerase domain in the C-terminal region. Among family A DNA polymerases, NtPolI-like proteins formed, together with other plant DNA polymerase I homologs, a phylogenetic group distinct from mitochondrial DNA polymerase gamma in animals and fungi, as well as eukaryotic cell nuclear-localized repair enzymes. In contrast to computer predictions, experiments with green fluorescent protein (GFP) fusion protein and Western blotting analysis suggested dual targeting of the gene products to both plastids and mitochondria. The recombinant NtPolI-like2 protein exhibited DNA polymerase activity in vitro. Their biochemical character roughly coincided with those of the 116 kDa DNA polymerases found in the plastid and mitochondrial nuclei (nucleoids) isolated from BY-2 cells. Pre-treatment of the organelle nuclear extracts with anti-NtPolI-like antibody removed most of the DNA polymerase activity. Reverse transcription-PCR (RT-PCR) and Western blotting analyses demonstrated transient activation of NtPolI-like gene expression in the initial phase of cell proliferation, exactly when the 116 kDa DNA polymerases in the isolated organelle nuclei were activated and preferential synthesis of organelle DNAs occurred. Taken together, our results suggest that NtPolI-like1/2 genes encode DNA polymerases engaged in DNA replication in both plastids and mitochondria.

  18. High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries.

    PubMed

    Malausa, Thibaut; Gilles, André; Meglécz, Emese; Blanquart, Hélène; Duthoy, Stéphanie; Costedoat, Caroline; Dubut, Vincent; Pech, Nicolas; Castagnone-Sereno, Philippe; Délye, Christophe; Feau, Nicolas; Frey, Pascal; Gauthier, Philippe; Guillemaud, Thomas; Hazard, Laurent; Le Corre, Valérie; Lung-Escarmant, Brigitte; Malé, Pierre-Jean G; Ferreira, Stéphanie; Martin, Jean-François

    2011-07-01

    Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.

  19. A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates.

    PubMed Central

    Li, C; Ferguson, D A; Ha, T; Chi, D S; Thomas, E

    1993-01-01

    HindIII-digested DNA fragments derived from an EcoRI-digested 6.5-kb fragment of chromosomal DNA prepared from Helicobacter pylori ATCC 43629 (type strain) were cloned into the pUC19 vector. A 0.86-kb insert was identified as a potential chromosomal DNA probe. The specificity of the probe was evaluated by testing 166 non-H. pylori bacterial strains representing 38 genera and 91 species which included aerobic, anaerobic, and microaerophilic flora of the upper and lower gastrointestinal tracts. None of the 166 non-H. pylori strains hybridized with this probe (100% specificity), and the sensitivity of this probe was also 100% when H. pylori isolates from 72 patients with gastritis and with the homologous ATCC type strain were tested by dot blot hybridization. The capability of this probe for differentiating between strains of H. pylori was evaluated by Southern blot hybridization of HaeIII-digested chromosomal DNA from 68 clinical isolates and the homologous ATCC type strain of H. pylori. Fifty-one unique hybridization patterns were seen among the 69 strains tested, demonstrating considerable genotypic variation among H. pylori clinical isolates. We propose that this probe would be of significant value for conducting epidemiologic studies. Images PMID:8370744

  20. Isolation of immunomodulatory triterpene acids from a standardized rose hip powder (Rosa canina L.).

    PubMed

    Saaby, Lasse; Jäger, Anna Katharina; Moesby, Lise; Hansen, Erik Wind; Christensen, Søren Brøgger

    2011-02-01

    A previously published systematic review and a metaanalysis have concluded that the consumption of standardized rose hip powder (Rosa canina L.) can reduce pain in osteoarthritis patients. Synovial inflammation has been suggested to play an important role in the pathogenesis of osteoarthritis and mainly to involve infiltration of the synovial membrane by macrophages. Therefore, the immunomodulatory effect of standardized rose hip powder of Rosa canina L. was investigated and active principles isolated using the Mono Mac 6 cell line as a model for human macrophages. Treatment of Mono Mac 6 cells with the residue of a crude dichloromethane extract of rose hip powder significantly and concentration dependently inhibited the lipopolysaccharide induced interleukin-6 release. Through bioassay-guided fractionation the immunomodulatory effect of the dichloromethane extract was correlated to a mixture of three triterpene acids; oleanolic acid, betulinic acid and ursolic acid (IC(50) 21 ± 6 µm). Further studies revealed that only oleanolic acid and ursolic acid, but not betulinic acid, could inhibit the lipopolysaccharide induced interleukin-6 release from Mono Mac 6 cells when tested separately. Combination of either oleanolic acid or ursolic acid with betulinic acid enhanced the immunomodulatory effect of the two triterpene acids.

  1. A novel method for isolating and analyzing organic acids in biological cultures

    SciTech Connect

    Polman, K.

    1995-12-31

    Fermentatively produced organic acids have significant potential as chemical feedstocks for the production of various commodity materials. Such acids include acetic and succinic acids. Fermentations frequently result in the simultaneous production of two or more organic acids, and often other fermentation products as well. This necessitates separation of these products from each other, so that quantification and purification can be achieved. A multitude of methodologies for the identification, purification, and quantitation of organic acids has been developed and described; both liquid and gas chromatography have been used for such separations. High-performance liquid chromatography (HPLC) media used for the separation of organic acids have included C18 columns, Aminex HPX-87H (ion-moderated partition resin), TEAP-Si 100 Polyol (strongly basic anion-exchange resin), Dowex 1 (cation-exchange column), Shodex Ionpak KC811, and others. Methodologies for HPLC analysis of organic acids also vary in these aspects: (1) Sample pretreatment (e.g., pretreatment with Sep-Pak C18 cartridges or with DEAE-Sephadex); (2) Mobile-phase composition (e.g., dilute sulfuric acid or formic acid); and (3) Method of organic acid detection (e.g., refractive index or light absorption). In this study, we present a methodology for isolating and quantifying organic acids found in fermentation broths. The methodology is simple, utilizes dual separation chemistries to effect an enhanced separation capacity, and is durable in terms of HPLC column life.

  2. Mechanisms of DNA strand breakage and interstrand cross-linking by diaziridinylbenzoquinone (diaziquone) in isolated nuclei from human cells.

    PubMed

    Szmigiero, L; Kohn, K W

    1984-10-01

    AZQ had been found to produce DNA strand breaks and interstrand cross-links in intact cells; evidence had indicated that these two DNA lesions arise by different chemical mechanisms and vary independently in degree in different cell types. In the present work, the mechanisms of the production of DNA strand breaks and interstrand cross-links by AZQ were studied in isolated cell nuclei. This system avoided the problem of poor penetration of test substances into cells. The DNA lesions were measured by means of the alkaline elution technique. It was found that the production of DNA strand breaks by AZQ in isolated nuclei required the addition of a reducing agent such as NADPH and was almost completely prevented by superoxide dismutase. This indicates that the mechanism of DNA strand breakage involves transfer of an electron from a reduced form of AZQ to molecular oxygen. Unexpectedly, interstrand cross-linking also was enhanced greatly by previous reduction of AZQ by NADPH or NaBH4. However, this reaction was not inhibited by superoxide dismutase. General alkylating activity of AZQ also was stimulated by reduction; the pH-dependence of this reaction was determined. The mechanism of DNA interstrand cross-linking by AZQ was surmised to stem from alkylation reactions of the two aziridine groups. The findings suggest the possibility that AZQ or related compounds may function as bioreductive alkylating agents which might be selectively toxic to hypoxic tissues.

  3. Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells

    SciTech Connect

    Stewart, B.W.; Kavallaris, M.; Catchpoole, D.; Norris, M.D. )

    1991-02-01

    In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to (3H)thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

  4. Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides.

    PubMed

    Japelaghi, Reza Heidari; Haddad, Raheem; Garoosi, Ghasem-Ali

    2011-10-01

    Isolation of high quality nucleic acids from plant tissues rich in polysaccharides and polyphenols is often difficult. The presence of these substances can affect the quality and/or quantity of the nucleic acids isolated. Here, we describe a rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high quality nucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees. The nucleic acids isolated with this protocol were successfully used for many functional genomic based experiments including polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), cloning, and semiquantitative RT-PCR.

  5. Amino acid profiles of lactic acid bacteria, isolated from kefir grains and kefir starter made from them.

    PubMed

    Simova, Emilina; Simov, Zhelyasko; Beshkova, Dora; Frengova, Ginka; Dimitrov, Zhechko; Spasov, Zdravko

    2006-03-15

    The characteristics of cell growth, lactic acid production, amino acid release and consumption by single-strain cultures of lactic acid bacteria (isolated from kefir grains), and by a multiple-strain kefir starter prepared from them, were studied. The change in the levels of free amino acids was followed throughout the kefir process: single-strain kefir bacteria and the kefir starter (Lactococcus lactis C15-1%+Lactobacillus helveticus MP12-3%+(Streptococcus thermophilus T15+Lactobacillus bulgaricus HP1 = 1:1)-3%) were cultivated in pasteurized (92 degrees C for 20 min) cow's milk (3% fat content) at 28 degrees C for 5 h (the kefir starter reached pH 4.7) and subsequently grown at 20 degrees C for 16 h; storage was at 4 degrees C for 168 h. The strain L. helveticus MP12 was unrivaled with respect to free amino acid production (53.38 mg (100 g)(-1)) and cell growth (17.8 x 10(8) CFU ml(-1)); however, it manifested the lowest acidification activity. L. bulgaricus HP1 released approximately 3.7 times less amino acids, nearly 5 times lower cell growth, and produced about 1.2 times more lactic acid. S. thermophilus T15 demonstrated dramatically complex amino acid necessities for growth and metabolism. With L. lactis C15, the highest levels of growth and lactic acid synthesis were recorded (18.3 x 10(8) CFU ml(-1) and 7.8 g l(-1) lactic acid at the 21st hour), and as for free amino acid production, it approximated L. bulgaricus HP1 (17.03 mg (100 g)(-1) maximum concentration). In the L. lactis C15 culture, the amino acids were used more actively throughout the first exponential growth phase (by the 10th hour) than during the second growth phase. The unique properties of the L. helveticus MP12 strain to produce amino acids were employed to create a symbiotic bioconsortium kefir culture, which, under conditions of kefir formation, enhanced lactic acid production and shortened the time required to reach pH 4.7; intensified cell growth activity, resulting in a respective 90

  6. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

    PubMed Central

    MacInnes, M A; Dickson, J A; Hernandez, R R; Learmonth, D; Lin, G Y; Mudgett, J S; Park, M S; Schauer, S; Reynolds, R J; Strniste, G F

    1993-01-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. We have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Our available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5'-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Our evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed from several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeasts, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Our analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. Images PMID:8413238

  7. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of saccharomyces cerevisiae and schizosaccharomyces pombe

    SciTech Connect

    MacInnes, M.A.; Dickson, J.A.; Hernandez, R.R.; Lin, G.Y.; Park, M.S.; Schauer, S.; Reynolds, R.J.; Strniste, G.F. ); Learmonth, D. ); Mudgett, J.S. ); Yu, J.Y. )

    1993-10-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. The authors have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Their available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5[prime]-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Their evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed for several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeast, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Their analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. 69 refs., 6 figs., 1 tab.

  8. Fatty acid profiles in Leishmania spp. isolates with natural resistance to nitric oxide and trivalent antimony.

    PubMed

    de Azevedo, Alana Freire; Dutra, Jorge Luís de Lisboa; Santos, Micheli Luize Barbosa; Santos, Darlisson de Alexandria; Alves, Péricles Barreto; de Moura, Tatiana Rodrigues; de Almeida, Roque Pacheco; Fernandes, Marcelo Ferreira; Scher, Ricardo; Fernandes, Roberta Pereira Miranda

    2014-01-01

    Fatty acids, especially those from phospholipids (PLFA), are essential membrane components that are present in relatively constant proportions in biological membranes under natural conditions. However, under harmful growth conditions, such as diseases, environmental changes, and chemical exposure, the fatty acid proportions might vary. If such changes could be identified and revealed to be specific for adverse situations, they could be used as biomarkers. Such biomarkers could facilitate the identification of virulence and resistance mechanisms to particular chemotherapeutic agents. Therefore, specific biomarkers could lead to better therapeutic decisions that would, in turn, enhance treatment effectiveness. The objective of this study was to compare the fatty acid profiles of trivalent antimony and nitric oxide (NO)-resistant and -sensitive Leishmania chagasi and Leishmania amazonensis isolates. Fatty acid methyl esters (FAMEs) were obtained from total lipids (MIDI), ester-linked lipids (ELFA), and ester-linked phospholipids (PLFA). FAMEs were analyzed by chromatography and mass spectrometry. Species- or resistance-associated differences in FAME profiles were assessed by nonmetric multidimensional scaling, multiresponse permutation procedures, and indicator species analyses. The isolate groups had different MIDI-FAME profiles. However, neither the ELFA nor PLFA profiles differed between the sensitive and resistant isolates. Levels of the fatty acid 18:1 Δ9c were increased in sensitive isolates (p < 0,001), whereas the fatty acid 20:4 Δ5,8,11,14 showed the opposite trend (p < 0.01). We conclude that these two fatty acids are potential biomarkers for NO and antimony resistance in L. chagasi and L. amazonensis and that they could be helpful in therapeutic diagnoses.

  9. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  10. Non-aflatoxigenic Aspergillus flavus isolates reduce aflatoxins, cyclopiazonic acid and fumonisin in corn (maize)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus strains vary widely in their production of aflatoxins and cyclopiazonic acid (CPA). A total of 500 Aspergillus strains isolated from a variety of sources showed 16.4% were negative for both aflatoxin and CPA, 41.3% were positive for both mycotoxins, 13.0% were positive only fo...

  11. Gibberellic Acid-Induced Synthesis of Protease by Isolated Aleurone Layers of Barley 1

    PubMed Central

    Jacobsen, John V.; Varner, J. E.

    1967-01-01

    The production of protease by isolated aleurone layers of barley in response to gibberellic acid has been examined. The protease arises in the aleurone layer and is mostly released from the aleurone cells. The courses of release of amylase and protease from aleurone layers, the dose responses to gibberellic acid and the effects of inhibitors on the production of both enzymes are parallel. As is the case for amylase, protease is made de novo in response to the hormone. These data give some credence to the hypothesis that the effect of gibberellic acid is to promote the simultaneous synthesis and secretion of a group of hydrolases. PMID:16656695

  12. The enhanced rate of transcription of methyl mercury-exposed DNA by RNA polymerase is not sufficient to explain the stimulatory effect of methyl mercury on RNA synthesis in isolated nuclei.

    PubMed

    Frenkel, G D; Ducote, J

    1987-10-01

    Previous work demonstrated two stimulatory effects of methyl mercury on nucleic acid synthesis: (1) in isolated nuclei, methyl mercury stimulates RNA synthesis which is catalyzed by RNA polymerase II [Frenkel and Randles, J. Biol. Chem. 257, 6275-6279 (1982)]. (2) Brief exposure of purified DNA to methyl mercury increases the rate of its transcription by purified RNA polymerase II [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)]. The latter effect was considered as a possible mechanism of the former. Two lines of evidence are presented here which demonstrate that the latter effect is not a sufficient explanation for the former. (1) Mercuric perchlorate has been found to increase the rate of DNA transcription by purified polymerase and the template properties of the mercuric perchlorate-exposed DNA have been found to resemble those of methyl mercury-exposed DNA. Nevertheless, mercuric perchlorate has been shown not to stimulate RNA synthesis in isolated HeLa nuclei. (2) In isolated nuclei of the B50 rat neuroblastoma cell line, RNA synthesis has been found to be stimulated only minimally by methyl mercury. Nevertheless, RNA polymerase II purified from the B50 cells has been found to transcribe methyl mercury-exposed DNA at a higher rate than unexposed control DNA.

  13. Hydroxy-oleic acid, but not oleic acid, inhibits vascular responsiveness in isolated aortic tissue

    EPA Science Inventory

    Oleic acid (OA) and other fatty acids can become abundant in circulation after air pollution exposure as endogenously released lipolysis byproducts or by entering the body as a component of air pollution. Vascular damage has been observed with OA infusion, but it is not yet estab...

  14. Semipreparative isolation of dehydrodiferulic and dehydrotriferulic acids as standard substances from maize bran.

    PubMed

    Bunzel, Mirko; Funk, Carola; Steinhart, Hans

    2004-09-01

    A method for the isolation of diferulic and triferulic acids in quantities and purity that comply with the requirements for their use as standard substances was developed. The procedure includes alkaline hydrolysis of destarched maize bran and ether extraction of liberated phenolic compounds. Following a first purification by liquid-liquid extraction Sephadex LH-20 chromatography is performed. This step is the core of the method and allows the separation of monomeric and dimeric/trimeric substances. A good pre-separation of di- and triferulic acids (purity in most cases >75%) is also achieved. Further separation and purification is carried out by semipreparative RP18-HPLC. Using this rapid, easy to handle, and moderately priced separation procedure it is possible to obtain approx. 41 mg 8-O-4'-diferulic acid, 27 mg 5-5'-diferulic acid, 12 mg 8-5'-diferulic acid (benzofuran form), 16 mg 8-5'-diferulic acid (open form), 11 mg 8-5'-diferulic acid (decarboxylated form), 7 mg 8-8'-diferulic acid (cyclic form), 5 mg 8-8'-diferulic acid (open form), and 10 mg 5-5',8'-O-4"-triferulic acid out of 20 g destarched maize bran. The incorporation of minor modifications allows a further upscaling of this procedure.

  15. Detection of Ribosomal DNA Sequence Polymorphisms in the Protist Plasmodiophora brassicae for the Identification of Geographical Isolates.

    PubMed

    Laila, Rawnak; Robin, Arif Hasan Khan; Yang, Kiwoung; Choi, Gyung Ja; Park, Jong-In; Nou, Ill-Sup

    2017-01-04

    Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY). The nuclear ribosomal DNA (rDNA) sequence of P. brassicae, comprising 6932 base pairs (bp), was cloned and sequenced and found to include the small subunits (SSUs) and a large subunit (LSU), internal transcribed spacers (ITS1 and ITS2), and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs), oligonucleotide polymorphisms, and insertions/deletions (InDels). A combination of three markers was able to distinguish the geographical isolates into two groups.

  16. Detection of Ribosomal DNA Sequence Polymorphisms in the Protist Plasmodiophora brassicae for the Identification of Geographical Isolates

    PubMed Central

    Laila, Rawnak; Robin, Arif Hasan Khan; Yang, Kiwoung; Choi, Gyung Ja; Park, Jong-In; Nou, Ill-Sup

    2017-01-01

    Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY). The nuclear ribosomal DNA (rDNA) sequence of P. brassicae, comprising 6932 base pairs (bp), was cloned and sequenced and found to include the small subunits (SSUs) and a large subunit (LSU), internal transcribed spacers (ITS1 and ITS2), and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs), oligonucleotide polymorphisms, and insertions/deletions (InDels). A combination of three markers was able to distinguish the geographical isolates into two groups. PMID:28054984

  17. Mammalian cell DNA damage and repair kinetics of monohaloacetic acid drinking water disinfection by-products.

    PubMed

    Komaki, Yukako; Pals, Justin; Wagner, Elizabeth D; Mariñas, Benito J; Plewa, Michael J

    2009-11-01

    Haloacetic acids (HAAs) are the second most common class of chlorinated water disinfection by-products (DBPs). The single cell gel electrophoresis genotoxicity assay using Chinese hamster ovary (CHO) cells was modified to include liquid holding recovery time to measure genomic DNA damage and repair kinetics of three monoHAAs: chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA). The rank order of genotoxic potency was IAA > BAA > CAA from previous research. The concentration of each HAA was chosen to generate approximately the same level of genotoxic damage. No cytotoxicity was expressed during the 24 h liquid holding period. Nuclei from CHO cells treated with BAA showed the lowest rate of DNA repair (t(50) = 296 min) compared to that of CAA or IAA (t(50) = 134 and 84 min, respectively). The different rates of genomic repair expressed by IAA or CAA versus BAA suggest that different distributions of DNA lesions are induced. The use of DNA repair coupled with genomic technologies may lead to the understanding of the biological and genetic mechanisms involved in toxic responses induced by DBPs.

  18. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin.

    Arsenic causes cancer in human skin, urinary bladder, lung, liver and kidney and is a significant world-wide public health problem. Although the metabolism of inorganic arsenic is ...

  19. Design and synthesis of N-benzoyl amino acid derivatives as DNA methylation inhibitors.

    PubMed

    Garella, Davide; Atlante, Sandra; Borretto, Emily; Cocco, Mattia; Giorgis, Marta; Costale, Annalisa; Stevanato, Livio; Miglio, Gianluca; Cencioni, Chiara; Fernández-de Gortari, Eli; Medina-Franco, José L; Spallotta, Francesco; Gaetano, Carlo; Bertinaria, Massimo

    2016-11-01

    The inhibition of human DNA Methyl Transferases (DNMT) is a novel promising approach to address the epigenetic dysregulation of gene expression in different diseases. Inspired by the validated virtual screening hit NSC137546, a series of N-benzoyl amino acid analogues was synthesized and obtained compounds were assessed for their ability to inhibit DNMT-dependent DNA methylation in vitro. The biological screening allowed the definition of a set of preliminary structure-activity relationships and the identification of compounds promising for further development. Among the synthesized compounds, L-glutamic acid derivatives 22, 23, and 24 showed the highest ability to prevent DNA methylation in a total cell lysate. Compound 22 inhibited DNMT1 and DNMT3A activity in a concentration-dependent manner in the micromolar range. In addition, compound 22 proved to be stable in human serum and it was thus selected as a starting point for further biological studies.

  20. DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

    PubMed

    Li, Yueran; Chen, Xifeng; Wang, Bidou; Liu, Guangxing; Tang, Yuguo; Miao, Peng

    2016-06-07

    Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection.

  1. A Concentrated Hydrochloric Acid-based Method for Complete Recovery of DNA from Bone.

    PubMed

    Huynen, Leon; Lambert, David M

    2015-11-01

    The successful extraction of DNA from historical or ancient animal bone is important for the analysis of discriminating genetic markers. Methods used currently rely on the digestion of bone with EDTA and proteinase K, followed by purification with phenol/chloroform and silica bed binding. We have developed a simple concentrated hydrochloric acid-based method that precludes the use of phenol/chloroform purification and can lead to a several-fold increase in DNA yield when compared to other commonly used methods. Concentrated hydrochloric acid was shown to dissolve most of the undigested bone and allowed the efficient recovery of DNA fragments <100 bases in length. This method should prove useful for the recovery of DNAs from highly degraded animal bone, such as that found in historical or ancient samples.

  2. Isolation, characterization and evaluation of probiotic lactic acid bacteria for potential use in animal production.

    PubMed

    García-Hernández, Yaneisy; Pérez-Sánchez, Tania; Boucourt, Ramón; Balcázar, José L; Nicoli, Jacques R; Moreira-Silva, João; Rodríguez, Zoraya; Fuertes, Héctor; Nuñez, Odalys; Albelo, Nereyda; Halaihel, Nabil

    2016-10-01

    In livestock production, lactic acid bacteria (LAB) are the most common microorganisms used as probiotics. For such use, these bacteria must be correctly identified and characterized to ensure their safety and efficiency. In the present study, LAB were isolated from broiler excreta, where a fermentation process was used. Nine among sixteen isolates were identified by biochemical and molecular (sequencing of the 16S rRNA gene) methods as Lactobacillus crispatus (n=1), Lactobacillus pentosus (n=1), Weissella cibaria (n=1), Pediococcus pentosaceus (n=2) and Enterococcus hirae (n=4). Subsequently, these bacteria were characterized for their growth capabilities, lactic acid production, acidic pH and bile salts tolerance, cell surface hydrophobicity, antimicrobial susceptibility and antagonistic activity. Lactobacillus pentosus strain LB-31, which showed the best characteristics, was selected for further analysis. This strain was administered to broilers and showed the ability of modulating the immune response and producing beneficial effects on morpho-physiological, productive and health indicators of the animals.

  3. All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

    PubMed

    So, Hongyun; Lee, Kunwoo; Murthy, Niren; Pisano, Albert P

    2014-12-10

    This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

  4. Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells.

    PubMed

    Csortos, C; Lazar, V; Garcia, J G

    1999-01-01

    The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

  5. Leojaponic acids A and B, two new homologous terpenoids, isolated from Leonurus japonicus.

    PubMed

    Wu, Han-Kui; Mao, Yan-Jun; Sun, Shan-Shan; Xu, Zhi-Yong; Ma, Ya; Cao, Jin-Xia; Qi, He; Wu, Zhi-Fu; Li, Gang; Yang, Wei-Hua

    2016-04-01

    The present study aimed at isolation and purification of the bioactive terpenoids from the herb of Leonurus japonicus by chromatographic separations such as silica gel, sephadex LH-20 and C18 reversed phase silica gel, as well as preparative HPLC. As a result, leojaponic acids A (1, C17H24O4) and B (2, C18H26O4), two homologous terpenoids, together with (-)-loliolide (3), 1-(3-ethylphenyl) ethane-1, 2-diol (4) and dibutyl phthalate (5), were isolated from the EtOH extract of L. japonicus. All the chemical structures of the isolates were elucidated on the basis of 1D and 2D NMR analyses. Compounds 1 and 2 were new terpenoids, and Compounds 3 and 4 were isolated and identified for the first time from this plant. In addition, the α-glucosidase and tyrosinase inhibitory activity of the new compounds were evaluated.

  6. Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase.

    PubMed

    Nabel, Christopher S; Lee, Jae W; Wang, Laura C; Kohli, Rahul M

    2013-08-27

    Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID's functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID's selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2'-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID's reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2'-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID's closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2'-fluoro-RNA substrates, AID's deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID's DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA.

  7. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    PubMed Central

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  8. Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

    PubMed Central

    Muratovska, Aleksandra; Lightowlers, Robert N.; Taylor, Robert W.; Turnbull, Douglass M.; Smith, Robin A. J.; Wilce, Jacqueline A.; Martin, Stephen W.; Murphy, Michael P.

    2001-01-01

    The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA

  9. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes

    PubMed Central

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2′-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of −22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL−1. In the presence of divalent Ca2+, the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca2+/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca2+ and the synthesized copolymer on DNA transfection was observed. The use of Ca2+ or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca2+/DNA polyplex could serve as a promising candidate for gene delivery. PMID:25767385

  10. Generation and characterization of isolates of Peronophythora litchii resistant to carboxylic acid amide fungicides.

    PubMed

    Wang, Hancheng; Sun, Haiyan; Stammler, Gerd; Ma, Jianxia; Zhou, Mingguo

    2010-05-01

    Four isolates of Peronophythora litchii with resistance to carboxylic acid amide (CAA) fungicides were selected on fungicide-amended agar. These isolates had various levels of resistance, as evidenced by their resistance factor (RF), which is the 50% effective concentration (EC(50)) value of a particular isolate divided by that of the wild-type parent. RF values to dimethomorph for the four isolates were 15, 24, 141, and >1,500. Resistance was stable for two isolates, while the EC(50) values decreased for the other two after repeated subculturing on fungicide-free medium. Cross-resistance occurred with all CAAs tested here (dimethomorph, mandipropamid, flumorph, and pyrimorph), but not with strobilurins (azoxystrobin and famoxadone) or other fungicides (metalaxyl, cymoxanil, and mancozeb). Studies on fitness parameters (mycelial growth, sporulation, spore germination, zoospore formation, aggressiveness, and temperature tolerance) in the parent wild-type and resistant isolates demonstrated that penalties in different parameters may be associated with CAA resistance, depending on the isolate. These studies show that Peronophythora litchii is able to express CAA resistance under laboratory conditions but it is not known if resistant strains could become established in the field and sensitivity monitoring studies are recommended.

  11. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

    PubMed

    Wagler, Patrick; Minero, Gabriel Antonio S; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S

    2015-10-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA