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Sample records for groupings usingbranched dna

  1. Directed assembly of discrete gold nanoparticle groupings usingbranched DNA scaffolds

    SciTech Connect

    Claridge, Shelley A.; Goh, Sarah L.; Frechet, Jean M.J.; Williams, Shara C.; Micheel, Christine M.; Alivisatos, A. Paul

    2004-09-14

    The concept of self-assembled dendrimers is explored for the creation of discrete nanoparticle assemblies. Hybridization of branched DNA trimers and nanoparticle-DNA conjugates results in the synthesis of nanoparticle trimer and tetramer complexes. Multiple tetramer architectures are investigated, utilizing Au-DNA conjugates with varying secondary structural motifs. Hybridization products are analyzed by gel electrophoresis, and discrete bands are observed corresponding to structures with increasing numbers of hybridization events. Samples extracted from each band are analyzed by transmission electron microscopy, and statistics compiled from micrographs are used to compare assembly characteristics for each architecture. Asymmetric structures are also produced in which both 5 and 10 nm Au particles are assembled on branched scaffolds.

  2. Xenorhabdus luminescens (DNA hybridization group 5) from human clinical specimens.

    PubMed Central

    Farmer, J J; Jorgensen, J H; Grimont, P A; Akhurst, R J; Poinar, G O; Ageron, E; Pierce, G V; Smith, J A; Carter, G P; Wilson, K L

    1989-01-01

    An unusual isolate from a human leg wound was identified as Xenorhabdus luminescens. This finding led to the discovery or isolation of four additional strains, two from blood and two from wounds. Three of the five strains were from patients in San Antonio, Tex. Three strains were studied by DNA-DNA hybridization (S1 nuclease-trichloroacetic acid method) and were 77 to 100% related to each other, 34% related to the type strain of X. luminescens, 35 to 40% related to three of Grimont's other DNA hybridization groups of X. luminescens, and 9% related to the type strain of Xenorhabdus nematophilus. The new group of five strains was designated X. luminescens DNA hybridization group 5. All five strains were very inactive biochemically and fermented only D-glucose and D-mannose. The key reactions for recognizing this new organism are yellow pigment production, negative test for nitrate reduction to nitrite, weak bioluminescence (10 to 15 min of dark adaptation is required to see the weak light produced), and a unique hemolytic reaction on sheep blood agar plates incubated at 25 degrees C. Two case histories of strains from wounds are given; these suggest that X. luminescens DNA hybridization group 5 may be a new bacterial agent that causes wound infections. The two cases of wound infection, along with the two blood isolates, suggest that the new organism is clinically significant. Images PMID:2768446

  3. Flexible automated platform for blood group genotyping on DNA microarrays.

    PubMed

    Paris, Sandra; Rigal, Dominique; Barlet, Valérie; Verdier, Martine; Coudurier, Nicole; Bailly, Pascal; Brès, Jean-Charles

    2014-05-01

    The poor suitability of standard hemagglutination-based assay techniques for large-scale automated screening of red blood cell antigens severely limits the ability of blood banks to supply extensively phenotype-matched blood. With better understanding of the molecular basis of blood antigens, it is now possible to predict blood group phenotype by identifying single-nucleotide polymorphisms in genomic DNA. Development of DNA-typing assays for antigen screening in blood donation qualification laboratories promises to enable blood banks to provide optimally matched donations. We have designed an automated genotyping system using 96-well DNA microarrays for blood donation screening and a first panel of eight single-nucleotide polymorphisms to identify 16 alleles in four blood group systems (KEL, KIDD, DUFFY, and MNS). Our aim was to evaluate this system on 960 blood donor samples with known phenotype. Study data revealed a high concordance rate (99.92%; 95% CI, 99.77%-99.97%) between predicted and serologic phenotypes. These findings demonstrate that our assay using a simple protocol allows accurate, relatively low-cost phenotype prediction at the DNA level. This system could easily be configured with other blood group markers for identification of donors with rare blood types or blood units for IH panels or antigens from other systems. PMID:24726279

  4. Mitochondrial DNA evolution in the Anaxyrus boreas species group

    USGS Publications Warehouse

    Goebel, A.M.; Ranker, T.A.; Corn, P.S.; Olmstead, R.G.

    2009-01-01

    The Anaxyrus boreas species group currently comprises four species in western North America including the broadly distributed A. boreas, and three localized species, Anaxyrus nelsoni, Anaxyrus exsul and Anaxyrus canorus. Phylogenetic analyses of the mtDNA 12S rDNA, cytochrome oxidase I, control region, and restriction sites data, identified three major haplotype clades. The Northwest clade (NW) includes both subspecies of A. boreas and divergent minor clades in the middle Rocky Mountains, coastal, and central regions of the west and Pacific Northwest. The Southwest (SW) clade includes A. exsul, A. nelsoni, and minor clades in southern California. Anaxyrus canorus, previously identified as paraphyletic, has populations in both the NW and SW major clades. The Eastern major clade (E) includes three divergent lineages from southern Utah, the southern Rocky Mountains, and north of the Great Basin at the border of Utah and Nevada. These results identify new genetic variation in the eastern portion of the toad's range and are consistent with previous regional studies from the west coast. Low levels of control region sequence divergence between major clades (2.2-4.7% uncorrected pair-wise distances) are consistent with Pleistocene divergence and suggest that the phylogeographic history of the group was heavily influenced by dynamic Pleistocene glacial and climatic changes, and especially pluvial changes, in western North America. Results reported here may impact conservation plans in that the current taxonomy does not reflect the diversity in the group. ?? 2008 Elsevier Inc.

  5. Mitochondrial DNA evolution in the Anaxyrus boreas species group.

    PubMed

    Goebel, Anna M; Ranker, Tom A; Corn, Paul Stephen; Olmstead, Richard G

    2009-02-01

    The Anaxyrus boreas species group currently comprises four species in western North America including the broadly distributed A. boreas, and three localized species, Anaxyrus nelsoni, Anaxyrusexsul and Anaxyrus canorus. Phylogenetic analyses of the mtDNA 12S rDNA, cytochrome oxidase I, control region, and restriction sites data, identified three major haplotype clades. The Northwest clade (NW) includes both subspecies of A. boreas and divergent minor clades in the middle Rocky Mountains, coastal, and central regions of the west and Pacific Northwest. The Southwest (SW) clade includes A. exsul, A. nelsoni, and minor clades in southern California. Anaxyrus canorus, previously identified as paraphyletic, has populations in both the NW and SW major clades. The Eastern major clade (E) includes three divergent lineages from southern Utah, the southern Rocky Mountains, and north of the Great Basin at the border of Utah and Nevada. These results identify new genetic variation in the eastern portion of the toad's range and are consistent with previous regional studies from the west coast. Low levels of control region sequence divergence between major clades (2.2-4.7% uncorrected pair-wise distances) are consistent with Pleistocene divergence and suggest that the phylogeographic history of the group was heavily influenced by dynamic Pleistocene glacial and climatic changes, and especially pluvial changes, in western North America. Results reported here may impact conservation plans in that the current taxonomy does not reflect the diversity in the group. PMID:18662792

  6. Mutant Cockayne syndrome group B protein inhibits repair of DNA topoisomerase I-DNA covalent complex.

    PubMed

    Horibata, Katsuyoshi; Saijo, Masafumi; Bay, Mui N; Lan, Li; Kuraoka, Isao; Brooks, Philip J; Honma, Masamitsu; Nohmi, Takehiko; Yasui, Akira; Tanaka, Kiyoji

    2011-01-01

    Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B. PMID:21143350

  7. Rapid antigen testing for group A Streptococcus by DNA probe.

    PubMed

    Heelan, J S; Wilbur, S; Depetris, G; Letourneau, C

    1996-02-01

    The Gen-probe group A Streptococcus direct test (GASD), a nucleic acid probe assay for detecting GAS from throat swabs, has recently been developed. The test uses an acridium ester-labeled DNA probe which is complementary to the rRNA of Streptococcus pyogenes. In this study, 318 single culturette throat swabs were tested by this method using culture as a "gold standard." After plating onto trypticase soy agar plates with 5% sheep blood, swabs were stored at 4 degrees C for no more than 72 h before the probe assay was performed. Our patient population consisted of symptomatic outpatients seen in the Memorial Hospital Emergency Department and in the Family Care Center. After discrepancy testing, sensitivity, specificity, and positive and negative predictive values were 91.4%, 97%, 91.4%, and 97%. The GASD is a rapid, easy-to-perform method for batch screening for streptococcal pharyngitis.

  8. Genetic Kinship Investigation from Blood Groups to DNA Markers.

    PubMed

    Geserick, Gunther; Wirth, Ingo

    2012-06-01

    The forensic application of hereditary characteristics became possible after the discovery of human blood groups by Karl Landsteiner in 1901. The foundation for their use in kinship investigation was laid by Emil von Dungern and Ludwig Hirschfeld in 1910 by clarification of the inheritance of the ABO groups. Up to the middle of the 20th century further red cell membrane systems were discovered. From the 1920s Fritz Schiff and Georg Strassmann fought for the introduction of blood groups into forensic kinship investigation. A new era of hemogenetics was opened from 1955 as genetic polymorphisms were described in serum proteins. Starting in 1958 there followed the complex HLA system of white blood cells, which from 1963 was joined by polymophisms in erythrocyte enzymes. Therefore, from the 1980s, it was possible to clarify the majority of kinship cases with a combination of conventional markers. From 1990 to 2000 the conventional markers were gradually replaced by the more effective DNA markers. Simultaneously typing shifted from the phenotype level to the genotype level. The genomic structure of conventional genetic markers could also now be explained. As a reflection of scientific progress the legal situation also changed, particularly in the form of the official guidelines for kinship investigation.

  9. Genetic Kinship Investigation from Blood Groups to DNA Markers

    PubMed Central

    Geserick, Gunther; Wirth, Ingo

    2012-01-01

    The forensic application of hereditary characteristics became possible after the discovery of human blood groups by Karl Landsteiner in 1901. The foundation for their use in kinship investigation was laid by Emil von Dungern and Ludwig Hirschfeld in 1910 by clarification of the inheritance of the ABO groups. Up to the middle of the 20th century further red cell membrane systems were discovered. From the 1920s Fritz Schiff and Georg Strassmann fought for the introduction of blood groups into forensic kinship investigation. A new era of hemogenetics was opened from 1955 as genetic polymorphisms were described in serum proteins. Starting in 1958 there followed the complex HLA system of white blood cells, which from 1963 was joined by polymophisms in erythrocyte enzymes. Therefore, from the 1980s, it was possible to clarify the majority of kinship cases with a combination of conventional markers. From 1990 to 2000 the conventional markers were gradually replaced by the more effective DNA markers. Simultaneously typing shifted from the phenotype level to the genotype level. The genomic structure of conventional genetic markers could also now be explained. As a reflection of scientific progress the legal situation also changed, particularly in the form of the official guidelines for kinship investigation. PMID:22851931

  10. Ribosomal and Mitochondrial DNA Analyses of Xiphinema americanum-Group Populations.

    PubMed

    Lazarova, Stela S; Malloch, Gaynor; Oliveira, Claudio M G; Hübschen, Judith; Neilson, Roy

    2006-12-01

    The 18S ribosomal DNA (rDNA) and cytochrome oxidase I region of mitochondrial DNA (mtDNA) were sequenced for 24 Xiphinema americanum-group populations sourced from a number of geographically disparate locations. Sequences were subjected to phylogenetic analysis and compared. 18S rDNA strongly suggested that only X. pachtaicum, X. simile (two populations) and a X. americanum s.l. population from Portugal were different from the other 20 populations studied, whereas mtDNA indicated some heterogeneity between populations. Phylogenetically, based on mtDNA, an apparent dichotomy existed amongst X. americanum-group populations from North America and those from Asia, South America and Oceania. Analyses of 18S rDNA and mtDNA sequences underpin the classical taxonomic issues of the X. americanum-group and cast doubt on the degree of speciation within the X. americanum-group.

  11. Ribosomal and Mitochondrial DNA Analyses of Xiphinema americanum-Group Populations

    PubMed Central

    Lazarova, Stela S.; Malloch, Gaynor; Oliveira, Claudio M.G.; Hübschen, Judith; Neilson, Roy

    2006-01-01

    The 18S ribosomal DNA (rDNA) and cytochrome oxidase I region of mitochondrial DNA (mtDNA) were sequenced for 24 Xiphinema americanum-group populations sourced from a number of geographically disparate locations. Sequences were subjected to phylogenetic analysis and compared. 18S rDNA strongly suggested that only X. pachtaicum, X. simile (two populations) and a X. americanum s.l. population from Portugal were different from the other 20 populations studied, whereas mtDNA indicated some heterogeneity between populations. Phylogenetically, based on mtDNA, an apparent dichotomy existed amongst X. americanum-group populations from North America and those from Asia, South America and Oceania. Analyses of 18S rDNA and mtDNA sequences underpin the classical taxonomic issues of the X. americanum-group and cast doubt on the degree of speciation within the X. americanum-group. PMID:19259456

  12. Biochemical Identification and Characterization of DNA Groups within the Proteus vulgaris Complex

    PubMed Central

    Janda, J. Michael; Abbott, Sharon L.; Khashe, Shideh; Probert, Will

    2001-01-01

    We placed 43 isolates belonging to the Proteus vulgaris complex into proposed DNA groups (genomovars) using five previously recommended tests (tests for esculin hydrolysis, production of acid from salicin, l-rhamnose fermentation, and elaboration of DNase and lipase). On the basis of the results of these five tests, 49% of the isolates fell into DNA groups 5 and 6, 37% fell into DNA group 2, and the remaining 14% fell into DNA groups 3 and 4. Sequencing of the 16S rRNA genes of 11 members of DNA groups 5 and 6 indicated that 10 of these isolates (91%) could be unambiguously assigned to one of these two genomospecies. Overall expression of selected enzymatic and virulence-associated characteristics did not differ significantly among DNA groups. PMID:11283033

  13. Short-step chemical synthesis of DNA by use of MMTrS group for protection of 5'-hydroxyl group.

    PubMed

    Shiraishi, Miyuki; Utagawa, Eri; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2007-01-01

    4-methoxytrithylthio (MMTrS) group was applied for the appropriately protected four canonical nucleosides. We prepared the phosphoroamidite units by use of these nucleosides and developed the synthesis of oligodeoxynucleotides without any acidic treatment. Moreover, the new DNA synthesis protocol was applied to an automated DNA synthesizer for the synthesis of longer oligodeoxynucleotides. PMID:18029620

  14. Hands on Group Work Paper Model for Teaching DNA Structure, Central Dogma and Recombinant DNA

    ERIC Educational Resources Information Center

    Altiparmak, Melek; Nakiboglu Tezer, Mahmure

    2009-01-01

    Understanding life on a molecular level is greatly enhanced when students are given the opportunity to visualize the molecules. Especially understanding DNA structure and function is essential for understanding key concepts of molecular biology such as DNA, central dogma and the manipulation of DNA. Researches have shown that undergraduate…

  15. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed Central

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-01-01

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region. PMID:3671088

  16. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-10-26

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region.

  17. Two high-mobility group box domains act together to underwind and kink DNA

    SciTech Connect

    Sánchez-Giraldo, R.; Acosta-Reyes, F. J.; Malarkey, C. S.; Saperas, N.; Churchill, M. E. A.; Campos, J. L.

    2015-06-30

    The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.

  18. Hydrogel with chains functionalized with carboxyl groups as universal 3D platform in DNA biosensors.

    PubMed

    Kowalczyk, Agata; Fau, Michal; Karbarz, Marcin; Donten, Mikolaj; Stojek, Zbigniew; Nowicka, Anna M

    2014-04-15

    Application of hydrogel based on N-isopropylacrylamide with carboxyl groups grafted to the chains enabled the immobilization of DNA at an extent exceeding that for flat surfaces by at least one order of magnitude. The probe DNA strands in the 3D platform were fully available for the hybridization process. The examination of the gels containing different amounts of grafted carboxyl groups (1-10%) was done using quartz crystal microbalance, electrochemical impedance spectroscopy, chronoamperometry and ionic coupled plasma with laser ablation. The optimal carboxyl group content was determined to be 5%. A very good agreement of the data obtained with independent techniques on content of DNA in the gel was obtained. In comparison to the other methods of immobilization of DNA the new platform enabled complete removal of DNA after the measurements and analysis and, therefore, could be used many times. After a 10-fold exchange of the DNA-sensing layer the efficiency of hybridization and analytical signal did not change by more than 5%. The sensor response increased linearly with logarithm of concentration of target DNA in the range 1×10(-13)-1×10(-6) M. The obtained detection limit was circa 8×10(-13) M of target DNA in the sample which is a substantial improvement over the planar sensing layers. PMID:24287408

  19. Mitochondrial DNA sequence diversity in two groups of Italian Veneto speakers from Veneto.

    PubMed

    Mogentale-Profizi, N; Chollet, L; Stévanovitch, A; Dubut, V; Poggi, C; Pradié, M P; Spadoni, J L; Gilles, A; Béraud-Colomb, E

    2001-03-01

    Although frequencies of mitochondrial DNA (mtDNA) haplogroups in the different European populations are rather homogenous, there are a few European populations or linguistic isolates that show different mtDNA haplogroup distributions; examples are the Saami and Ladin speakers from the eastern Italian Alps. MtDNA sequence diversity was analysed from subjects from two villages in Veneto. The first, Posina, is situated in the Venetian Alps near Vicenza. The second, Barco di Pravisdomini is a village on the plains near Venice. In spite of their common Veneto dialect, the two group populations have not preserved a genetic homogeneity; particularly, they show differences in T and J haplogroups frequencies. MtDNA diversity in these two groups seems to depend more on their geographic situation.

  20. A mitochondrial DNA based phylogeny of weakfish species of the Cynoscion group (Pisces: Sciaenidae).

    PubMed

    Vergara-Chen, Carlos; Aguirre, Windsor E; González-Wangüemert, Mercedes; Bermingham, Eldredge

    2009-11-01

    We infer the phylogeny of fishes in the New World Cynoscion group (Cynoscion, Isopisthus, Macrodon, Atractoscion, Plagioscion) using 1603bp of DNA sequence data from three mitochondrial genes. With the exception of Plagioscion, whose position was ambiguous, the Cynoscion group is monophyletic. However, several genera examined are not monophyletic. Atlantic and Pacific species of Cynoscion are interspersed in the tree and geminate species pairs are identified. Intergeneric relationships in the group are clarified. Our analysis is the first comprehensive phylogeny for the Cynoscion group based on molecular data and provides a baseline for future comparative studies of this important group.

  1. Spy: a new group of eukaryotic DNA transposons without target site duplications.

    PubMed

    Han, Min-Jin; Xu, Hong-En; Zhang, Hua-Hao; Feschotte, Cédric; Zhang, Ze

    2014-06-24

    Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution. Most DNA transposons belong to the cut-and-paste types, which are relatively simple elements characterized by terminal-inverted repeats (TIRs) flanking a single gene encoding a transposase. All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion. Here, we report a new group of evolutionarily related DNA transposons called Spy, which also include TIRs and DDE motif-containing transposase but surprisingly do not create TSDs upon insertion. Instead, Spy transposons appear to transpose precisely between 5'-AAA and TTT-3' host nucleotides, without duplication or modification of the AAATTT target sites. Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches. Phylogenetic analyses indicate that Spy transposases are distantly related to IS5, ISL2EU, and PIF/Harbinger transposases. However, Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference. Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties.

  2. Spy: a new group of eukaryotic DNA transposons without target site duplications.

    PubMed

    Han, Min-Jin; Xu, Hong-En; Zhang, Hua-Hao; Feschotte, Cédric; Zhang, Ze

    2014-07-01

    Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution. Most DNA transposons belong to the cut-and-paste types, which are relatively simple elements characterized by terminal-inverted repeats (TIRs) flanking a single gene encoding a transposase. All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion. Here, we report a new group of evolutionarily related DNA transposons called Spy, which also include TIRs and DDE motif-containing transposase but surprisingly do not create TSDs upon insertion. Instead, Spy transposons appear to transpose precisely between 5'-AAA and TTT-3' host nucleotides, without duplication or modification of the AAATTT target sites. Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches. Phylogenetic analyses indicate that Spy transposases are distantly related to IS5, ISL2EU, and PIF/Harbinger transposases. However, Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference. Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties. PMID:24966181

  3. Coumestan inhibits radical-induced oxidation of DNA: is hydroxyl a necessary functional group?

    PubMed

    Xi, Gao-Lei; Liu, Zai-Qun

    2014-06-18

    Coumestan is a natural tetracycle with a C═C bond shared by a coumarin moiety and a benzofuran moiety. In addition to the function of the hydroxyl group on the antioxidant activity of coumestan, it is worth exploring the influence of the oxygen-abundant scaffold on the antioxidant activity as well. In this work, seven coumestans containing electron-withdrawing and electron-donating groups were synthesized to evaluate the abilities to trap 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(•+)), 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical, respectively, and to inhibit the oxidations of DNA mediated by (•)OH, Cu(2+)/glutathione (GSH), and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), respectively. It was found that all of the coumestans used herein can quench the aforementioned radicals and can inhibit (•)OH-, Cu(2+)/GSH-, and AAPH-induced oxidations of DNA. In particular, substituent-free coumestan exhibits higher ability to quench DPPH and to inhibit AAPH-induced oxidation of DNA than Trolox. In addition, nonsubstituted coumestan shows a similar ability to inhibit (•)OH- and Cu(2+)/GSH-induced oxidations of DNA relative to that of Trolox. The antioxidant effectiveness of the coumestan can be attributed to the lactone in the coumarin moiety and, therefore, a hydroxyl group may not be a necessary functional group for coumestan to be an antioxidant.

  4. Effects of Trehalose Polycation End-group Functionalization on Plasmid DNA Uptake and Transfection

    PubMed Central

    Anderson, Kevin; Sizovs, Antons; Cortez, Mallory; Waldron, Chris; Haddleton, D.M.; Reineke, Theresa M.

    2012-01-01

    In this study, we have synthesized six analogs of a trehalose-pentaethylenehexamine glycopolymer (Tr4) that contain (1A) adamantane, (1B) carboxy, (1C) alkynyl-oligoethyleneamine, (1D) azido trehalose, (1E) octyl, or (1F) oligoethyleneamine end groups and evaluated the effects of polymer end group chemistry on the ability of these systems to bind, compact, and deliver pDNA in cultured HeLa cells. The polymers were synthesized in one-pot azide-alkyne cycloaddition reactions with an adaptation of the Carothers equation for step-growth polymerization to produce a series of polymers with similar degrees of polymerization. An excess of end-capping monomer was added at the end of the polymerizations to maximize functionalization efficiency, which was evaluated with GPC, NMR and MALDI-TOF. The polymers were all found to bind and compact pDNA at similarly low N/P ratios and form polyplexes with plasmid DNA. The effects of the different end group structures were most evident in the polyplex internalization and transfection assays completed in the presence of serum, as determined by flow cytometry and luciferase gene expression respectively. The Tr4 polymers end-capped with carboxyl groups (1B) (N/P = 7), octyne (1E) (N/P = 7), and oligoethyleneamine (1F) (N/P = 7), were taken into cells as polyplex and exhibited the highest levels of fluorescence, resulting from labeled reporter plasmid. Similarly, the polymers end-functionalized with the carboxyl groups (1E at N/P = 7), octyl groups (1E at N/P = 15) and, in particular, the oligoethyleneamine groups (F at N/P = 15) yielded dramatically higher reporter gene expression in the presence of serum. This study yields insight into how very subtle structural changes in the polymer chemistry such as end groups can yield very significant differences in the biological delivery efficiency and transgene expression of polymers used for pDNA delivery. PMID:22616977

  5. Effects of trehalose polycation end-group functionalization on plasmid DNA uptake and transfection.

    PubMed

    Anderson, Kevin; Sizovs, Antons; Cortez, Mallory; Waldron, Chris; Haddleton, D M; Reineke, Theresa M

    2012-08-13

    In this study, we have synthesized six analogs of a trehalose-pentaethylenehexamine glycopolymer (Tr4) that contain (1A) adamantane, (1B) carboxy, (1C) alkynyl-oligoethyleneamine, (1D) azido trehalose, (1E) octyl, or (1F) oligoethyleneamine end groups and evaluated the effects of polymer end group chemistry on the ability of these systems to bind, compact, and deliver pDNA to cultured HeLa cells. The polymers were synthesized in one-pot azide-alkyne cycloaddition reactions with an adaptation of the Carothers equation for step-growth polymerization to produce a series of polymers with similar degrees of polymerization. An excess of end-capping monomer was added at the end of the polymerizations to maximize functionalization efficiency, which was evaluated with GPC, NMR, and MALDI-TOF. The polymers were all found to bind and compact pDNA at similarly low N/P ratios and form polyplexes with plasmid DNA. The effects of the different end group structures were most evident in the polyplex internalization and transfection assays in the presence of serum as determined by flow cytometry and luciferase gene expression, respectively. The Tr4 polymers end-capped with carboxyl groups (1B) (N/P = 7), octyne (1E) (N/P = 7), and oligoethyleneamine (1F) (N/P = 7), were taken into cells as polyplex and exhibited the highest levels of fluorescence, resulting from labeled plasmid. Similarly, the polymers end-functionalized with carboxyl groups (1E at N/P = 7), octyl groups (1E at N/P = 15), and in particular oligoethyleneamine groups (1F at N/P = 15) yielded dramatically higher reporter gene expression in the presence of serum. This study yields insight into how very subtle structural changes in polymer chemistry, such as end groups can yield very significant differences in the biological delivery efficiency and transgene expression of polymers used for pDNA delivery.

  6. Maternal Methyl-Group Donor Intake and Global DNA (Hydroxy)Methylation before and during Pregnancy

    PubMed Central

    Pauwels, Sara; Duca, Radu Corneliu; Devlieger, Roland; Freson, Kathleen; Straetmans, Dany; Van Herck, Erik; Huybrechts, Inge; Koppen, Gurdun; Godderis, Lode

    2016-01-01

    It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxyl)methylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring’s Epigenome) study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxy)methylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline) using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxy)methylation levels were highest pre-pregnancy and at weeks 18–22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04) and hydroxymethylation (p = 0.04). A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxy)methylation percentage in weeks 11–13 and weeks 18–22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxy)methylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy. PMID:27509522

  7. Maternal Methyl-Group Donor Intake and Global DNA (Hydroxy)Methylation before and during Pregnancy.

    PubMed

    Pauwels, Sara; Duca, Radu Corneliu; Devlieger, Roland; Freson, Kathleen; Straetmans, Dany; Van Herck, Erik; Huybrechts, Inge; Koppen, Gurdun; Godderis, Lode

    2016-01-01

    It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxyl)methylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring's Epigenome) study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxy)methylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline) using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxy)methylation levels were highest pre-pregnancy and at weeks 18-22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04) and hydroxymethylation (p = 0.04). A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxy)methylation percentage in weeks 11-13 and weeks 18-22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxy)methylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy. PMID:27509522

  8. Human cDNA clones that modify radiomimetic sensitivity of Ataxia-telangiectasia (Group A) cells

    SciTech Connect

    Ziv, Y.; Bar-Shira, A.; Sartiel, A.

    1995-03-01

    Genes responsible for genetic diseases with increased sensitivity to DNA-damaging agents can be identified using complementation cloning. This strategy is based on in vitro complementation of the cellular sensitivity by gene transfer. Ataxia-telangiectasia (A-T) is a multisystem autosomal recessive disorder involving cellular sensitivity to ionizing radiation and radiomimetic drugs. A-T is genetically heterogeneous, with four complementation groups. We attempted to identify cDNA clones that modify the radiomimetic sensitivity of A-T cells assigned to complementation group [A-T(A)]. The cells were transfected with human cDNA libraries clones in episomal vectors, and various protocols by radiomimetic selection were applied. Thirteen cDNAs rescued from survivor cells were found to confer various degrees of radiomimetic resistance to A-T(A) cells upon repeated introduction, and one of them also partially influenced another feature of the A-T phenotype, radioresistant DNA synthesis. None of the clones mapped to the A-T locus on chromosome 11q22-23. Nine of the clones were derived from known genes, some of which are involved in cellular stress responses. We concluded that a number of different genes, not necessarily associated with A-T, can influence the response of A-T cells to radiomimetic drugs, and hence the complementation cloning approach may be less applicable to A-T than to other diseases involving abnormal processing of DNA damage. 57 refs., 5 figs., 2 tabs.

  9. Direct immobilization and hybridization of DNA on group III nitride semiconductors

    NASA Astrophysics Data System (ADS)

    Xu, Xiaobin; Jindal, Vibhu; Shahedipour-Sandvik, Fatemeh; Bergkvist, Magnus; Cady, Nathaniel C.

    2009-03-01

    A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.

  10. Phylogenetic reconstruction of the Drosophila obscura group, on the basis of mitochondrial DNA.

    PubMed

    Barrio, E; Latorre, A; Moya, A; Ayala, F J

    1992-07-01

    We have constructed restriction-site maps of the mtDNAs in 13 species and one subspecies of the Drosophila obscura group. The traditional division of this group into two subgroups (affinis and obscura) does not correspond to the phylogeny of the group, which shows two well-defined clusters (the Nearctic affinis and pseudoobscura subgroups) plus a very heterogeneous set of anciently diverged species (the Palearctic obscura subgroup). The mtDNA of Drosophila exhibits a tendency to evolve toward high A+T values. This leads to a "saturation" effect that (1) begets an apparent decrease in the rate of evolution as the time since the divergence of taxa increases and (2) reduces the value that mtDNA restriction analysis has for the phylogenetic reconstruction of Drosophila species that are not closely related.

  11. Distinct Structural Features of the Peroxide Response Regulator from Group A Streptococcus Drive DNA Binding

    PubMed Central

    Hammel, Michal; Nix, Jay C.; Tseng, Hsiao-Ling; Tsou, Chih-Cheng; Fei, Chun-Hsien; Chiou, Huo-Sheng; Jeng, U-Ser; Lin, Yee-Shin; Chuang, Woei-Jer; Wu, Jiunn-Jong; Wang, Shuying

    2014-01-01

    Group A streptococcus (GAS, Streptococcus pyogenes) is a strict human pathogen that causes severe, invasive diseases. GAS does not produce catalase, but has an ability to resist killing by reactive oxygen species (ROS) through novel mechanisms. The peroxide response regulator (PerR), a member of ferric uptake regulator (Fur) family, plays a key role for GAS to cope with oxidative stress by regulating the expression of multiple genes. Our previous studies have found that expression of an iron-binding protein, Dpr, is under the direct control of PerR. To elucidate the molecular interactions of PerR with its cognate promoter, we have carried out structural studies on PerR and PerR-DNA complex. By combining crystallography and small-angle X-ray scattering (SAXS), we confirmed that the determined PerR crystal structure reflects its conformation in solution. Through mutagenesis and biochemical analysis, we have identified DNA-binding residues suggesting that PerR binds to the dpr promoter at the per box through a winged-helix motif. Furthermore, we have performed SAXS analysis and resolved the molecular architecture of PerR-DNA complex, in which two 30 bp DNA fragments wrap around two PerR homodimers by interacting with the adjacent positively-charged winged-helix motifs. Overall, we provide structural insights into molecular recognition of DNA by PerR and define the hollow structural arrangement of PerR-30bpDNA complex, which displays a unique topology distinct from currently proposed DNA-binding models for Fur family regulators. PMID:24586487

  12. Ternary DNA chip based on a novel thymine spacer group chemistry.

    PubMed

    Yang, Yanli; Yildiz, Umit Hakan; Peh, Jaime; Liedberg, Bo

    2015-01-01

    A novel thymine-based surface chemistry suitable for label-free electrochemical DNA detection is described. It involves a simple two-step sequential process: immobilization of 9-mer thymine-terminated probe DNAs followed by backfilling with 9-mer thymine-based spacers (T9). As compared to commonly used organic spacer groups like 2-mercaptoethanol, 3-mercapto-1-propanol and 6-mercapto-1-hexanol, the 9-mer thymine-based spacers offer a 10-fold improvement in discriminating between complementary and non-complementary target hybridization, which is due mainly to facilitated transport of the redox probes through the probe-DNA/T9 layers. Electrochemical measurements, complemented with Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM-D) binding analyses, reveal that optimum selectivity between complementary and non-complementary hybridization is obtained for a sensing surface prepared using probe-DNA and backfiller T9 at equimolar concentration (1:1). At this particular ratio, the probe-DNAs are preferentially oriented and easily accessible to yield a sensing surface with favorable hybridization and electron transfer characteristics. Our findings suggest that oligonucleotide-based spacer groups offer an attractive alternative to short organic thiol spacers in the design of future DNA biochips. PMID:25465760

  13. Mitochondrial DNA control region analysis of three ethnic groups in the Republic of Macedonia.

    PubMed

    Jankova-Ajanovska, Renata; Zimmermann, Bettina; Huber, Gabriela; Röck, Alexander W; Bodner, Martin; Jakovski, Zlatko; Janeska, Biljana; Duma, Aleksej; Parson, Walther

    2014-11-01

    A total of 444 individuals representing three ethnic groups (Albanians, Turks and Romanies) in the Republic of Macedonia were sequenced in the mitochondrial control region. The mtDNA haplogroup composition differed between the three groups. Our results showed relatively high frequencies of haplogroup H12 in Albanians (8.8%) and less in Turks (3.3%), while haplogroups M5a1 and H7a1a were dominant in Romanies (13.7% and 10.3%, respectively) but rare in the former two. This highlights the importance of regional sampling for forensic mtDNA databasing purposes. These population data will be available on EMPOP under accession numbers EMP00644 (Albanians), EMP00645 (Romanies) and EMP00646 (Turks).

  14. Mitochondrial DNA control region analysis of three ethnic groups in the Republic of Macedonia

    PubMed Central

    Jankova-Ajanovska, Renata; Zimmermann, Bettina; Huber, Gabriela; Röck, Alexander W.; Bodner, Martin; Jakovski, Zlatko; Janeska, Biljana; Duma, Aleksej; Parson, Walther

    2014-01-01

    A total of 444 individuals representing three ethnic groups (Albanians, Turks and Romanies) in the Republic of Macedonia were sequenced in the mitochondrial control region. The mtDNA haplogroup composition differed between the three groups. Our results showed relatively high frequencies of haplogroup H12 in Albanians (8.8%) and less in Turks (3.3%), while haplogroups M5a1 and H7a1a were dominant in Romanies (13.7% and 10.3%, respectively) but rare in the former two. This highlights the importance of regional sampling for forensic mtDNA databasing purposes. These population data will be available on EMPOP under accession numbers EMP00644 (Albanians), EMP00645 (Romanies) and EMP00646 (Turks). PMID:25051224

  15. Targeting radiosensitizers to DNA by attachment of an intercalating group: Nitroimidazole-linked phenanthridines

    SciTech Connect

    Cowan, D.S.; Panicucci, R.; McClelland, R.A.; Rauth, A.M. )

    1991-07-01

    The nitroimidazole-linked phenanthridine series of compounds (NLP-1, 2, and 3) were synthesized under the assumption that it should be possible to enhance the molar efficiency of 2-nitroimidazoles as hypoxic cell radiosensitizers and cytotoxins by targeting them to their likely site of action, DNA. The targeting group chosen was the phenanthridine moiety, the major component of the classical DNA intercalating compound, ethidium bromide. The sole difference between the compounds is the length of the hydrocarbon chain linking the nitroimidazole to the phenanthridine. The phenanthridine group with a three-carbon side chain, P-1, was also synthesized to allow studies on the effect of the targeting group by itself. The ability of the compounds to bind to DNA is inversely proportional to their linker chain length with binding constant values ranging from approximately 1 {times} 10(5) mol-1 for NLP-2 to 6 {times} 10(5) mol-1 for NLP-3. The NLP compounds show selective toxicity to hypoxic cells at 37 degrees C at external drug concentrations 10-40 times lower than would be required for untargeted 2-nitroimidazoles such as misonidazole in vitro. Toxicity to both hypoxic and aerobic cells is dependent on the linker chain: the shorter the chain, the greater the toxicity. In addition, the NLP compounds radiosensitize hypoxic cells at external drug concentrations as low as 0.05 mM with almost the full oxygen effect being observed at a concentration of 0.5 mM. These concentrations are 10-100 times lower than would be required for similar radiosensitization using misonidazole. Radiosensitizing ability is independent of linker chain length. The present compounds represent prototypes for further studies of the efficacy and mechanism of action of 2-nitroimidazoles targeted to DNA by linkage to an intercalating group.

  16. Divergent histories of rDNA group I introns in the lichen family Physciaceae.

    PubMed

    Simon, Dawn; Moline, Jessica; Helms, Gert; Friedl, Thomas; Bhattacharya, Debashish

    2005-04-01

    The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene.

  17. DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus Crataegus

    PubMed Central

    Zarrei, Mehdi; Talent, Nadia; Kuzmina, Maria; Lee, Jeanette; Lund, Jensen; Shipley, Paul R.; Stefanović, Saša; Dickinson, Timothy A.

    2015-01-01

    DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication

  18. DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus Crataegus.

    PubMed

    Zarrei, Mehdi; Talent, Nadia; Kuzmina, Maria; Lee, Jeanette; Lund, Jensen; Shipley, Paul R; Stefanović, Saša; Dickinson, Timothy A

    2015-01-01

    DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication

  19. Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

    NASA Technical Reports Server (NTRS)

    Tsang, Joyce; Joyce, Gerald F.

    1996-01-01

    In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

  20. High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

    PubMed Central

    Singh, Indrabahadur; Ozturk, Nihan; Cordero, Julio; Mehta, Aditi; Hasan, Diya; Cosentino, Claudia; Sebastian, Carlos; Krüger, Marcus; Looso, Mario; Carraro, Gianni; Bellusci, Saverio; Seeger, Werner; Braun, Thomas; Mostoslavsky, Raul; Barreto, Guillermo

    2015-01-01

    The eukaryotic genome is organized into chromatins, the physiological template for DNA-dependent processes including replication, recombination, repair, and transcription. Chromatin-mediated transcription regulation involves DNA methylation, chromatin remodeling, and histone modifications. However, chromatin also contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Although it is known that HMG proteins induce structural changes of chromatin, the processes underlying transcription regulation by HMG proteins are poorly understood. Here we decipher the molecular mechanism of transcription regulation mediated by the HMG AT-hook 2 protein (HMGA2). We combined proteomic, ChIP-seq, and transcriptome data to show that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX) mediated by the protein kinase ataxia telangiectasia mutated (ATM). Furthermore, we demonstrate the biological relevance of this mechanism within the context of TGFβ1 signaling. The interplay between HMGA2, ATM, and H2AX is a novel mechanism of transcription initiation. Our results link H2AXS139ph to transcription, assigning a new function for this DNA damage marker. Controlled chromatin opening during transcription may involve intermediates with DNA breaks that may require mechanisms that ensure the integrity of the genome. PMID:26045162

  1. Xeroderma Pigmentosum Group A Protein Loads as a Separate Factor onto DNA Lesions

    PubMed Central

    Rademakers, Suzanne; Volker, Marcel; Hoogstraten, Deborah; Nigg, Alex L.; Moné, Martijn J.; van Zeeland, Albert A.; Hoeijmakers, Jan H. J.; Houtsmuller, Adriaan B.; Vermeulen, Wim

    2003-01-01

    Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient (∼5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair. PMID:12897146

  2. Mitochondrial DNA variability among six South American Amerindian villages from the Pano linguistic group.

    PubMed

    Mendes-Junior, Celso T; Simoes, Aguinaldo L

    2014-01-01

    Although scattered throughout a large geographic area, the members of the Pano linguistic group present strong ethnic, linguistic, and cultural homogeneity, a feature that causes them to be considered components of a same "Pano" tribe. Nevertheless, the genetic homogeneity between Pano villages has not yet been examined. To study the genetic structure of the Pano linguistic group, four major Native American mitochondrial DNA (mtDNA) founder haplogroups were analyzed in 77 Amerindians from six villages of four Pano tribes (Katukina, Kaxináwa, Marúbo, and Yaminawa) located in the Brazilian Amazon. The central position of these tribes in the continent makes them relevant for attempts to reconstruct population movements in South America. Except for a single individual that presented an African haplogroup L, all remaining individuals presented one of the four Native American haplogroups. Significant heterogeneity was observed across the six Pano villages. Although Amerindian populations are usually characterized by considerable interpopulational diversity, the high heterogeneity level observed is unexpected if the strong ethnic, linguistic, and cultural homogeneity of the Pano linguistic group is taken into account. The present findings indicate that the ethnic, linguistic, and cultural homogeneity does not imply genetic homogeneity. Even though the genetic heterogeneity uncovered may be a female-specific process, the most probable explanation for that is the joint action of isolation and genetic drift as major factors influencing the genetic structure of the Pano linguistic group.

  3. Conserved primary sequences of the DNA terminal proteins of five different human adenovirus groups.

    PubMed

    Green, M; Brackmann, K; Wold, W S; Cartas, M; Thornton, H; Elder, J H

    1979-09-01

    The 31 human adenoviruses (Ad) from five groups (A-E) whose DNAs are <20% homologous by molecular hybridization. Ad5 (group C) DNA contains a 55,000-dalton protein probably covalently bound to each 5' terminus. This covalently bound protein may be analogous to polypeptides found in other viral and nonviral systems that are covalently bound to genomic DNAs or RNAs and that are thought to function in DNA or RNA replication. Because of the importance of proteins linked to nucleic acids, we have investigated whether DNAs from all five groups of human adenoviruses have terminal proteins, as well as the peptide relationships among the different terminal proteins. We show here that DNAs from Ad12, 7, 2, 19, and 4, representing Ad groups A-E, respectively, all contain covalently bound proteins of about 55,000 daltons. To investigate the peptide relatedness among the terminal proteins, we prepared microgram quantities of covalently bound protein from Ads in groups A-E and compared their chymotryptic and tryptic (125)I-labeled peptide maps. We find that the covalently bound protein maps of the five Ad groups are highly related and possibly identical. On the other hand, the tryptic and chymotryptic peptide maps of the major virion protein II and the core proteins V and VII of groups B, C, and E Ads show considerable heterology. Assuming that the covalently bound protein is virally coded, the conserved primary sequence of these proteins suggests a major functional role for the protein in Ad replication. Because the genetic origin of the Ad covalently bound proteins is not established, our data are also consistent with the possibility that the protein is coded by a cellular gene.

  4. The Cockayne syndrome group B DNA repair protein as an anti-cancer target.

    PubMed

    Lu, Y; Mani, S; Kandimalla, E R; Yu, D; Agrawal, S; States, J C; Bregman, D B

    2001-12-01

    Cells from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair (TCR), which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS (of which there are two complementation groups, CSA and CSB) do not demonstrate an elevated incidence of cancer. Recently, we demonstrated that disruption of the CSB gene reduces the spontaneous tumor rate in cancer predisposed Ink4a/ARF-/- mice as well as causing their embryo fibroblasts to proliferate more slowly and be more sensitive to UV-induced apoptosis. In the present study we characterized phosphorothioate backbone antisense oligodeoxynucleotides (AOs) that reduced the levels of CSB mRNA in A2780/CP70 ovarian carcinoma cells. The AOs caused the cells to proliferate more slowly and made them more sensitive to either cisplatin or oxaliplatin. The AOs also enhanced the cytotoxicity of hydrogen peroxide and gamma-radiation, both of which can induce oxidative DNA lesions, which are subject to TCR. The AOs did not potentiate the cytotoxicity of topotecan, which induces DNA strand breaks. Chemically modified () AOs (MBOs) targeting CSB were able to potentiate the anti-tumor effect of cisplatin against A2780/CP70 tumor xenografts formed in nude mice. The MBOs enabled a non-toxic (3 mg/kg) dose of cisplatin to have the same degree of anti-tumor efficacy as a more toxic (5 mg/kg) cisplatin dose. Collectively, these results suggest that the CSB gene product may be viewed as an anti-cancer target. PMID:11713576

  5. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    NASA Technical Reports Server (NTRS)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  6. Increased levels of chromosomal aberrations and DNA damage in a group of workers exposed to formaldehyde.

    PubMed

    Costa, Solange; Carvalho, Sandra; Costa, Carla; Coelho, Patrícia; Silva, Susana; Santos, Luís S; Gaspar, Jorge F; Porto, Beatriz; Laffon, Blanca; Teixeira, João P

    2015-07-01

    Formaldehyde (FA) is a commonly used chemical in anatomy and pathology laboratories as a tissue preservative and fixative. Because of its sensitising properties, irritating effects and cancer implication, FA accounts probably for the most important chemical-exposure hazard concerning this professional group. Evidence for genotoxic effects and carcinogenic properties in humans is insufficient and conflicting, particularly in regard to the ability of inhaled FA to induce toxicity on other cells besides first contact tissues, such as buccal and nasal cells. To evaluate the effects of exposure to FA in human peripheral blood lymphocytes, a group of 84 anatomy pathology laboratory workers exposed occupationally to FA and 87 control subjects were tested for chromosomal aberrations (CAs) and DNA damage (comet assay). The level of exposure to FA in the workplace air was evaluated. The association between genotoxicity biomarkers and polymorphic genes of xenobiotic-metabolising and DNA repair enzymes were also assessed. The estimated mean level of FA exposure was 0.38±0.03 ppm. All cytogenetic endpoints assessed by CAs test and comet assay % tail DNA (%TDNA) were significantly higher in FA-exposed workers compared with controls. Regarding the effect of susceptibility biomarkers, results suggest that polymorphisms in CYP2E1 and GSTP1 metabolic genes, as well as, XRCC1 and PARP1 polymorphic genes involved in DNA repair pathways are associated with higher genetic damage in FA-exposed subjects. Data obtained in this study show a potential health risk situation of anatomy pathology laboratory workers exposed to FA (0.38 ppm). Implementation of security and hygiene measures may be crucial to decrease risk. The obtained information may also provide new important data to be used by health care programs and by governmental agencies responsible for occupational health and safety.

  7. Capture-recapture of white-tailed deer using DNA from fecal pellet-groups

    USGS Publications Warehouse

    Goode, Matthew J; Beaver, Jared T; Muller, Lisa I; Clark, Joseph D.; van Manen, Frank T.; Harper, Craig T; Basinger, P Seth

    2014-01-01

    Traditional methods for estimating white-tailed deer population size and density are affected by behavioral biases, poor detection in densely forested areas, and invalid techniques for estimating effective trapping area. We evaluated a noninvasive method of capture—recapture for white-tailed deer (Odocoileus virginianus) density estimation using DNA extracted from fecal pellets as an individual marker and for gender determination, coupled with a spatial detection function to estimate density (spatially explicit capture—recapture, SECR). We collected pellet groups from 11 to 22 January 2010 at randomly selected sites within a 1-km2 area located on Arnold Air Force Base in Coffee and Franklin counties, Tennessee. We searched 703 10-m radius plots and collected 352 pellet-group samples from 197 plots over five two-day sampling intervals. Using only the freshest pellets we recorded 140 captures of 33 different animals (15M:18F). Male and female densities were 1.9 (SE = 0.8) and 3.8 (SE = 1.3) deer km-2, or a total density of 5.8 deer km-2 (14.9 deer mile-2). Population size was 20.8 (SE = 7.6) over a 360-ha area, and sex ratio was 1.0 M: 2.0 F (SE = 0.71). We found DNA sampling from pellet groups improved deer abundance, density and sex ratio estimates in contiguous landscapes which could be used to track responses to harvest or other management actions.

  8. Novel groups of cyanobacterial podovirus DNA polymerase (pol) genes exist in paddy waters in northeast China.

    PubMed

    Wang, Xinzhen; Liu, Junjie; Yu, Zhenhua; Jin, Jian; Liu, Xiaobing; Wang, Guanghua

    2016-12-01

    In this study, we surveyed cyanopodovirus DNA polymerase (pol) sequences in paddy waters using the culture-independent PCR and Sanger sequencing methods. Four paddy waters generated from a pot experiment with different soil types collected from op E: n paddy fields in northeast China were used in this study. A total of 438 DNA pol clones were identified as cyanopodoviruses. The clones from the paddy waters formed nine unique groups of cyanopodoviruses either exclusively or with clones from East Lake in China (subclusters α-1 to α-8 and cluster β). None of the clones from open oceans or coastal waters fell into these unique groups. Additionally, the distribution proportions of the clones into different cyanopodovirus groups varied among paddy water samples, which suggested that the cyanopodovirus compositions were spatially distributed in the paddy fields. The comparison of clone libraries in different studies indicated that the diversity of cyanopodoviruses in paddy waters was comparable to the diversity in the open oceans but was less than the diversity in the coastal estuary of Chesapeake Bay. Non-metric multidimensional scaling analysis indicated that the cyanopodovirus communities in paddy waters were similar to those in lake freshwater but distinct from the communities in marine and coastal waters. PMID:27612493

  9. Genetic Variation Among Vegetative Compatibility Groups of Fusarium oxysporum f. sp. cubense Analyzed by DNA Fingerprinting.

    PubMed

    Bentley, S; Pegg, K G; Moore, N Y; Davis, R D; Buddenhagen, I W

    1998-12-01

    ABSTRACT Genetic variation within a worldwide collection of 208 isolates of Fu-sarium oxysporum f. sp. cubense, representing physiological races 1, 2, 3, and 4 and the 20 reported vegetative compatibility groups (VCGs), was analyzed using modified DNA amplification fingerprinting. Also characterized were 133 isolates that did not belong to any of the reported VCGs of F. oxysporum f. sp. cubense including race 3 isolates from a Heliconia species and isolates from a symptomatic wild banana species growing in the jungle in peninsular Malaysia. The DNA fingerprint patterns were generally VCG specific, irrespective of geographic or host origin. A total of 33 different genotypes were identified within F. oxysporum f. sp. cu-bense; 19 genotypes were distinguished among the isolates that belonged to the 20 reported VCGs, and 14 new genotypes were identified among the isolates that did not belong to any of the existing VCGs. DNA fingerprinting analysis also allowed differentiation of nine clonal lineages within F. oxysporum f. sp. cubense. Five of these lineages each contained numerous closely related VCGs and genotypes, and the remaining four lineages each contained a single genotype. The genetic diversity and geographic distribution of several of these lineages of F. oxysporum f. sp. cubense suggests that they have coevolved with edible bananas and their wild diploid progenitors in Asia. DNA fingerprinting analysis of isolates from the wild pathosystem provides further evidence for the coevolution hypothesis. The genetic isolation and limited geographic distribution of four of the lineages of F. oxysporum f. sp. cubense suggests that the pathogen has also arisen independently, both within and outside of the center of origin of the host.

  10. Adsorption and desorption of DNA tuned by hydroxyl groups in graphite oxides-based solid extraction material.

    PubMed

    Akceoglu, Garbis Atam; Li, Oi Lun; Saito, Nagahiro

    2015-12-01

    The extraction of DNA is the most crucial method used in molecular biology. Up to date silica matrices has been widely applied as solid support for selective DNA adsorption and extraction. However, since adsorption force of SiOH functional groups is much greater than that of desorption force, the DNA extraction efficiency of silica surfaces is limited. In order to increase the DNA extraction yield, a new surface with different functional groups which possess of greater desorption property is required. In this study, we proposed cellulose/graphite oxide (GO) composite as an alternative material for DNA adsorption and extraction. GO/Cellulose composite provides the major adsorption and desorption of DNA by COH, which belongs to alkyl or phenol type of OH functional group. Compared to SiOH, COH is less polarized and reactive, therefore the composite might provide a higher desorption of DNA during the elution process. The GO/cellulose composite were prepared in spherical structure by mixing urea, cellulose, NaOH, Graphite oxide and water. The concentration of GO within the composites were controlled to be 0-4.15 wt.%. The extraction yield of DNA increased with increasing weight percentage of GO. The highest yield was achieved at 4.15 wt.% GO, where the extraction efficiency was reported as 660.4 ng/μl when applying 2M GuHCl as the binding buffer. The absorbance ratios between 260 nm and 280 nm (A260/A280) of the DNA elution was demonstrated as 1.86, indicating the extracted DNA consisted of high purity. The results proved that GO/cellulose composite provides a simple method for selective DNA extraction with high extraction efficiency of pure DNA.

  11. Rapid discrimination and classification of the Lactobacillus plantarum group based on a partial dnaK sequence and DNA fingerprinting techniques.

    PubMed

    Huang, Chien-Hsun; Lee, Fwu-Ling; Liou, Jong-Shian

    2010-03-01

    The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.

  12. Cryptons: a group of tyrosine-recombinase-encoding DNA transposons from pathogenic fungi.

    PubMed

    Goodwin, Timothy J D; Butler, Margaret I; Poulter, Russell T M

    2003-11-01

    A new group of transposable elements, which the authors have named cryptons, was detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. These elements are unlike any previously described transposons. An archetypal member of the group, crypton Cn1, is 4 kb in length and is present at a low but variable copy number in a variety of C. neoformans strains. It displays interstrain variations in its insertion sites, suggesting recent mobility. The internal region contains a long gene, interrupted by several introns. The product of this gene contains a putative tyrosine recombinase near its middle, and a region similar in sequence to the DNA-binding domains of several fungal transcription factors near its C-terminus. The element contains no long repeat sequences, but is bordered by short direct repeats which may have been produced by its insertion into the host genome by recombination. Many of the structural features of crypton Cn1 are conserved in the other known cryptons, suggesting that these elements represent the functional forms. The presence of cryptons in ascomycetes and basidiomycetes suggests that this is an ancient group of elements (>400 million years old). Sequence comparisons suggest that cryptons may be related to the DIRS1 and Ngaro1 groups of tyrosine-recombinase-encoding retrotransposons. PMID:14600222

  13. Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

    PubMed Central

    McCauley, Micah J.; Rueter, Emily M.; Rouzina, Ioulia; Maher, L. James; Williams, Mark C.

    2013-01-01

    Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (∼10 s−1) are higher than those observed for wild-type proteins (∼0.1–1.0 s−1), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB–DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility. PMID:23143110

  14. Conducting polymer based DNA biosensor for the detection of the Bacillus cereus group species

    NASA Astrophysics Data System (ADS)

    Velusamy, Vijayalakshmi; Arshak, Khalil; Korostynska, Olga; Oliwa, Kamila; Adley, Catherine

    2009-05-01

    Biosensor designs are emerging at a significant rate and play an increasingly important role in foodborne pathogen detection. Conducting polymers are excellent tools for the fabrication of biosensors and polypyrrole has been used in the detection of biomolecules due to its unique properties. The prime intention of this paper was to pioneer the design and fabrication of a single-strand (ss) DNA biosensor for the detection of the Bacillus cereus (B.cereus) group species. Growth of B. cereus, results in production of several highly active toxins. Therefore, consumption of food containing >106 bacteria/gm may results in emetic and diarrhoeal syndromes. The most common source of this bacterium is found in liquid food products, milk powder, mixed food products and is of particular concern in the baby formula industry. The electrochemical deposition technique, such as cyclic voltammetry, was used to develop and test a model DNA-based biosensor on a gold electrode electropolymerized with polypyrrole. The electrically conducting polymer, polypyrrole is used as a platform for immobilizing DNA (1μg) on the gold electrode surface, since it can be more easily deposited from neutral pH aqueous solutions of pyrrolemonomers. The average current peak during the electrodeposition event is 288μA. There is a clear change in the current after hybridization of the complementary oligonucleotide (6.35μA) and for the noncomplementary oligonucleotide (5.77μA). The drop in current after each event was clearly noticeable and it proved to be effective.

  15. The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair

    PubMed Central

    Costantini, Silvia; Pegoraro, Silvia; Ros, Gloria; Penzo, Carlotta; Triolo, Gianluca; Demarchi, Francesca; Sgarra, Riccardo; Vindigni, Alessandro; Manfioletti, Guidalberto

    2016-01-01

    The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens. PMID:27723831

  16. DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar

    PubMed Central

    Smith, M. Alex; Fisher, Brian L; Hebert, Paul D.N

    2005-01-01

    The role of DNA barcoding as a tool to accelerate the inventory and analysis of diversity for hyperdiverse arthropods is tested using ants in Madagascar. We demonstrate how DNA barcoding helps address the failure of current inventory methods to rapidly respond to pressing biodiversity needs, specifically in the assessment of richness and turnover across landscapes with hyperdiverse taxa. In a comparison of inventories at four localities in northern Madagascar, patterns of richness were not significantly different when richness was determined using morphological taxonomy (morphospecies) or sequence divergence thresholds (Molecular Operational Taxonomic Unit(s); MOTU). However, sequence-based methods tended to yield greater richness and significantly lower indices of similarity than morphological taxonomy. MOTU determined using our molecular technique were a remarkably local phenomenon—indicative of highly restricted dispersal and/or long-term isolation. In cases where molecular and morphological methods differed in their assignment of individuals to categories, the morphological estimate was always more conservative than the molecular estimate. In those cases where morphospecies descriptions collapsed distinct molecular groups, sequence divergences of 16% (on average) were contained within the same morphospecies. Such high divergences highlight taxa for further detailed genetic, morphological, life history, and behavioral studies. PMID:16214741

  17. Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae).

    PubMed

    Zhang, Cai-Yun; Wang, Feng-Ying; Yan, Hai-Fei; Hao, Gang; Hu, Chi-Ming; Ge, Xue-Jun

    2012-01-01

    It has been suggested that rbcL and matK are the core barcodes in plants, but they are not powerful enough to distinguish between closely related plant groups. Additional barcodes need to be evaluated to improve the level of discrimination between plant species. Because of their well-studied taxonomy and extreme diversity, we used Chinese Lysimachia (Myrsinaceae) species to test the performance of core barcodes (rbcL and matK) and two additional candidate barcodes (trnH-psbA and the nuclear ribosomal ITS); 97 accessions from four subgenus representing 34 putative Lysimachia species were included in this study. And many closely related species pairs in subgen. Lysimachia were covered to detect their discriminatory power. The inefficiency of rbcL and matK alone or combined in closely related plant groups was validated in this study. TrnH-psbA combined with rbcL + matK did not yet perform well in Lysimachia groups. In contrast, ITS, alone or combined with rbcL and/or matK, revealed high resolving ability in Lysimachia. We support ITS as a supplementary barcode on the basis of core barcode rbcL and matK. Besides, this study also illustrates several mistakes or underlying evolutionary events in Lysimachia detected by DNA barcoding. PMID:21967641

  18. Chloroplast DNA variation and geographical structure of the Aristolochia kaempferi group (Aristolochiaceae).

    PubMed

    Watanabe, Kana; Kajita, Tadashi; Murata, Jin

    2006-03-01

    The present study documents cpDNA variation in the Aristolochia kaempferi group (Aristolochiaceae), which consists of one Chinese and all Japanese and Taiwanese species of the subgenus Siphisia. In a phylogenetic analysis based on the nucleotide sequences of the matK gene, and the atpB-rbcL and trnS-trnG intergenic spacer regions, 38 haplotypes were recognized in the A. kaempferi group and as many as 24 within A. kaempferi. This is the most haplotypes reported for a single species to date. Although six highly significant major clades were identified in the phylogenetic analysis, they were not congruent with previous classifications. This might be attributed to the specific speciation process, such as convergent evolution, incomplete lineage sorting, and/or reticulate evolution. The six major clades had a clear geographical distribution pattern and were significantly associated with geographical distribution of haplotypes in a nested clade analysis and AMOVA. The results allow us to deduce a scenario in which multiple contractions and expansions of the geographical ranges brought about by Quaternary climatic oscillations affected the patterns of genetic diversity. The present geographic patterns of haplotype distribution within the A. kaempferi group can be explained by the last postglacial range expansion from different refugia, and the boundaries may be suture zones. PMID:21646203

  19. Genetic analysis of 15 mtDNA SNP loci in Chinese Yi ethnic group using SNaPshot minisequencing.

    PubMed

    Hu, Chun-Ting; Yan, Jiang-Wei; Chen, Feng; Zhang, Qing-Xia; Wang, Hong-Dan; Yin, Cai-Yong; Fan, Han-Ting; Hu, Ling-Li; Shen, Chun-Mei; Meng, Hao-Tian; Zhang, Yu-Dang; Wang, Hui; Zhu, Bo-Feng

    2016-01-15

    SNaPshot minisequencing is a rapid and robust methodology based on a single base extension with a labeled ddNTP. The present study detected 15 selected SNPs in the mitochondrial DNA (mtDNA) control and coding regions by minisequencing methodology using SNaPshot for forensic purpose. The samples were collected from 99 unrelated individuals of the Yi ethnic minority group in Yunnan Province. We have predominantly found high-frequency transitions (91.7%) and a significantly lower frequency of transversions (8.3%). The nt152, 489, 8701, 10,398, 16,183, and 16,362 loci were highly polymorphic, while the nt231, 473 and 581 loci were not polymorphic in the studied population. Based on these 15 SNPs, a total of 28 mtDNA haplotypes were defined in 99 individuals with the haplotype diversity of 0.9136. Also, we compared the mtDNA sequences of Yi group and other 9 populations worldwide and drew a Neighbor-Joining tree based on the shared 12 mtDNA SNP loci, which demonstrated a close relationship between Yi and Bai groups. In conclusion, the analysis of the 15 selected SNPs increases considerably the discrimination power of mtDNA. Moreover, the SNaPshot minisequencing method could quickly detect mtDNA SNPs, and is economical and sensitive. The set of selected 15 SNPs is highly informative and is capable for anthropology genetic analysis.

  20. Genetic analysis of 15 mtDNA SNP loci in Chinese Yi ethnic group using SNaPshot minisequencing.

    PubMed

    Hu, Chun-Ting; Yan, Jiang-Wei; Chen, Feng; Zhang, Qing-Xia; Wang, Hong-Dan; Yin, Cai-Yong; Fan, Han-Ting; Hu, Ling-Li; Shen, Chun-Mei; Meng, Hao-Tian; Zhang, Yu-Dang; Wang, Hui; Zhu, Bo-Feng

    2016-01-15

    SNaPshot minisequencing is a rapid and robust methodology based on a single base extension with a labeled ddNTP. The present study detected 15 selected SNPs in the mitochondrial DNA (mtDNA) control and coding regions by minisequencing methodology using SNaPshot for forensic purpose. The samples were collected from 99 unrelated individuals of the Yi ethnic minority group in Yunnan Province. We have predominantly found high-frequency transitions (91.7%) and a significantly lower frequency of transversions (8.3%). The nt152, 489, 8701, 10,398, 16,183, and 16,362 loci were highly polymorphic, while the nt231, 473 and 581 loci were not polymorphic in the studied population. Based on these 15 SNPs, a total of 28 mtDNA haplotypes were defined in 99 individuals with the haplotype diversity of 0.9136. Also, we compared the mtDNA sequences of Yi group and other 9 populations worldwide and drew a Neighbor-Joining tree based on the shared 12 mtDNA SNP loci, which demonstrated a close relationship between Yi and Bai groups. In conclusion, the analysis of the 15 selected SNPs increases considerably the discrimination power of mtDNA. Moreover, the SNaPshot minisequencing method could quickly detect mtDNA SNPs, and is economical and sensitive. The set of selected 15 SNPs is highly informative and is capable for anthropology genetic analysis. PMID:26432004

  1. Genetic Diversity within Schistosoma haematobium: DNA Barcoding Reveals Two Distinct Groups

    PubMed Central

    Webster, Bonnie L.; Emery, Aiden M.; Webster, Joanne P.; Gouvras, Anouk; Garba, Amadou; Diaw, Oumar; Seye, Mohmoudane M.; Tchuente, Louis Albert Tchuem; Simoonga, Christopher; Mwanga, Joseph; Lange, Charles; Kariuki, Curtis; Mohammed, Khalfan A.; Stothard, J. Russell; Rollinson, David

    2012-01-01

    Background Schistosomiasis in one of the most prevalent parasitic diseases, affecting millions of people and animals in developing countries. Amongst the human-infective species S. haematobium is one of the most widespread causing urogenital schistosomiasis, a major human health problem across Africa, however in terms of research this human pathogen has been severely neglected. Methodology/Principal Findings To elucidate the genetic diversity of Schistosoma haematobium, a DNA ‘barcoding’ study was performed on parasite material collected from 41 localities representing 18 countries across Africa and the Indian Ocean Islands. Surprisingly low sequence variation was found within the mitochondrial cytochrome oxidase subunit I (cox1) and the NADH-dehydrogenase subunit 1 snad1). The 61 haplotypes found within 1978 individual samples split into two distinct groups; one (Group 1) that is predominately made up of parasites from the African mainland and the other (Group 2) that is made up of samples exclusively from the Indian Ocean Islands and the neighbouring African coastal regions. Within Group 1 there was a dominance of one particular haplotype (H1) representing 1574 (80%) of the samples analyzed. Population genetic diversity increased in samples collected from the East African coastal regions and the data suggest that there has been movement of parasites between these areas and the Indian Ocean Islands. Conclusions/Significance The high occurrence of the haplotype (H1) suggests that at some point in the recent evolutionary history of S. haematobium in Africa the population may have passed through a genetic ‘bottleneck’ followed by a population expansion. This study provides novel and extremely interesting insights into the population genetics of S. haematobium on a large geographic scale, which may have consequence for control and monitoring of urogenital schistosomiasis. PMID:23145200

  2. Doping Level of Boron-Doped Diamond Electrodes Controls the Grafting Density of Functional Groups for DNA Assays.

    PubMed

    Švorc, Ĺubomír; Jambrec, Daliborka; Vojs, Marian; Barwe, Stefan; Clausmeyer, Jan; Michniak, Pavol; Marton, Marián; Schuhmann, Wolfgang

    2015-09-01

    The impact of different doping levels of boron-doped diamond on the surface functionalization was investigated by means of electrochemical reduction of aryldiazonium salts. The grafting efficiency of 4-nitrophenyl groups increased with the boron levels (B/C ratio from 0 to 20,000 ppm). Controlled grafting of nitrophenyldiazonium was used to adjust the amount of immobilized single-stranded DNA strands at the surface and further on the hybridization yield in dependence on the boron doping level. The grafted nitro functions were electrochemically reduced to the amine moieties. Subsequent functionalization with a succinic acid introduced carboxyl groups for subsequent binding of an amino-terminated DNA probe. DNA hybridization significantly depends on the probe density which is in turn dependent on the boron doping level. The proposed approach opens new insights for the design and control of doped diamond surface functionalization for the construction of DNA hybridization assays.

  3. Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis.

    PubMed

    Fang, Jing Yuan; Xiao, Shu Dong

    2003-01-01

    DNA methylation is the main epigenetic modification after replication in humans. DNA (cytosine-5)-methyltransferase (DNMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to C5 of cytosine within CpG dinucleotide sequences in the genomic DNA of higher eukaryotes. There is considerable evidence that aberrant DNA methylation plays an integral role in carcinogenesis. Folic acid or folate is crucial for normal DNA synthesis and can regulate DNA methylation, and through this, it affects cellular SAM levels. Folate deficiency results in DNA hypomethylation. Epidemiological studies have indicated that folic acid protects against gastrointestinal (GI) cancers. Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are the enzymes involved in folate metabolism and are thought to influence DNA methylation. MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level. Two common MTHFR polymorphisms, 677CT (or 677TT) and A1298C, and an MS polymorphism, A-->G at 2756, have been identified. Most studies support an inverse association between folate status and the rate of colorectal adenomas and carcinomas. During human GI carcinogenesis, MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level, as well as aberrant methylation.

  4. Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A.

    PubMed

    Rapić Otrin, V; Kuraoka, I; Nardo, T; McLenigan, M; Eker, A P; Stefanini, M; Levine, A S; Wood, R D

    1998-06-01

    Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB- XP-E cell extracts, but microinjection of the protein into DDB- XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin. PMID:9584159

  5. Pyramidal and Chiral Groupings of Gold Nanocrystals Assembled Using DNA Scaffolds

    SciTech Connect

    Mastroianni, Alexander; Claridge, Shelley; Alivisatos, A. Paul

    2009-03-30

    Nanostructures constructed from metal and semiconductor nanocrystals conjugated to, and organized by DNA are an emerging class of material with collective optical properties. We created discrete pyramids of DNA with gold nanocrystals at the tips. By taking small angle X-ray scattering (SAXS) measurments from solutions of these pyramids we confirmed that this pyramidal geometry creates structures which are more rigid in solution than linear DNA. We then took advantage of the tetrahedral symmetry to demonstrate construction of chiral nanostructures.

  6. Primary and secondary structure analyses of the rDNA group-I introns of the Zygnematales (Charophyta).

    PubMed

    Bhattacharya, D; Damberger, S; Surek, B; Melkonian, M

    1996-02-01

    The Zygnematales (Charophyta) contain a group-I intron (subgroupIC1) within their nuclear-encoded small subunit ribosomal DNA (SSU rDNA) coding region. This intron, which is inserted after position 1506 (relative to the SSU rDNA of Escherichia coli), is proposed to have been vertically inherited since the origin of the Zygnematales approximately 350-400 million years ago. Primary and secondary structure analyses were carried out to model group-I intron evolution in the Zygnematales. Secondary structure analyses support genetic data regarding sequence conservation within regions known to be functionally important for in vitro self-splicing of group-I introns. Comparisons of zygnematalean group-I intron secondary structures also provided some new insights into sequences that may have important roles in in vivo RNA splicing. Sequence analyses showed that sequence divergence rates and the nucleotide compositions of introns and coding regions within any one taxon varied widely, suggesting that the "1506" group-I introns and rDNA coding regions in the Zygnematales evolve independently.

  7. Molecular phylogeny of fire ants of the Solenopsis saevissima species-group based on mtDNA sequences.

    PubMed

    Shoemaker, D DeWayne; Ahrens, Michael E; Ross, Kenneth G

    2006-01-01

    The systematics of South American fire ants (Solenopsis saevissima species-group) has been plagued by difficulties in recognizing species and their relationships on the basis of morphological characters. We surveyed mtDNA sequences from 623 individuals representing 13 described and undescribed species within the species-group and 18 individuals representing other major Solenopsis lineages to generate a phylogeny of the mitochondrial genome. Our analyses support the monophyly of the S. saevissima species-group, consistent with a single Neotropical origin and radiation of this important group of ants, as well as the monophyly of the socially polymorphic species within the group, consistent with a single origin of polygyny (multiple queens per colony) as a derived form of social organization. The mtDNA sequences of the inquiline social parasite S. daguerrei form a clade that appears to be distantly related to sequences from the several host species, consistent with the view that advanced social parasitism did not evolve via sympatric speciation of intraspecific parasites. An important general finding is that species-level polyphyly of the mtDNA appears to be the rule in this group of ants. The existence of multiple divergent mtDNA lineages within several nominal species (including the pest S. invicta) suggests that the pattern of widespread polyphyly often stems from morphological delimitation that overcircumscribes species. However, in two cases the mtDNA polyphyly likely results from recent interspecific hybridization. While resolving species boundaries and relationships is important for understanding general patterns of diversification of South American fire ants, these issues are of added importance because invasive fire ants are emerging as global pests and becoming important model organisms for evolutionary research.

  8. Four new species of the Stegana ornatipes species group (Diptera: Drosophilidae) from Yunnan, China, with DNA barcoding information.

    PubMed

    Zhang, Yuan; Chen, Hong-Wei

    2015-01-09

    Fore new species of Stegana (Steganina) ornatipes species group are found from Yunnan, China: S. (S.) angustifoliacea sp. nov., S. (S.) crinata sp. nov., S. (S.) nigripes sp. nov. and S. (S.) polysphyra sp. nov. The DNA sequences of the mitochondrial COI gene with BOLD Process ID and GenBank accession numbers are provided for the Chinese species. 

  9. Systematic and phylogeographical assessment of the Acanthodactylus erythrurus group (Reptilia: Lacertidae) based on phylogenetic analyses of mitochondrial and nuclear DNA.

    PubMed

    Fonseca, Miguel M; Brito, José C; Paulo, Octávio S; Carretero, Miguel A; Harris, D James

    2009-05-01

    We have used mitochondrial 12S rRNA, 16S rRNA and nuclear beta-fibrinogen (intron 7) sequences to investigate the phylogenetic and phylogeographic relationships between Acanthodactylus erythrurus group species (except for A. boueti). The phylogenetic analyses of the Acanthodactylus genus did not cluster A. guineensis and A. savignyi with the remaining species of the group (A. blanci, A. lineomaculatus and A. erythrurus). Within the A. erythrurus group, the results of the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) showed a complex phylogeny with geographic structure, but it was not congruent with the present taxonomy. Some taxonomic units, such as A. blanci, A. lineomaculatus, A. e. atlanticus and A. e. belli did not form monophyletic genetic units. The application of a molecular clock suggested that the uplift of the Atlas Mountains in the mid-late Miocene and the reopening of the Strait of Gibraltar could be major biogeographic events responsible for the genetic differentiation in the group. Additionally, diverse micro-evolutionary patterns due to the recent contraction/expansion phases of the habitats in North Africa associated with the high dispersal capabilities of these lizards could be related to the complex phylogenetic patterns observed.

  10. DNA polymorphism analysis of candidate genes for type 2 diabetes mellitus in a Mexican ethnic group.

    PubMed

    Flores-Martínez, S E; Islas-Andrade, S; Machorro-Lazo, M V; Revilla, M C; Juárez, R E; Mújica-López, K I; Morán-Moguel, M C; López-Cardona, M G; Sánchez-Corona, J

    2004-01-01

    Type 2 diabetes mellitus is a complex metabolic disorder resulting from the action and interaction of many genetic and environmental factors. It has been reported that polymorphisms in genes involved in the metabolism of glucose are associated with the susceptibility to develop type 2 diabetes mellitus. Although the risk of developing type 2 diabetes mellitus increases with age, as well as with obesity and hypertension, its prevalence and incidence are different among geographical regions and ethnic groups. In Mexico, a higher prevalence and incidence has been described in the south of the country, and differences between urban and rural communities have been observed. We studied 73 individuals from Santiago Jamiltepec, a small indigenous community from Oaxaca State, Mexico. This population has shown a high prevalence of type 2 diabetes mellitus, and the aim of this study was to analyze the relationship between the Pst I (insulin gene), Nsi I (insulin receptor gene) and Gly972Arg (insulin receptor substrate 1 gene) polymorphisms and type 2 diabetes mellitus, obesity and hypertension in this population. Clinical evaluation consisted of BMI and blood pressure measurements, and biochemical assays consisted of determination of fasting plasma insulin and glucose levels. PCR and restriction enzyme digestion analysis were applied to genomic DNA to identify the three polymorphisms. From statistical analysis carried out here, individually, the Pst I, Nsi I and Gly972Arg polymorphisms were not associated with the type 2 diabetes, obese or hypertensive phenotypes in this population. Nevertheless, there was an association between the Nsi I and Pst I polymorphisms and increased serum insulin levels.

  11. Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani.

    PubMed

    Kuninaga, S; Natsuaki, T; Takeuchi, T; Yokosawa, R

    1997-09-01

    Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66-100% for isolates of different subgroups within an AG, and 55-96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.

  12. 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)

  13. Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

    PubMed Central

    Hseu, R S; Wang, H H; Wang, H F; Moncalvo, J M

    1996-01-01

    Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated. PMID:8919797

  14. Efimov-like phase of a three-stranded DNA and the renormalization-group limit cycle

    NASA Astrophysics Data System (ADS)

    Pal, Tanmoy; Sadhukhan, Poulomi; Bhattacharjee, Somendra M.

    2015-04-01

    A three-stranded DNA with short range base pairings only is known to exhibit a classical analog of the quantum Efimov effect, viz., a three-chain bound state at the two-chain melting point where no two are bound. By using a nonperturbative renormalization-group method for a rigid duplex DNA and a flexible third strand, with base pairings and strand exchange, we show that the Efimov-DNA is associated with a limit cycle type behavior of the flow of an effective three-chain interaction. The analysis also shows that thermally generated bubbles play an essential role in producing the effect. A toy model for the flow equations shows the limit cycle in an extended three-dimensional parameter space of the two-chain coupling and a complex three-chain interaction.

  15. Efimov-like phase of a three-stranded DNA and the renormalization-group limit cycle.

    PubMed

    Pal, Tanmoy; Sadhukhan, Poulomi; Bhattacharjee, Somendra M

    2015-04-01

    A three-stranded DNA with short range base pairings only is known to exhibit a classical analog of the quantum Efimov effect, viz., a three-chain bound state at the two-chain melting point where no two are bound. By using a nonperturbative renormalization-group method for a rigid duplex DNA and a flexible third strand, with base pairings and strand exchange, we show that the Efimov-DNA is associated with a limit cycle type behavior of the flow of an effective three-chain interaction. The analysis also shows that thermally generated bubbles play an essential role in producing the effect. A toy model for the flow equations shows the limit cycle in an extended three-dimensional parameter space of the two-chain coupling and a complex three-chain interaction.

  16. Efimov-like phase of a three-stranded DNA and the renormalization-group limit cycle.

    PubMed

    Pal, Tanmoy; Sadhukhan, Poulomi; Bhattacharjee, Somendra M

    2015-04-01

    A three-stranded DNA with short range base pairings only is known to exhibit a classical analog of the quantum Efimov effect, viz., a three-chain bound state at the two-chain melting point where no two are bound. By using a nonperturbative renormalization-group method for a rigid duplex DNA and a flexible third strand, with base pairings and strand exchange, we show that the Efimov-DNA is associated with a limit cycle type behavior of the flow of an effective three-chain interaction. The analysis also shows that thermally generated bubbles play an essential role in producing the effect. A toy model for the flow equations shows the limit cycle in an extended three-dimensional parameter space of the two-chain coupling and a complex three-chain interaction. PMID:25974437

  17. Early postnatal ataxia and abnormal cerebellar development in mice lacking Xeroderma pigmentosum Group A and Cockayne syndrome Group B DNA repair genes.

    PubMed

    Murai, M; Enokido, Y; Inamura, N; Yoshino, M; Nakatsu, Y; van der Horst, G T; Hoeijmakers, J H; Tanaka, K; Hatanaka, H

    2001-11-01

    Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are rare autosomal recessive disorders associated with a defect in the nucleotide excision repair (NER) pathway required for the removal of DNA damage induced by UV light and distorting chemical adducts. Although progressive neurological dysfunction is one of the hallmarks of CS and of some groups of XP patients, the causative mechanisms are largely unknown. Here we show that mice lacking both the XPA (XP-group A) and CSB (CS-group B) genes in contrast to the single mutants display severe growth retardation, ataxia, and motor dysfunction during early postnatal development. Their cerebella are hypoplastic and showed impaired foliation and stunted Purkinje cell dendrites. Reduced neurogenesis and increased apoptotic cell death occur in the cerebellar external granular layer. These findings suggest that XPA and CSB have additive roles in the mouse nervous system and support a crucial role for these genes in normal brain development. PMID:11687625

  18. Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

    PubMed Central

    Lovell, C R; Hui, Y

    1991-01-01

    The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed. Images PMID:1768134

  19. Retrovirus-mediated gene transfer corrects DNA repair defect of xeroderma pigmentosum cells of complementation groups A, B and C.

    PubMed

    Zeng, L; Quilliet, X; Chevallier-Lagente, O; Eveno, E; Sarasin, A; Mezzina, M

    1997-10-01

    With the aim to devise a long-term gene therapy protocol for skin cancers in individuals affected by the inherited autosomal recessive xeroderma pigmentosum we transferred the human DNA repair XPA, XPB/ERCC3 and XPC cDNAs, by using the recombinant retroviral vector LXSN, into primary and immortalized fibroblasts obtained from two XP-A, one XP-B (associated with Cockayne's syndrome) and two XP-C patients. After transduction, the complete correction of DNA repair deficiency and functional expression of the transgenes were monitored by UV survival, unscheduled DNA synthesis and recovery of RNA synthesis, and Western blots. The results show that the recombinant retroviruses are highly efficient vectors to transfer and stably express the human DNA repair genes in XP cells and correct the defect of DNA repair of group A, B and C. With our previous results with XPD/ERCC2, the present work extends further promising issues for the gene therapy strategy for most patients suffering from this cancer-prone syndrome. PMID:9415314

  20. Quaternary ammonium groups exposed at the surface of silica nanoparticles suitable for DNA complexation in the presence of cationic lipids.

    PubMed

    Reinhardt, Nora; Adumeau, Laurent; Lambert, Olivier; Ravaine, Serge; Mornet, Stéphane

    2015-05-28

    The production of silica nanoparticles (NPs) exposing quaternary ammonium groups (NPQ(+)) has been achieved using an optimized chemical surface functionalization protocol. The procedures of surface modification and quaternization of amino groups were validated by diffuse reflectance infrared Fourier transform (DRIFT) and (1)H NMR spectroscopies. Compared to nonquaternized aminated NP, the colloidal stability of NPQ(+) was improved for various pH and salt conditions as assessed by ζ potential and light scattering measurements. In the context of their use for nucleic acid delivery, DNA efficiently bound to NPQ(+) analyzed by cosedimentation assays for a large pH range and various NaCl concentrations and exhibited a better efficacy at basic pH than nonquaternized NP. The study of NPQ(+)/DNA/cationic lipids ternary complexes was carried out with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and analyzed by cryo-electron microscopy (cryo-EM). Cryo-EM images showed ternary assemblies where condensed DNA strands are sandwiched between the NPQ(+) surface and the cationic lipid bilayer. Because of an unusual electrostatic colloidal stability of NPQ(+) and a high propensity to bind DNA molecules particularly at high salt concentrations, a novel type of ternary assembly has been formed that might impact the delivery properties of these complexes including their stability in biological environment.

  1. Quaternary ammonium groups exposed at the surface of silica nanoparticles suitable for DNA complexation in the presence of cationic lipids.

    PubMed

    Reinhardt, Nora; Adumeau, Laurent; Lambert, Olivier; Ravaine, Serge; Mornet, Stéphane

    2015-05-28

    The production of silica nanoparticles (NPs) exposing quaternary ammonium groups (NPQ(+)) has been achieved using an optimized chemical surface functionalization protocol. The procedures of surface modification and quaternization of amino groups were validated by diffuse reflectance infrared Fourier transform (DRIFT) and (1)H NMR spectroscopies. Compared to nonquaternized aminated NP, the colloidal stability of NPQ(+) was improved for various pH and salt conditions as assessed by ζ potential and light scattering measurements. In the context of their use for nucleic acid delivery, DNA efficiently bound to NPQ(+) analyzed by cosedimentation assays for a large pH range and various NaCl concentrations and exhibited a better efficacy at basic pH than nonquaternized NP. The study of NPQ(+)/DNA/cationic lipids ternary complexes was carried out with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and analyzed by cryo-electron microscopy (cryo-EM). Cryo-EM images showed ternary assemblies where condensed DNA strands are sandwiched between the NPQ(+) surface and the cationic lipid bilayer. Because of an unusual electrostatic colloidal stability of NPQ(+) and a high propensity to bind DNA molecules particularly at high salt concentrations, a novel type of ternary assembly has been formed that might impact the delivery properties of these complexes including their stability in biological environment. PMID:25950202

  2. Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5' terminus.

    PubMed Central

    Kremsky, J N; Wooters, J L; Dougherty, J P; Meyers, R E; Collins, M; Brown, E L

    1987-01-01

    A general method for the immobilization of DNA through its 5'-end has been developed. A synthetic oligonucleotide, modified at its 5'-end with an aldehyde or carboxylic acid, was attached to latex microspheres containing hydrazide residues. Using T4 polynucleotide ligase and an oligonucleotide splint, a single stranded 98mer was efficiently joined to the immobilized synthetic fragment. After impregnation of the latex microspheres with the fluorescent dye, Nile Red and attachment of an aldehyde 16mer, 5 X 10(5) bead-DNA conjugates could be detected with a conventional fluorimeter. Images PMID:3562241

  3. Genetic polymorphism of Malassezia furfur isolates from Han and Tibetan ethnic groups in China using DNA fingerprinting.

    PubMed

    Zhang, Hao; Zhang, Ruifeng; Ran, Yuping; Dai, Yaling; Lu, Yao; Wang, Peng

    2010-12-01

    Reported isolation rates of Malassezia yeast from human skin show geographic variations. In China, the populations of the Han (1,182.95 million) and Tibetan (5.41 million) ethnic groups are distributed over 9.6 and 3.27 million square kilometers respectively, making biodiversity research feasible and convenient. Malassezia furfur clinical strains (n = 29) isolated from different individuals, with or without associated dermatoses, of these two ethnic groups (15 Han and 12 Tibetan) were identified and analyzed with DNA fingerprinting using single primers specific to minisatellites. Using the Bionumerics software, we found that almost all M. furfur clinical isolates and type strains formed five distinct group clusters according to their associated skin diseases and the ethnic groups of the patients. These findings are the first to focus on the genetic diversity and relatedness of M. furfur in the Tibetan and Han ethnic groups in China and reveal genetic variation associated with related diseases, host ethnicity and geographic origin.

  4. A Randomly Amplified Polymorphic DNA Marker Specific for the Bacillus cereus Group Is Diagnostic for Bacillus anthracis

    PubMed Central

    Daffonchio, Daniele; Borin, Sara; Frova, Giuseppe; Gallo, Romina; Mori, Elena; Fani, Renato; Sorlini, Claudia

    1999-01-01

    Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group. PMID:10049896

  5. Temperature-sensitive mutants of herpes simplex virus type 2: description of three new complementation groups and studies on the inhibition of host cell DNA synthesis.

    PubMed

    Halliburton, I W; Timbury, M C

    1976-02-01

    Three new complementation groups of type 2 herpes simplex virus are described bringing the total number of complementation groups characterized to 13. Of the three new groups, ts 11 fails to make virus DNA at non-permissive temperature (38 degrees C) whereas ts 12 and ts 13 synthesize only very small amounts of virus or cellular DNA at 38 degrees C. ts 11, like ts 9 (Halliburton & Timbury, 1973) fails to switch off host cell DNA synthesis at 38 degrees C. That this is a failure to switch off cell DNA rather than a stimulation of cell DNA synthesis was confirmed in experiments using resting cells. Both the inability to make virus DNA and the inability to switch off cell DNA are reversed in temperature shift-down experiments with cells infected with ts 9 or ts 11. In temperature shift-up experiments, cellular DNA synthesis is inhibited after the shift but virus DNA is only made in very small amounts, probably due to the continuing functioning of a protein made at permissive temperature (31 degrees C) before the shift but which cannot be made at 38 degrees C. The shift-down experiments and the fact that ts 9 and ts 11 complement one another, suggest that the switch-off of host cell DNA synthesis may involve more than one virus specified function. U.v. irradiated virus fails to switch off host cell DNA synthesis.

  6. A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses

    PubMed Central

    2012-01-01

    Background Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis. Results Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar to those found only in single-stranded RNA viruses. The presence and circular configuration of the complete virus genome was confirmed by inverse PCR amplification from native DNA extracted from lake sediment. The virus genome appears to be the result of a RNA-DNA recombination event between two ostensibly unrelated virus groups. Environmental sequence databases were examined for homologous genes arranged in similar configurations and three similar putative virus genomes from marine environments were identified. This result indicates the existence of a widespread but previously undetected group of viruses. Conclusions This unique viral genome carries implications for theories of virus emergence and evolution, as no mechanism for interviral RNA-DNA recombination has yet been identified, and only scant evidence exists that genetic exchange occurs between such distinct virus lineages. Reviewers This article was reviewed by EK, MK (nominated by PF) and AM. For the full reviews, please go to the Reviewers' comments section. PMID:22515485

  7. Complex interactions of the Eastern and Western Slavic populations with other European groups as revealed by mitochondrial DNA analysis.

    PubMed

    Grzybowski, Tomasz; Malyarchuk, Boris A; Derenko, Miroslava V; Perkova, Maria A; Bednarek, Jarosław; Woźniak, Marcin

    2007-06-01

    Mitochondrial DNA sequence variation was examined by the control region sequencing (HVS I and HVS II) and RFLP analysis of haplogroup-diagnostic coding region sites in 570 individuals from four regional populations of Poles and two Russian groups from northwestern part of the country. Additionally, sequences of complete mitochondrial genomes representing K1a1b1a subclade in Polish and Polish Roma populations have been determined. Haplogroup frequency patterns revealed in Poles and Russians are similar to those characteristic of other Europeans. However, there are several features of Slavic mtDNA pools seen on the level of regional populations which are helpful in the understanding of complex interactions of the Eastern and Western Slavic populations with other European groups. One of the most important is the presence of subhaplogroups U5b1b1, D5, Z1 and U8a with simultaneous scarcity of haplogroup K in populations of northwestern Russia suggesting the participation of Finno-Ugrian tribes in the formation of mtDNA pools of Russians from this region. The results of genetic structure analyses suggest that Russians from Velikii Novgorod area (northwestern Russia) and Poles from Suwalszczyzna (northeastern Poland) differ from all remaining Polish and Russian samples. Simultaneously, northwestern Russians and northeastern Poles bear some similarities to Baltic (Latvians) and Finno-Ugrian groups (Estonians) of northeastern Europe, especially on the level of U5 haplogroup frequencies. The occurrence of K1a1b1a subcluster in Poles and Polish Roma is one of the first direct proofs of the presence of Ashkenazi-specific mtDNA lineages in non-Jewish European populations.

  8. The Polycomb Group Protein EZH2 Impairs DNA Damage Repair Gene Expression in Human Uterine Fibroids.

    PubMed

    Yang, Qiwei; Nair, Sangeeta; Laknaur, Archana; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-03-01

    Uterine fibroids are benign, smooth muscle tumors that occur in approximately 70%-80% of women by age 50 yr. The cellular and molecular mechanism(s) by which uterine fibroids (UFs) develop are not fully understood. Accumulating evidence demonstrates that several genetic abnormalities, including deletions, rearrangements, translocations, as well as mutations, have been found in UFs. These genetic anomalies suggest that low DNA damage repair capacity may be involved in UF formation. The objective of this study was to determine whether expression levels of DNA damage repair-related genes were altered, and how they were regulated in the pathogenesis of UFs. Expression levels of DNA repair-related genes RAD51 and BRCA1 were deregulated in fibroid tissues as compared to adjacent myometrial tissues. Expression levels of chromatin protein enhancer of zeste homolog 2 (EZH2) were higher in a subset of fibroids as compared to adjacent myometrial tissues by both immunohistochemistry and Western blot analysis. Treatment with an inhibitor of EZH2 markedly increased expression levels of RAD51 and BRCA1 in fibroid cells and inhibited cell proliferation paired with cell cycle arrest. Restoring the expression of RAD51 and BRCA1 by treatment with EZH2 inhibitor was dependent on reducing the enrichment of trimethylation of histone 3 lysine 27 epigenetic mark in their promoter regions. This study reveals the important role of EZH2-regulated DNA damage-repair genes via histone methylation in fibroid biology, and may provide novel therapeutic targets for the medical treatment of women with symptomatic UFs. PMID:26888970

  9. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation

    PubMed Central

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-01-01

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation. PMID:25820262

  10. The electrokinetic characterization of gold nanoparticles, functionalized with cationic functional groups, and its' interaction with DNA.

    PubMed

    Lazarus, Geraldine Genevive; Revaprasadu, Neerish; López-Viota, Julián; Singh, Moganavelli

    2014-09-01

    Gold nanoparticles have attracted strong biomedical interest for drug delivery due to their low toxic nature, surface plasmon resonance and capability of increasing the stability of the payload. However, gene transfection represents another important biological application. Considering that cellular barriers keep enclosed their secret to deliver genes using nanoparticles, an important step can be achieved by studying the functionalization of nanoparticles with DNA. In the present contribution the synthesis of nanoparticles consisting of a gold core coated with one or more layers of amino acid (l-lysine), and cationic polyelectrolytes (poly-ethyleneimine and poly-l-lysine) is reported. All nanoparticles were subjected to dynamic light scattering, electrophoretic mobility measurements, UV-vis optical spectrophotometry analysis and transmission electron microscopy imaging. In addition, the adsorption of DNA plasmid (pSGS) with linear and supercoiled configurations was studied for those gold nanoparticles under the most suitable surface modifications. Preliminary results showed that the gold nanoparticles functionalized with poly-ethyleneimine and poly-l-lysine, respectively, and bound to linear DNA configurations, present in absolute value a higher electrophoretic mobility irrespective of the pH of the media, compared to the supercoiled and nicked configuration. The findings from this study suggest that poly-ethyleneimine and poly-l-lysine functionalized gold nanoparticles are biocompatible and may be promising in the chemical design and future optimization of nanostructures for biomedical applications such as gene and drug delivery.

  11. Expression of human histo-blood group ABO genes is dependent upon DNA methylation of the promoter region.

    PubMed

    Kominato, Y; Hata, Y; Takizawa, H; Tsuchiya, T; Tsukada, J; Yamamoto, F

    1999-12-24

    We have investigated the regulatory role of DNA methylation in the expression of the human histo-blood group ABO genes. The ABO gene promoter region contains a CpG island whose methylation status correlates well with gene expression in the cell lines tested. The CpG island was found hypomethylated in some cell lines that expressed ABO genes, whereas the other cell lines that did not express ABO genes were hypermethylated. Whereas constitutive transcriptional activity of the ABO gene promoter was demonstrated in both expressor and nonexpressor cell lines by transient transfection of reporter constructs containing the ABO gene promoter sequence, HhaI methylase-catalyzed in vitro methylation of the promoter region prior to DNA transfection suppressed the promoter activity when introduced into the expressor gastric cancer cell line KATOIII cells. On the other hand, in the nonexpressor gastric cancer cell line MKN28 cells, treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation of the ABO gene promoter and appearance of A-transferase messages, as well as A-antigens synthesized by A-transferase. Taken together, these studies suggest that DNA methylation of the ABO gene promoter may play an important role in the regulation of ABO gene expression. PMID:10601288

  12. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  13. Single nucleotide polymorphisms of mitochondrial DNA HVS-I and HVS-II in Chinese Bai ethnic group.

    PubMed

    Chen, Feng; Yin, Cai-Yong; Qian, Xiao-Qin; Fan, Han-Ting; Deng, Ya-Jun; Zhang, Yu-Dang; Meng, Hao-Tian; Shen, Chun-Mei; Yang, Chun-Hua; Jin, Rui; Zhu, Bo-Feng; Xu, Peng

    2015-03-01

    For forensic and population genetic purposes, a total of 125 unrelated volunteers' blood samples were collected from Chinese Bai ethnic minority group to analyze sequence variation of two hypervariable segments (HVS-I and HVS-II) in the mitochondrial DNA control region. Comparing the HVS-I and HVS-II sequences of the 125 Chinese Bais to the Anderson reference sequence, we found 86 polymorphic loci in HVS-I and 40 in HVS-II in mitochondrial DNA sequences of the Chinese Bai ethnic minority group, which defined 93 and 53 different haplotypes, respectively. Haplotype diversity and the mean pairwise differences were 0.992 ± 0.003 and 6.553 in HVS-I, and 0.877 ± 0.027 and 2.407 in HVS-II, respectively. We defined four macrohaplogroups R, M, N and D with the proportions ranging from 9.6% to 40.0%. With the analysis of the hypervariable domain from nucleotide 16 180-16 193 in HVS-I, our study revealed new haplotypes of sequence variations. In addition, the Fst metric, phylogenetic tree, and principal component analysis demonstrated a close genetic relationship between the Bai group and Chinese Han populations from South China, Changsha, and Guangdong. The results support that the Bai group is a multiorigin ethnic minority that has merged with the Chinese Han population.

  14. Comprehensive phylogenetic analysis of bacterial group II intron-encoded ORFs lacking the DNA endonuclease domain reveals new varieties.

    PubMed

    Toro, Nicolás; Martínez-Abarca, Francisco

    2013-01-01

    Group II introns are self-splicing RNAs that act as mobile retroelements in the organelles of plants, fungi and protists. They are also widely distributed in bacteria, and are generally assumed to be the ancestors of nuclear spliceosomal introns. Most bacterial group II introns have a multifunctional intron-encoded protein (IEP) ORF within the ribozyme domain IV (DIV). This ORF encodes an N-terminal reverse transcriptase (RT) domain, followed by a putative RNA-binding domain with RNA splicing or maturase activity and, in some cases, a C-terminal DNA-binding (D) region followed by a DNA endonuclease (En) domain. In this study, we focused on bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region (classes A, C, D, E, F and unclassified introns). On the basis of phylogenetic analyses of the maturase domain and its C-terminal extension, which appears to be a signature characteristic of ORF phylogenetic class, with support from the phylogeny inferred from the RT domain, we have revised the proposed new class F, defining new intron ORF varieties. Our results increase knowledge of the lineage of group II introns encoding proteins lacking the En-domain.

  15. Identification of DNA homologies among H incompatibility group plasmids by restriction enzyme digestion and Southern transfer hybridization.

    PubMed Central

    Whiteley, M; Taylor, D E

    1983-01-01

    Plasmids belonging to the three HI plasmid incompatibility subgroups were characterized by the use of restriction enzymes and Southern transfer hybridization. A diversity of restriction enzyme patterns was noted among the HI subgroups, and a small amount of DNA homology was observed by probing these digests with a nick-translated HI1 plasmid. Within a single subgroup (HI1 and HI2), similar restriction enzyme patterns were noted. Plasmids of all three HI subgroups and the HII group had a guanine plus cytosine content of 49 to 50 mol%. The IncHII plasmid pHH1508a also showed some homology with the HI1 probe. The DNA homology observed is probably responsible for common phenotypic properties encoded by these plasmids. Images PMID:6314885

  16. Three Group-I introns in 18S rDNA of Endosymbiotic Algae of Paramecium bursaria from Japan

    NASA Astrophysics Data System (ADS)

    Hoshina, Ryo; Kamako, Shin-ichiro; Imamura, Nobutaka

    2004-08-01

    In the nuclear encoded small subunit ribosomal DNA (18S rDNA) of symbiotic alga of Paramecium bursaria (F36 collected in Japan) possesses three intron-like insertions (Hoshina et al., unpubl. data, 2003). The present study confirmed these exact lengths and insertion sites by reverse transcription-PCR. Two of them were inserted at Escherichia coli 16S rRNA genic position 943 and 1512 that are frequent intron insertion positions, but another insertion position (nearly 1370) was the first finding. Their secondary structures suggested they belong to Group-I intron; one belongs to subgroup IE, others belong to subgroup IC1. Similarity search indicated these introns are ancestral ones.

  17. Comparison of base composition analysis and Sanger sequencing of mitochondrial DNA for four U.S. population groups.

    PubMed

    Kiesler, Kevin M; Coble, Michael D; Hall, Thomas A; Vallone, Peter M

    2014-01-01

    A set of 711 samples from four U.S. population groups was analyzed using a novel mass spectrometry based method for mitochondrial DNA (mtDNA) base composition profiling. Comparison of the mass spectrometry results with Sanger sequencing derived data yielded a concordance rate of 99.97%. Length heteroplasmy was identified in 46% of samples and point heteroplasmy was observed in 6.6% of samples in the combined mass spectral and Sanger data set. Using discrimination capacity as a metric, Sanger sequencing of the full control region had the highest discriminatory power, followed by the mass spectrometry base composition method, which was more discriminating than Sanger sequencing of just the hypervariable regions. This trend is in agreement with the number of nucleotides covered by each of the three assays.

  18. Development of cDNA probes for typing group A bovine rotaviruses on the basis of VP4 specificity.

    PubMed Central

    Parwani, A V; Rosen, B I; McCrae, M A; Saif, L J

    1992-01-01

    Dot and Northern (RNA) blot hybridization assays were developed for the P typing of group A bovine rotaviruses (BRV) by using cDNA probes prepared from gene segment 4. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions (nucleotides 211 to 686) of BRV strain UK, IND, NCDV, and Cr VP4 cDNA by using specific oligonucleotide primers. The probes were P type specific (VP4) and exhibited little or no cross-reactivity with double-stranded RNA from heterologous rotavirus P types. Our studies indicate that at least three P types, as defined by polymerase chain reaction-derived VP4 gene probes from the UK, NCDV, and Cr strains, exist among the seven BRV isolates tested. Images PMID:1383267

  19. Synthesis and biological studies of pyrazolyl-diamine Pt(II) complexes containing polyaromatic DNA-binding groups.

    PubMed

    Gama, Sofia; Mendes, Filipa; Esteves, Teresa; Marques, Fernanda; Matos, António; Rino, José; Coimbra, Joana; Ravera, Mauro; Gabano, Elisabetta; Santos, Isabel; Paulo, António

    2012-11-01

    New [PtCl(pz*NN)](n+) complexes anchored by pyrazolyl-diamine (pz*NN) ligands incorporating anthracenyl or acridine orange DNA-binding groups have been synthesized so as to obtain compounds that would display synergistic effects between platination and intercalation of DNA. Study of their interaction with supercoiled DNA indicated that the anthracenyl-containing complex L(2) Pt displays a covalent type of binding, whereas the acridine orange counterpart L(3) Pt shows a combination of intercalative and covalent binding modes with a strong contribution from the former. L(2) Pt showed a very strong cytotoxic effect on ovarian carcinoma cell lines A2780 and A2780cisR, which are, respectively, sensitive to and resistant to cisplatin. In these cell lines, L(2) Pt is nine to 27 times more cytotoxic than cisplatin. In the sensitive cell line, L(3) Pt showed a cytotoxic activity similar to that of cisplatin, but like L(2) Pt was able significantly to overcome cisplatin cross-resistance. Cell-uptake studies showed that L(2) Pt accumulates preferentially in the cytoplasm, whereas L(3) Pt reaches the cell nucleus more easily, as clearly visualized by time-lapse confocal imaging of live A2870 cells. Altogether, these findings seem to indicate that interaction with biological targets other than DNA might be involved in the mechanism of action of L(2) Pt because this compound, despite having a weaker ability to target the cell nucleus than L(3) Pt, as well as an inferior DNA affinity, is nevertheless more cytotoxic. Furthermore, ultrastructural studies of A2870 cells exposed to L(2) Pt and L(3) Pt revealed that these complexes induce different alterations in cell morphology, thus indicating the involvement of different modes of action in cell death.

  20. [Sequence polymorphism of mtDNA HVR Iand HVR II of Oroqen ethnic group in Inner Mongolia].

    PubMed

    Yan, Chun-Xia; Chen, Feng; Dang, Yong-Hui; Li, Tao; Zheng, Hai-Bo; Chen, Teng; Li, Sheng-Bin

    2008-04-01

    Venous blood samples from 50 unrelated Oroqen individuals living in Inner Mongolia were collected and their mtDNA HVR I and HVR II sequences were detected by using ABI PRISM377 sequencers. The number of polymorphic loci, haplotype, haplotype frequence, average nucleotide variability and other polymorphic parameters were calculated. Based on Oroqen mtDNA sequence data obtained in our experiments and published data, genetic distance between Oroqen ethnic group and other populations were computered by Nei's measure. Phylogenetic tree was constructed by Neighbor Joining method. Comparing with Anderson sequence, 52 polymorphic loci in HVR I and 24 loci in HVR II were found in Oroqen mtDNA sequence, 38 and 27 haplotypes were defined herewith. Haplotype diversity and average nucleotide variability were 0.964+/-0.018 and 7.379 in HVR I, 0.929+/-0.019 and 2.408 in HVR II respectively. Fst and dA genetic distance between 12 populations were calculated based on HVR I sequence, and their relative coefficients were 0.993(P < 0.01). A phylogenetic tree was constructed based on genetic distances and included Oroqen, Taiwan and South Han population in a clade, which indicated near genetic relation between them, and far relation with northern Han, Mongolian and other foreign populations. The genetic polymorphism of mtDNA HVR I and HVR II in Oroqen ethnic group has some specificities compared with that of other populations. These data provide a useful tool in forensic identification, population genetic study and other research fields.

  1. Phylogeny of the Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analyses.

    PubMed

    Siering, P L; Ghiorse, W C

    1996-01-01

    Phase-contrast light microscopy revealed that only one of eight cultivated strains belonging to the Sphaerotilus-Leptothrix group of sheathed bacteria actually produced a sheath in standard growth media. Two Sphaerotilus natans strains produced branched cells, but other morphological characteristics that were used to identify these bacteria were consistent with previously published descriptions. Genomic fingerprints, which were obtained by performing PCR amplification with primers corresponding to enterobacterial repetitive intergenic consensus sequences, were useful for distinguishing between the genera Sphaerotilus and Leptothrix, as well as among individual strains. The complete 16S ribosomal DNA (rDNA) sequences of two strains of "Leptothrix discophora" (strains SP-6 and SS-1) were determined. In addition, partial sequences (approximately 300 nucleotides) of one strain of Leptothrix cholodnii (strain LMG 7171), an unidentified Leptothrix strain (strain NC-1), and four strains of Sphaerotilus natans (strains ATCC 13338T [T = type strain], ATCC 15291, ATCC 29329, and ATCC 29330) were determined. We found that two of the S. natans strains (ATCC 15291 and ATCC 13338T), which differed in morphology and in their genomic fingerprints, had identical sequences in the 300-nucleotide region sequenced. Both parsimony and distance matrix methods were used to infer the evolutionary relationships of the eight strains in a comparison of the 16S rDNA sequences of these organisms with 16S rDNA sequences obtained from ribosomal sequence databases. All of the strains clustered in the Rubrivivax subdivision of the beta subclass of the Proteobacteria, which confirmed previously published conclusions concerning selected individual strains. Additional analyses revealed that all of the S. natans strains clustered in one closely related group, while the Leptothrix strains clustered in two separate lineages that were approximately equidistant from the S. natans cluster. This finding

  2. Phylogeny of the Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analyses.

    PubMed

    Siering, P L; Ghiorse, W C

    1996-01-01

    Phase-contrast light microscopy revealed that only one of eight cultivated strains belonging to the Sphaerotilus-Leptothrix group of sheathed bacteria actually produced a sheath in standard growth media. Two Sphaerotilus natans strains produced branched cells, but other morphological characteristics that were used to identify these bacteria were consistent with previously published descriptions. Genomic fingerprints, which were obtained by performing PCR amplification with primers corresponding to enterobacterial repetitive intergenic consensus sequences, were useful for distinguishing between the genera Sphaerotilus and Leptothrix, as well as among individual strains. The complete 16S ribosomal DNA (rDNA) sequences of two strains of "Leptothrix discophora" (strains SP-6 and SS-1) were determined. In addition, partial sequences (approximately 300 nucleotides) of one strain of Leptothrix cholodnii (strain LMG 7171), an unidentified Leptothrix strain (strain NC-1), and four strains of Sphaerotilus natans (strains ATCC 13338T [T = type strain], ATCC 15291, ATCC 29329, and ATCC 29330) were determined. We found that two of the S. natans strains (ATCC 15291 and ATCC 13338T), which differed in morphology and in their genomic fingerprints, had identical sequences in the 300-nucleotide region sequenced. Both parsimony and distance matrix methods were used to infer the evolutionary relationships of the eight strains in a comparison of the 16S rDNA sequences of these organisms with 16S rDNA sequences obtained from ribosomal sequence databases. All of the strains clustered in the Rubrivivax subdivision of the beta subclass of the Proteobacteria, which confirmed previously published conclusions concerning selected individual strains. Additional analyses revealed that all of the S. natans strains clustered in one closely related group, while the Leptothrix strains clustered in two separate lineages that were approximately equidistant from the S. natans cluster. This finding

  3. Rapid plant identification using species- and group-specific primers targeting chloroplast DNA.

    PubMed

    Wallinger, Corinna; Juen, Anita; Staudacher, Karin; Schallhart, Nikolaus; Mitterrutzner, Evi; Steiner, Eva-Maria; Thalinger, Bettina; Traugott, Michael

    2012-01-01

    Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory. PMID:22253728

  4. Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

    PubMed Central

    Staudacher, Karin; Schallhart, Nikolaus; Mitterrutzner, Evi; Steiner, Eva-Maria; Thalinger, Bettina; Traugott, Michael

    2012-01-01

    Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory. PMID:22253728

  5. Are clownfish groups composed of close relatives? An analysis of microsatellite DNA variation in Amphiprion percula.

    PubMed

    Buston, Peter M; Bogdanowicz, Steven M; Wong, Alex; Harrison, Richard G

    2007-09-01

    A central question of evolutionary ecology is: why do animals live in groups? Answering this question requires that the costs and benefits of group living are measured from the perspective of each individual in the group. This, in turn, requires that the group's genetic structure is elucidated, because genetic relatedness can modulate the individuals' costs and benefits. The clown anemonefish, Amphiprion percula, lives in groups composed of a breeding pair and zero to four nonbreeders. Both breeders and nonbreeders stand to gain by associating with relatives: breeders might prefer to tolerate nonbreeders that are relatives because there is little chance that relatives will survive to breed elsewhere; nonbreeders might prefer to associate with breeders that are relatives because of the potential to accrue indirect genetic benefits by enhancing anemone and, consequently, breeder fitness. Given the potential benefits of associating with relatives, we use microsatellite loci to investigate whether or not individuals within groups of A. percula are related. We develop seven polymorphic microsatellite loci, with a number of alleles (range 2-24) and an observed level of heterozygosity (mean = 0.5936) sufficient to assess fine-scale genetic structure. The mean coefficient of relatedness among group members is 0.00 +/- 0.10 (n = 9 groups), and there are no surprising patterns in the distribution of pairwise relatedness. We conclude that A. percula live in groups of unrelated individuals. This study lays the foundation for further investigations of behavioural, population and community ecology of anemonefishes which are emerging as model systems for evolutionary ecology in the marine environment.

  6. Complete mtDNA genomes of Filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the Asia-Pacific region

    PubMed Central

    Delfin, Frederick; Min-Shan Ko, Albert; Li, Mingkun; Gunnarsdóttir, Ellen D; Tabbada, Kristina A; Salvador, Jazelyn M; Calacal, Gayvelline C; Sagum, Minerva S; Datar, Francisco A; Padilla, Sabino G; De Ungria, Maria Corazon A; Stoneking, Mark

    2014-01-01

    The Philippines is a strategic point in the Asia-Pacific region for the study of human diversity, history and origins, as it is a cross-road for human migrations and consequently exhibits enormous ethnolinguistic diversity. Following on a previous in-depth study of Y-chromosome variation, here we provide new insights into the maternal genetic history of Filipino ethnolinguistic groups by surveying complete mitochondrial DNA (mtDNA) genomes from a total of 14 groups (11 groups in this study and 3 groups previously published) including previously published mtDNA hypervariable segment (HVS) data from Filipino regional center groups. Comparison of HVS data indicate genetic differences between ethnolinguistic and regional center groups. The complete mtDNA genomes of 14 ethnolinguistic groups reveal genetic aspects consistent with the Y-chromosome, namely: diversity and heterogeneity of groups, no support for a simple dichotomy between Negrito and non-Negrito groups, and different genetic affinities with Asia-Pacific groups that are both ancient and recent. Although some mtDNA haplogroups can be associated with the Austronesian expansion, there are others that associate with South Asia, Near Oceania and Australia that are consistent with a southern migration route for ethnolinguistic group ancestors into the Asia-Pacific, with a timeline that overlaps with the initial colonization of the Asia-Pacific region, the initial colonization of the Philippines and a possible separate post-colonization migration into the Philippine archipelago. PMID:23756438

  7. Function of high-mobility group A proteins in the DNA damage signaling for the induction of apoptosis

    PubMed Central

    Fujikane, Ryosuke; Komori, Kayoko; Sekiguchi, Mutsuo; Hidaka, Masumi

    2016-01-01

    O6-Methylguanine produced in DNA can pair with thymine during DNA replication, thus leading to a G-to-A transition mutation. To prevent such outcomes, cells harboring O6-methylguanine-containing mispair undergo apoptosis that requires the function of mismatch repair (MMR) protein complex. To identify the genes involved in the induction of apoptosis, we performed gene-trap mutagenesis and isolated a clone of mouse cells exhibiting an increased resistance to the killing effect of an alkylating agent, N-methyl-N-nitrosourea (MNU). The mutant carries an insertion in the Hmga2 gene, which belongs to a gene family encoding the high-mobility group A non-histone chromatin proteins. To elucidate the function of HMGA proteins in the apoptosis pathway, we introduced siRNAs for HMGA1 and/or HMGA2 into human HeLa MR cells defective in O6-methylguanine-DNA methyltransferase. HMGA1- and HMGA2-single knockdown cells showed an increased resistance to MNU, and HMGA1/HMGA2-double knockdown cells exhibited further increased tolerance compared to the control. The phosphorylation of ATR and CHK1, the appearance of a sub-G1 population, and caspase-9 activation were suppressed in the knockdown cells, although the formation of mismatch recognition complex was unaffected. These results suggest that HMGA family proteins function at the step following the damage recognition in the process of apoptosis triggered by O6-methylguanine. PMID:27538817

  8. Inclusion of methoxy groups inverts the thermodynamic stabilities of DNA-RNA hybrid duplexes: A molecular dynamics simulation study.

    PubMed

    Suresh, Gorle; Priyakumar, U Deva

    2015-09-01

    Modified nucleic acids have found profound applications in nucleic acid based technologies such as antisense and antiviral therapies. Previous studies on chemically modified nucleic acids have suggested that modifications incorporated in furanose sugar especially at 2'-position attribute special properties to nucleic acids when compared to other modifications. 2'-O-methyl modification to deoxyribose sugars of DNA-RNA hybrids is one such modification that increases nucleic acid stability and has become an attractive class of compounds for potential antisense applications. It has been reported that modification of DNA strands with 2'-O-methyl group reverses the thermodynamic stability of DNA-RNA hybrid duplexes. Molecular dynamics simulations have been performed on two hybrid duplexes (DR and RD) which differ from each other and 2'-O-methyl modified counterparts to investigate the effect of 2'-O-methyl modification on their duplex stability. The results obtained suggest that the modification drives the conformations of both the hybrid duplexes towards A-RNA like conformation. The modified hybrid duplexes exhibit significantly contrasting dynamics and hydration patterns compared to respective parent duplexes. In line with the experimental results, the relative binding free energies suggest that the introduced modifications stabilize the less stable DR hybrid, but destabilize the more stable RD duplex. Binding free energy calculations suggest that the increased hydrophobicity is primarily responsible for the reversal of thermodynamic stability of hybrid duplexes. Free energy component analysis further provides insights into the stability of modified duplexes.

  9. Species discrimination in the subfamily Ostertagiinae of Northern China: assessment of DNA barcode in a taxonomically challenging group.

    PubMed

    Lv, Jizhou; Zhang, Yongning; Feng, Chunyan; Yuan, Xiangfen; Sun, Degang; Deng, Junhua; Wang, Caixia; Wu, Shaoqiang; Lin, Xiangmei

    2016-03-01

    Gastrointestinal nematodes within the subfamily Ostertagiinae (Teladorsagia, Ostertagia, and Marshallagia et al.) are among the most common infections of domesticated livestock. These parasites are of particular interest, as many of the species within this group are of economic importance worldwide. Traditionally, nematode species designations have been based on morphological criteria. However, this group possesses poorly defined species. There is an urgent need to develop a reliable technique that can distinguish species of Ostertagiinae. DNA barcoding has been proved to be a powerful tool to identify species of birds, mammals, and arthropods, but this technique has not yet been examined for identifying species of Ostertagiinae. In this study, a total of 138 mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) sequences from individuals representing 11 species of Ostertagiinae were acquired by PCR for the first time. The specimens were collected from pastoral area of northern China. Genetic divergence analyses showed that mean interspecific Kimura two-parameter distances of COI (13.61 %) were about four times higher than the mean value of the intraspecific divergence (3.69 %). Then, the performance of the COI to identify species of Ostertagiinae was evaluated by identification success rates using nearest neighbor (NN) and BLASTn. The results indicated that the rates of correct sequence identification for COI were high (>80 %) when using the NN and BLASTn methods. Besides, the deep lineage divergences are detected in Teladorsagia circumcincta. Meanwhile, the analyses also detected no genetic differentiation between some species such as Ostertagia hahurica and Ostertagia buriatica. These results indicate that the traditional status of species within Ostertagiinae should be closely examined based on the molecular data.

  10. Microsatellite DNA analysis shows that greater sage grouse leks are not kin groups.

    PubMed

    Gibson, Robert M; Pires, Debra; Delaney, Kathleen S; Wayne, Robert K

    2005-12-01

    The spectacular social courtship displays of lekking birds are thought to evolve via sexual selection, but this view does not easily explain the participation of many males that apparently fail to mate. One of several proposed solutions to this 'lek skew paradox' is that kin selection favours low-ranking males joining leks to increase the fitness of closely related breeders. We investigated the potential for kin selection to operate in leks of the greater sage grouse, Centrocercus urophasianus, by estimating relatedness between lekking males using microsatellite DNA markers. We also calibrated these estimates using data from known families. Mean relatedness within leks was statistically indistinguishable from zero. We also found no evidence for local clustering of kin during lek display, although males tended to range closer to kin when off the lek. These results make kin selection an unlikely solution to the lek skew paradox in sage grouse. Together with other recent studies, they also raise the question of why kin selection apparently promotes social courtship in some lekking species, but not in others.

  11. Critical effect of the N2 amino group on structure, dynamics, and elasticity of DNA polypurine tracts.

    PubMed Central

    Lankas, Filip; Cheatham, Thomas E; Spacková, Nad'a; Hobza, Pavel; Langowski, Jörg; Sponer, Jirí

    2002-01-01

    Unrestrained 5-20-ns explicit-solvent molecular dynamics simulations using the Cornell et al. force field have been carried out for d[GCG(N)11GCG]2 (N, purine base) considering guanine*cytosine (G*C), adenine*thymine (A*T), inosine*5-methyl-cytosine (I*mC), and 2-amino-adenine*thymine (D*T) basepairs. The simulations unambiguously show that the structure and elasticity of N-tracts is primarily determined by the presence of the amino group in the minor groove. Simulated A-, I-, and AI-tracts show almost identical structures, with high propeller twist and minor groove narrowing. G- and D-tracts have small propeller twisting and are partly shifted toward the A-form. The elastic properties also differ between the two groups. The sequence-dependent electrostatic component of base stacking seems to play a minor role. Our conclusions are entirely consistent with available experimental data. Nevertheless, the propeller twist and helical twist in the simulated A-tract appear to be underestimated compared to crystallographic studies. To obtain further insight into the possible force field deficiencies, additional multiple simulations have been made for d(A)10, systematically comparing four major force fields currently used in DNA simulations and utilizing B and A-DNA forms as the starting structure. This comparison shows that the conclusions of the present work are not influenced by the force field choice. PMID:11964246

  12. DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA.

    PubMed

    Amaradasa, B S; Horvath, B J; Lakshman, D K; Warnke, S E

    2013-01-01

    Rhizoctonia blight is a common and serious disease of many turfgrass species. The most widespread causal agent, Thanatephorus cucumeris (anamorph: R. solani), consists of several genetically different subpopulations. In addition, Waitea circinata varieties zeae, oryzae and circinata (anamorph: Rhizoctonia spp.) also can cause the disease. Accurate identification of the causal pathogen is important for effective management of the disease. It is challenging to distinguish the specific causal pathogen based on disease symptoms or macroscopic and microscopic morphology. Traditional methods such as anastomosis reactions with tester isolates are time consuming and sometimes difficult to interpret. In the present study universally primed PCR (UP-PCR) fingerprinting was used to assess genetic diversity of Rhizoctonia spp. infecting turfgrasses. Eighty-four Rhizoctonia isolates were sampled from diseased turfgrass leaves from seven distinct geographic areas in Virginia and Maryland. Rhizoctonia isolates were characterized by ribosomal DNA internal transcribed spacer (rDNA-ITS) region and UP-PCR. The isolates formed seven clusters based on ITS sequences analysis and unweighted pair group method with arithmetic mean (UPGMA) clustering of UP-PCR markers, which corresponded well with anastomosis groups (AGs) of the isolates. Isolates of R. solani AG 1-IB (n = 18), AG 2-2IIIB (n = 30) and AG 5 (n = 1) clustered separately. Waitea circinata var. zeae (n = 9) and var. circinata (n = 4) grouped separately. A cluster of six isolates of Waitea (UWC) did not fall into any known Waitea variety. The binucleate Rhizoctonia-like fungi (BNR) (n = 16) clustered into two groups. Rhizoctonia solani AG 2-2IIIB was the most dominant pathogen in this study, followed by AG 1-IB. There was no relationship between the geographic origin of the isolates and clustering of isolates based on the genetic associations. To our knowledge this is the first time UP-PCR was used to characterize Rhizoctonia

  13. Two group I ribozymes with different functions in a nuclear rDNA intron.

    PubMed Central

    Decatur, W A; Einvik, C; Johansen, S; Vogt, V M

    1995-01-01

    DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self-splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS-1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self-splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS-1. Much of the P2 stem and all of the associated loop could be deleted without effect on self-splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5' splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1-mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron-encoded endonuclease I-DirI. Images PMID:7556099

  14. Importance of minor groove functional groups for the stability of DNA duplexes.

    PubMed

    Sun, Zhenhua; Chen, Dongli; Lan, Tao; McLaughlin, Larry W

    2002-11-01

    Eight oligonucleotide duplexes have been prepared with four pairs of selected complementary pairs of native/analogue heterocyclic bases incorporated at a selected test site. The base pairs vary in the nature of their functionality in the minor groove. Each pair has a minor groove purine amino group present or absent, and correspondingly has a minor grove pyrimidine carbonyl present or absent. Loss of duplex stability is most notable when the minor groove pyrimidine carbonyl is absent although in other respects normal Watson-Crick hydrogen bonding is maintained in these sequences. These differences in stability are discussed in terms of possible variations in minor groove hydration.

  15. Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae.

    PubMed Central

    Zelenaya-Troitskaya, O; Newman, S M; Okamoto, K; Perlman, P S; Butow, R A

    1998-01-01

    Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein.These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination. PMID:9581629

  16. Identification of aphids of Aphis frangulae-group living on Lamiaceae species through DNA barcode.

    PubMed

    Cocuzza, Giuseppe E Massimino; Cavalieri, Vincenzo

    2014-05-01

    The genus Aphis frangulae-group living on Lamiaceae includes several postulate species, which are morphologically indistinguishable. As a consequence, identification is possible only on the basis of the host plant or life cycle. This study tested the utility of a fragment (614 bp) of mitochondrial cytochrome oxidase 1 (COI) with the aim of identifying the species and/or to confirm the previous classification based on host plant and data reported in the literature. Although the general nucleotide variability found was rather low, the analysis enabled the separation and identification of all the specimens collected. In some cases, the lack of nucleotide variability among postulated taxa indicates the limits of identification based on biological traits. Consequently, based on the molecular analysis, the postulate species A. symphyti, A. stachydis and A. lamiorum should be regarded as synonymous of A. frangulae.

  17. DNA polymerase gene sequences indicate western and forest tent caterpillar viruses form a new taxonomic group within baculoviruses.

    PubMed

    Nielsen, Cydney B; Cooper, Dawn; Short, Steven M; Myers, Judith H; Suttle, Curtis A

    2002-11-01

    Baculoviruses infect larval lepidopterans, and thus have potential value as microbial controls of agricultural and forest pests. Understanding their genetic relatedness and host specificity is relevant to the risk assessment of viral insecticides if non-target impacts are to be avoided. DNA polymerase gene sequences have been demonstrated to be useful for inferring genetic relatedness among dsDNA viruses. We have adopted this approach to examine the relatedness among natural isolates of two uncharacterized caterpillar-infecting baculoviruses, Malacosoma californicum pluviale nucleopolyhedrovirus (McplMNPV) and Malacosoma disstria nucleopolyhedrovirus (MadiMNPV), which infect two closely related host species with little to no cross-infectivity. We designed two degenerate primers (BVP1 and BVP2) based on protein motifs conserved among baculoviruses. McplMNPV and MadiMNPV viral DNA was obtained from naturally infected caterpillars collected from geographically distinct sites in the Southern Gulf Islands and Prince George regions of British Columbia, Canada. Sequencing of 0.9 kb PCR amplicons from six McplMNPV and six MadiMNPV isolates obtained from a total of eight sites, revealed very low nucleotide variation among McplMNPV isolates (99.2-100% nucleotide identity) and among MadiMNPV isolates (98.9-100% nucleotide identity). Greater nucleotide variation was observed between viral isolates from the two different caterpillar species (only 84.7-86.1% nucleotide identity). Both maximum parsimony and maximum likelihood phylogenetic analyses support placement of McplMNPV and MadiMNPV in a clade that is distinct from other groups of baculoviruses.

  18. Japanese Wolves are Genetically Divided into Two Groups Based on an 8-Nucleotide Insertion/Deletion within the mtDNA Control Region.

    PubMed

    Ishiguro, Naotaka; Inoshima, Yasuo; Yanai, Tokuma; Sasaki, Motoki; Matsui, Akira; Kikuchi, Hiroki; Maruyama, Masashi; Hongo, Hitomi; Vostretsov, Yuri E; Gasilin, Viatcheslav; Kosintsev, Pavel A; Quanjia, Chen; Chunxue, Wang

    2016-02-01

    The mitochondrial DNA (mtDNA) control region (198- to 598-bp) of four ancient Canis specimens (two Canis mandibles, a cranium, and a first phalanx) was examined, and each specimen was genetically identified as Japanese wolf. Two unique nucleotide substitutions, the 78-C insertion and the 482-G deletion, both of which are specific for Japanese wolf, were observed in each sample. Based on the mtDNA sequences analyzed, these four specimens and 10 additional Japanese wolf samples could be classified into two groups- Group A (10 samples) and Group B (4 samples)-which contain or lack an 8-bp insertion/deletion (indel), respectively. Interestingly, three dogs (Akita-b, Kishu 25, and S-husky 102) that each contained Japanese wolf-specific features were also classified into Group A or B based on the 8-bp indel. To determine the origin or ancestor of the Japanese wolf, mtDNA control regions of ancient continental Canis specimens were examined; 84 specimens were from Russia, and 29 were from China. However, none of these 113 specimens contained Japanese wolf-specific sequences. Moreover, none of 426 Japanese modern hunting dogs examined contained these Japanese wolf-specific mtDNA sequences. The mtDNA control region sequences of Groups A and B appeared to be unique to grey wolf and dog populations. PMID:26853868

  19. Japanese Wolves are Genetically Divided into Two Groups Based on an 8-Nucleotide Insertion/Deletion within the mtDNA Control Region.

    PubMed

    Ishiguro, Naotaka; Inoshima, Yasuo; Yanai, Tokuma; Sasaki, Motoki; Matsui, Akira; Kikuchi, Hiroki; Maruyama, Masashi; Hongo, Hitomi; Vostretsov, Yuri E; Gasilin, Viatcheslav; Kosintsev, Pavel A; Quanjia, Chen; Chunxue, Wang

    2016-02-01

    The mitochondrial DNA (mtDNA) control region (198- to 598-bp) of four ancient Canis specimens (two Canis mandibles, a cranium, and a first phalanx) was examined, and each specimen was genetically identified as Japanese wolf. Two unique nucleotide substitutions, the 78-C insertion and the 482-G deletion, both of which are specific for Japanese wolf, were observed in each sample. Based on the mtDNA sequences analyzed, these four specimens and 10 additional Japanese wolf samples could be classified into two groups- Group A (10 samples) and Group B (4 samples)-which contain or lack an 8-bp insertion/deletion (indel), respectively. Interestingly, three dogs (Akita-b, Kishu 25, and S-husky 102) that each contained Japanese wolf-specific features were also classified into Group A or B based on the 8-bp indel. To determine the origin or ancestor of the Japanese wolf, mtDNA control regions of ancient continental Canis specimens were examined; 84 specimens were from Russia, and 29 were from China. However, none of these 113 specimens contained Japanese wolf-specific sequences. Moreover, none of 426 Japanese modern hunting dogs examined contained these Japanese wolf-specific mtDNA sequences. The mtDNA control region sequences of Groups A and B appeared to be unique to grey wolf and dog populations.

  20. Contribution to the study of Acanthodactylus (Sauria: Lacertidae) mtDNA diversity focusing on the A. boskianus species group.

    PubMed

    Psonis, N; Lymberakis, P; Poursanidis, D; Poulakakis, N

    2016-09-01

    The Acanthodactylus boskianus species group includes three species (A. boskianus, A. nilsoni, and A. schreiberi) of unclear phylogeny and phylogeographic history. By sequencing fragments of two mtDNA genes and performing phylogenetic, demographic, and chronophylogenetic analyses, we aimed at identifying their phylogenetic relationships while unravelling their biogeographic history. The analyses indicated that A. boskianus is a species complex, while A. s. schreiberi and A. s. ataturi show, both, low intraspecific genetic diversity. From a biogeographic point of view, the ancestor of A. s. schreiberi colonized Cyprus from the Middle East through overseas dispersal during the Pleistocene, whereas A. s. ataturi is considered to be a relict of a previously wider distribution. PMID:27402069

  1. The effect of leaving groups on binding and reactivity in enzyme-free copying of DNA and RNA.

    PubMed

    Kervio, Eric; Sosson, Marilyne; Richert, Clemens

    2016-07-01

    The template-directed incorporation of nucleotides at the terminus of a growing primer is the basis of the transmission of genetic information. Nature uses polymerases-catalyzed reactions, but enzyme-free versions exist that employ nucleotides with organic leaving groups. The leaving group affects yields, but it was not clear whether inefficient extensions are due to poor binding, low reactivity toward the primer, or rapid hydrolysis. We have measured the binding of a total of 15 different activated nucleotides to DNA or RNA sequences. Further, we determined rate constants for the chemical step of primer extension involving methylimidazolides or oxyazabenzotriazolides of deoxynucleotides or ribonucleotides. Binding constants range from 10 to >500 mM and rate constants from 0.1 to 370 M(-1) h(-1) For aminoterminal primers, a fast covalent step and slow hydrolysis are the main factors leading to high yields. For monomers with weakly pairing bases, the leaving group can improve binding significantly. A detailed mechanistic picture emerges that explains why some enzyme-free primer extensions occur in high yield, while others remain recalcitrant to copying without enzymatic catalysis. A combination of tight binding and rapid extension, coupled with slow hydrolysis induces efficient enzyme-free copying. PMID:27235418

  2. The effect of leaving groups on binding and reactivity in enzyme-free copying of DNA and RNA

    PubMed Central

    Kervio, Eric; Sosson, Marilyne; Richert, Clemens

    2016-01-01

    The template-directed incorporation of nucleotides at the terminus of a growing primer is the basis of the transmission of genetic information. Nature uses polymerases-catalyzed reactions, but enzyme-free versions exist that employ nucleotides with organic leaving groups. The leaving group affects yields, but it was not clear whether inefficient extensions are due to poor binding, low reactivity toward the primer, or rapid hydrolysis. We have measured the binding of a total of 15 different activated nucleotides to DNA or RNA sequences. Further, we determined rate constants for the chemical step of primer extension involving methylimidazolides or oxyazabenzotriazolides of deoxynucleotides or ribonucleotides. Binding constants range from 10 to >500 mM and rate constants from 0.1 to 370 M−1 h−1. For aminoterminal primers, a fast covalent step and slow hydrolysis are the main factors leading to high yields. For monomers with weakly pairing bases, the leaving group can improve binding significantly. A detailed mechanistic picture emerges that explains why some enzyme-free primer extensions occur in high yield, while others remain recalcitrant to copying without enzymatic catalysis. A combination of tight binding and rapid extension, coupled with slow hydrolysis induces efficient enzyme-free copying. PMID:27235418

  3. Mitochondrial DNA diversity in two ethnic groups in southeastern Kenya: perspectives from the northeastern periphery of the Bantu expansion

    PubMed Central

    Batai, Ken; Babrowski, Kara B.; Arroyo, Juan Pablo; Kusimba, Chapurukha M.; Williams, Sloan R.

    2013-01-01

    The Bantu languages are widely distributed throughout sub-Saharan Africa. Genetic research supports linguists and historians who argue that migration played an important role in the spread of this language family, but the genetic data also indicates a more complex process involving substantial gene flow with resident populations. In order to understand the Bantu expansion process in east Africa, mtDNA hypervariable region I variation in 352 individuals from the Taita and Mijikenda ethnic groups was analyzed, and we evaluated the interactions that took place between the Bantu- and non-Bantu-speaking populations in east Africa. The Taita and Mijikenda are Bantu-speaking agropastoralists from southeastern Kenya, at least some of whose ancestors probably migrated into the area as part of Bantu migrations that began around 3,000 BCE. Our analyses indicate that they show some distinctive differences that reflect their unique cultural histories. The Taita are genetically more diverse than the Mijikenda with larger estimates of genetic diversity. The Taita cluster with other east African groups, having high frequencies of haplogroups from that region, while the Mijikenda have high frequencies of central African haplogroups and cluster more closely with central African Bantu-speaking groups. The non-Bantu speakers who lived in southeastern Kenya before Bantu speaking groups arrived were at least partially incorporated into what are now Bantu-speaking Taita groups. In contrast, gene flow from non-Bantu speakers into the Mijikenda was more limited. These results suggest a more complex demographic history where the nature of Bantu and non-Bantu interactions varied throughout the area. PMID:23382080

  4. Phylogeny and chromosomal variations in East Asian Carex, Siderostictae group (Cyperaceae), based on DNA sequences and cytological data.

    PubMed

    Yano, Okihito; Ikeda, Hiroshi; Jin, Xiao-Feng; Hoshino, Takuji

    2014-01-01

    Carex (Cyperaceae) is one of the largest genera of the flowering plants, and comprises more than 2,000 species. In Carex, section Siderostictae with broader leaves distributed in East Asia is thought to be an ancestral group. We aimed to clarify the phylogenetic relationships and chromosomal variations within the section Siderostictae, and to examine the relationship of broad-leaved species of the sections Hemiscaposae and Surculosae from East Asia, inferred from DNA sequences and cytological data. Our results indicate that a monophyletic Siderostictae clade, including the sections Hemiscaposae, Siderostictae and Surculosae, as the earliest diverging group in the tribe Cariceae. Low chromosome numbers, 2n = 12 or 24, with large sizes were observed in these three sections. Our results suggest that the genus Carex might have originated or relictly restricted in the East Asia. Geographical distributions of diploid species are restricted in narrower areas, while those of tetraploid species are wider in East Asia. It is concluded that chromosomal variations in Siderostictae clade may have been caused by polyploidization and that tetraploid species may have been able to exploit their habitats by polyploidization.

  5. Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties.

    PubMed

    Kulaksiz, Gülnihal; Reardon, Joyce T; Sancar, Aziz

    2005-11-01

    Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease. PMID:16260596

  6. DNA sequence of the serum opacity factor of group A streptococci: identification of a fibronectin-binding repeat domain.

    PubMed Central

    Rakonjac, J V; Robbins, J C; Fischetti, V A

    1995-01-01

    The serum opacity factor (SOF) is a group A streptococcal protein that induces opacity of mammalian serum. The serum opacity factor 22 gene (sof22) from an M type 22 strain was cloned from an EMBL4 library by screening for plaques exhibiting serum opacity activity. DNA sequencing yielded an open reading frame of 3,075 bp. Its deduced amino acid sequence predicts a protein of 1,025 residues with a molecular weight of 112,735, a size that approximates that of the SOF22 protein isolated from both the original streptococcal strain and Escherichia coli harboring the cloned sof22 gene. The molecule is composed of three domains: an N-terminal domain responsible for the opacity reaction (opacity domain), a repeat domain with fibronectin-binding (Fn-binding) activity, and a C-terminal cell attachment domain. The C-terminal end of SOF22 is characterized by a hexameric LPXTGX motif, an adjacent hydrophobic region, and a charged C terminus, which are the hallmarks of cell-bound surface proteins found on nearly all gram-positive bacteria. Immediately upstream of this cell anchor region, SOF22 contains four tandem repeat sequence blocks, flanked by prolinerich segments. The repeats share up to 50% identity with a repeated motif found in other group A streptococcal Fn-binding proteins and exhibit Fn-binding activity, as shown by subcloning experiments. According to deletion analysis, the opacity domain is confined to the region N terminal to the repeat segment. Thus, SOF22 is unique among the known Fn-binding proteins from gram-positive bacteria in containing an independent module with a defined function in its N-terminal portion. Southern blot analysis with a probe from this N-terminal region indicates that the opacity domain of SOF varies extensively among different SOF-producing M types. PMID:7822031

  7. Spectroscopic study on the interaction of ct-DNA with manganese Salen complex containing triphenyl phosphonium groups

    NASA Astrophysics Data System (ADS)

    Dehkordi, Maryam Nejat; Bordbar, Abdol-Khalegh; Lincoln, Per; Mirkhani, Valiollah

    2012-05-01

    The DNA binding properties of a bulky and hydrophobic Schiff base complex of manganese(III) [N,N'-bis(5-(triphenyl phosphonium methyl)salicylidene)-1,2-ethylene diamine chloride Mn(III) acetate] was examined by spectroscopic techniques. UV-vis titration data indicate both hypo and hyperchromic effect with addition of DNA to complex. A competitive binding study showed that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by adding Mn Salen complex. This finding indicates that Mn Salen complex displaces EB from its binding site in DNA. Helix melting studies indicate improvement in the helix stability, and an increase in the melting temperature. The analysis of CD spectra represents the structural changes in DNA due to the binding of Mn Salen complex. The binding constant has been calculated using absorbance and fluorescence data. The results also represent that the binding process proceeds by strong electrostatic and hydrophobic interactions.

  8. New pyridinium-based fluorescent dyes: A comparison of symmetry and side-group effects on G-Quadruplex DNA binding selectivity and application in live cell imaging.

    PubMed

    Lu, Yu-Jing; Hu, Dong-Ping; Zhang, Kun; Wong, Wing-Leung; Chow, Cheuk-Fai

    2016-07-15

    A series of C1-, C2-and C3-symmetric pyridinium conjugates with different styrene-like side groups were synthesized and were utilized as G-quadruplex selective fluorescent probes. The new compounds were well-characterized. Their selectivity, sensitivity, and stability towards G-quadruplex were studied by fluorescence titration, native PAGE experiments, FRET and circular dichroism (CD) analyses. These new compounds investigated in the fluorescence assays were preferentially bound with G-quadruplex DNA compared with other type of nucleic acids and it is fascinating to realize the effects of molecular symmetry and associated side groups showing unexpectedly great influence on the fluorescent signal enhancement for the discrimination of G-quadruplexes DNA from other nucleic acids. This may correlate with the pocket symmetry and shape of the G-quadruplex DNA inherently. Among the compounds, a C2-symmetric dye (2,6-bis-((E)-2-(1H-indol-3-yl)-vinyl)-1-methylpyridin-1-ium iodide) with indolyl-groups substituted was screened out from the series giving the best selectivity and sensitivity towards G-quadruplexes DNA, particularly telo21, due to its high equilibrium binding constant (K=2.17×10(5)M(-1)). In addition, the limit of detection (LOD) of the dye to determine telo21 DNA in bioassays was found as low as 33nM. The results of the study give insight and certain crucial factors, such as molecular symmetry and the associated side groups, on developing of effective fluorescent dyes for G-quadruplex DNA applications including G-quadruplex structure stabilization, biosensing and clinical applications. The compound was also demonstrated as a very selective G-quadruplex fluorescent agent for living cell staining and imaging.

  9. Human mtDNA hypervariable regions, HVR I and II, hint at deep common maternal founder and subsequent maternal gene flow in Indian population groups.

    PubMed

    Sharma, Swarkar; Saha, Anjana; Rai, Ekta; Bhat, Audesh; Bamezai, Ramesh

    2005-01-01

    We have analysed the hypervariable regions (HVR I and II) of human mitochondrial DNA (mtDNA) in individuals from Uttar Pradesh (UP), Bihar (BI) and Punjab (PUNJ), belonging to the Indo-European linguistic group, and from South India (SI), that have their linguistic roots in Dravidian language. Our analysis revealed the presence of known and novel mutations in both hypervariable regions in the studied population groups. Median joining network analyses based on mtDNA showed extensive overlap in mtDNA lineages despite the extensive cultural and linguistic diversity. MDS plot analysis based on Fst distances suggested increased maternal genetic proximity for the studied population groups compared with other world populations. Mismatch distribution curves, respective neighbour joining trees and other statistical analyses showed that there were significant expansions. The study revealed an ancient common ancestry for the studied population groups, most probably through common founder female lineage(s), and also indicated that human migrations occurred (maybe across and within the Indian subcontinent) even after the initial phase of female migration to India.

  10. Comparison of a modified DNA hybridization assay with standard culture enrichment for detecting group B streptococci in obstetric patients.

    PubMed Central

    Kircher, S M; Meyer, M P; Jordan, J A

    1996-01-01

    Infection with group B streptococcus (GBS) results in 12,000 to 15,000 cases of neonatal sepsis annually in the United States. GBS is transmitted vertically in up to 70% of infants born to colonized women. Early-onset GBS disease (septicemia, pneumonia, or meningitis occurring within 7 days of life) has a mortality rate of up to 50%, with permanent neurologic sequelae occurring in 15 to 50% of infants surviving meningeal infection. Because of the fulminant nature of neonatal infection, it would be useful to have a rapid assay for determining the GBS status of laboring women. This study illustrated how a commercially available DNA probe-based test was modified to achieve this goal. Modifications included the use of mixed cultures rather than pure isolates for detecting GBS, along with a shorter culture enrichment time and a sample concentration step. To this end, vaginal and rectal swabs from 402 pregnant women during their third trimester were cocultured and tested for GBS rRNA. The 8-h enrichment protocol resulted in an assay with a sensitivity of 95% and specificity of 98%, while the 3-h enrichment protocol revealed a sensitivity of 73% and specificity of 99%. In summary, GBS was detected in the majority of colonized women in less than 4 h. This study illustrated the usefulness of the approach in identifying the most heavily colonized women, who are at the highest risk of transmitting GBS to their neonates. The modified test would have a significant impact on both the medical management and antibiotic therapy for these women and their newborns. PMID:8789012

  11. Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-induced DNA Damage via Physical Interaction with Xeroderma Pigmentosum Group A*

    PubMed Central

    Shell, Steven M.; Li, Zhengke; Shkriabai, Nikolozi; Kvaratskhelia, Mamuka; Brosey, Chris; Serrano, Moises A.; Chazin, Walter J.; Musich, Phillip R.; Zou, Yue

    2009-01-01

    In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation. PMID:19586908

  12. Quantitative estimation of the contribution of pyrrolcarboxamide groups of the antibiotic distamycin A into specificity of its binding to DNA AT pairs.

    PubMed Central

    Krylov, A S; Grokhovsky, S L; Zasedatelev, A S; Zhuze, A L; Gursky, G V; Gottikh, B P

    1979-01-01

    Interaction of DNA with the analogs of the antibiotic distamycin A having different numbers of pyrrolcarboxamide groups and labeled with dansyl was studied. The binding isoterms of the analogs to synthetic polydeoxyribonucleotides were obtained. Analysis of the experimental data leads to the following conclusions: (1) the free energy of binding of the analogs to poly(dA).poly(dT) depends linearly on the number of amide groups in the molecule of the analog whereas attachment of each pyrrolcarboxamide group produces changes of 2 kcal/mole in the free energy; (2) attachment of a pyrrolcarboxamide unit to the GC pair results in the free energy change of 0.95 kcal/mole; (3) the binding of analogs to poly(dA).poly(dT) is a cooperative process, presumbly, dependent on conformational changes induced by the binding of analogs to DNA. PMID:424293

  13. Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea).

    PubMed

    Tarcz, Sebastian; Potekhin, Alexey; Rautian, Maria; Przyboś, Ewa

    2012-05-01

    This is the first phylogenetic study of the intraspecific variability within Paramecium multimicronucleatum with the application of two-loci analysis (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) carried out on numerous strains originated from different continents. The species has been shown to have a complex structure of several sibling species within taxonomic species. Our analysis revealed the existence of 10 haplotypes for the rDNA fragment and 15 haplotypes for the COI fragment in the studied material. The mean distance for all of the studied P. multimicronucleatum sequence pairs was p=0.025/0.082 (rDNA/COI). Despite the greater variation of the COI fragment, the COI-derived tree topology is similar to the tree topology constructed on the basis of the rDNA fragment. P. multimicronucleatum strains are divided into three main clades. The tree based on COI fragment analysis presents a greater resolution of the studied P. multimicronucleatum strains. Our results indicate that the strains of P. multimicronucleatum that appear in different clades on the trees could belong to different syngens.

  14. A feasible approach to evaluate the relative reactivity of NHS-ester activated group with primary amine-derivatized DNA analogue and non-derivatized impurity.

    PubMed

    Dou, Shuping; Virostko, John; Greiner, Dale L; Powers, Alvin C; Liu, Guozheng

    2015-01-01

    Synthetic DNA analogues with improved stability are widely used in life science. The 3'and/or 5' equivalent terminuses are often derivatized by attaching an active group for further modification, but a certain amount of non-derivatized impurity often remains. It is important to know to what extent the impurity would influence further modification. The reaction of an NHS ester with primary amine is one of the most widely used options to modify DNA analogues. In this short communication, a 3'-(NH2-biotin)-derivatized morpholino DNA analogue (MORF) was utilized as the model derivatized DNA analogue. Inclusion of a biotin concomitant with the primary amine at the 3'-terminus allows for the use of streptavidin to discriminate between the products from the derivatized MORF and non-derivatized MORF impurity. To detect the MORF reaction with NHS ester, S-acetyl NHS-MAG3 was conjugated to the DNA analogue for labeling with (99m)Tc, a widely used nuclide in the clinic. It was found that the non-derivatized MORF also reacted with the S-acetyl NHS-MAG3. Radiolabeling of the product yielded an equally high labeling efficiency. Nevertheless, streptavidin binding indicated that under the conditions of this investigation, the non-derivatized MORF was five times less reactive than the amine-derivatized MORF. PMID:25621701

  15. A FEASIBLE APPROACH TO EVALUATE THE RELATIVE REACTIVITY OF NHS-ESTER ACTIVATED GROUP WITH PRIMARY AMINE-DERIVATIZED DNA ANALOGUE AND NON-DERIVATIZED IMPURITY

    PubMed Central

    Dou, Shuping; Virostko, John; Greiner, Dale L.; Powers, Alvin C.; Liu, Guozheng

    2015-01-01

    Synthetic DNA analogues with improved stability are widely used in life science. The 3′ and/or 5′ equivalent terminuses are often derivatized by attaching an active group for further modification, but a certain amount of non-derivatized impurity often remains. It is important to know to what extent the impurity would influence further modification. The reaction of an NHS ester with primary amine is one of the most widely used options to modify DNA analogues. In this short communication, a 3′-(NH2-biotin)-derivatized morpholino DNA analogue (MORF) was utilized as the model derivatized DNA analogue. Inclusion of a biotin concomitant with the primary amine at the 3′-terminus allows for the use of streptavidin to discriminate between the products from the derivatized MORF and non-derivatized MORF impurity. To detect the MORF reaction with NHS ester, S-acetyl NHS-MAG3 was conjugated to the DNA analogue for labeling with 99mTc, a widely used nuclide in the clinic. It was found that the non-derivatized MORF also reacted with the S-acetyl NHS-MAG3. Radiolabeling of the product yielded an equally high labeling efficiency. Nevertheless, streptavidin binding indicated that under the conditions of this investigation, the non-derivatized MORF was five times less reactive than the amine-derivatized MORF. PMID:25621701

  16. A distinct group of CpG islands shows differential DNA methylation between replicas of the same cell line in vitro

    PubMed Central

    2013-01-01

    Background CpG dinucleotide-rich genomic DNA regions, known as CpG islands (CGIs), can be methylated at their cytosine residues as an epigenetic mark that is stably inherited during cell mitosis. Differentially methylated regions (DMRs) are genomic regions showing different degrees of DNA methylation in multiple samples. In this study, we focused our attention on CGIs showing different DNA methylation between two culture replicas of the same cell line. Results We used methylation data of 35 cell lines from the Encyclopedia of DNA Elements (ENCODE) consortium to identify CpG islands that were differentially methylated between replicas of the same cell line and denoted them Inter Replicas Differentially Methylated CpG islands (IRDM-CGIs). We identified a group of IRDM-CGIs that was consistently shared by different cell lines, and denoted it common IRDM-CGIs. X chromosome CGIs were overrepresented among common IRDM-CGIs. Autosomal IRDM-CGIs were preferentially located in gene bodies and intergenic regions had a lower G + C content, a smaller mean length, and a reduced CpG percentage. Functional analysis of the genes associated with autosomal IRDM-CGIs showed that many of them are involved in DNA binding and development. Conclusions Our results show that several specific functional and structural features characterize common IRDM-CGIs. They may represent a specific subset of CGIs that are more prone to being differentially methylated for their intrinsic characteristics. PMID:24106769

  17. A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood.

    PubMed

    Burke, Rachael M; McKenna, James P; Cox, Ciara; Coyle, Peter V; Shields, Michael D; Fairley, Derek J

    2016-10-01

    Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidase pre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6-85.1, p<0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2-8.9, p<0.001), compared with no pre-lysis. Differences in yield due to pre-lysis were 2-3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture. PMID:27546716

  18. [Genomic fingerprints of organisms from different taxonomic groups: the use of phage M13 DNA as a hybridization probe].

    PubMed

    Ryskov, A P; Dzhincharadze, A G; Prosnik, M I; Ivanov, P L; Limborskaia, S A

    1988-02-01

    Hypervariable polymorphic patterns were detected using wild-type M13 DNA as a probe in genomic DNAs of very different organisms ranging from procaryotes and lower eucaryotes to upper plants and animals, including human beings. Due to somatic stability of highly polymorphic patterns and their discrete inheritance, individual-specific restriction pattern analysis ("DNA fingerprinting") with this test probe was found to be useful in applied human genetics, in particular, for identifying paternity and maternity, and mapping of human genomes. The data obtained also demonstrate some possibilities of the DNA fingerprinting technology in genetics and selection of agricultural plants and animals, such as variety analysis, classification and registration of individual inbred lines and strains, as well as identification of bacterial strains.

  19. Preparation of a DNA matrix via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group.

    PubMed Central

    Livache, T; Roget, A; Dejean, E; Barthet, C; Bidan, G; Téoule, R

    1994-01-01

    A new methodology for the preparation of addressed DNA matrices is described. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5' end a pyrrole moiety introduced by phosphoramidite chemistry. The electro-controlled synthesis of the copolymer (poly-pyrrole) gives, in one step, a solid conducting film deposited on the surface of an electrode. The resulting polymer consists of pyrrole chains bearing covalently linked oligonucleotide. The polymer growth is limited to the electrode surface, so that it is possible to prepare a DNA matrix on a multiple electrode device by successive copolymerizations. A support bearing four oligonucleotides was used to detect three ras mutations on a synthetic DNA fragment. PMID:8065902

  20. [Genetic diversity and relatives of the goitered gazelle (Gazella subgutturosa) groups from Uzbekistan, Turkmenistan, and Azerbaijan: analysis of the D-loop of mitochondrial DNA].

    PubMed

    Sorokin, P A; Soldatova, N V; Lukarevskiĭ, V S; Kholodova, M V

    2011-01-01

    Polymorphism of the nucleotide sequence of a hypervariable fragment of the D-loop (985 bp) of mtDNA in 76 Goitered gazelles of subspecies Gazella subgutturosa subgutturosa from Uzbekistan, Turkmenistan, and Azerbaijan was studied. The genetic similarity of gazelles from Turkmenistan and Uzbekistan has been identified. The population of gazelles from Shirvanskaya steppe reserve (Azerbaijan) is unique and strictly isolated from other groups studied. A high haplotypic (H = 0.9649 +/- 0.0091) and relatively low nucleotide diversity (pi = 0.0212 +/- 0.0105) were noted for all investigated groups of gazelle based on this mtDNA fragment, which is probably related to ecological peculiarities of the species and the history of formation of regional populations. PMID:22292288

  1. Identification of a group of cryptic marine limpet species, Cellana karachiensis (Mollusca: Patellogastropoda) off Veraval coast, India, using mtDNA COI sequencing.

    PubMed

    Joseph, Sneha; Poriya, Paresh; Vakani, Bhavik; Singh, S P; Kundu, Rahul

    2016-01-01

    Present communication reports the phylogenetic relationship between three groups of a marine limpet having different color banding patterns using COI sequencing. Samples were sequenced for mtDNA COI gene using universal primer. Comparative BLAST revealed that all three types were around 99.59% identical with Cellana karachiensis, first record of this species from Indian coasts. Apart from the morphological variations, the mtDNA COI gene analysis revealed around 1% nucleotide variations between these three types. The observed dissimilarity in COI sequences was possibly too little to consider these types as three different species. The derivation of amino acid positions indicated that these types could possibly be a complex of three cryptic species of C. karachiensis. The study proposes that the Oman and Indian populations of C. karachiensis might have derived by allopatric speciation due to geographical isolation. The group of these three cryptic species, sharing same habitat between themselves, possibly showed sympatric speciation.

  2. Quantifying male-biased dispersal among social groups in the collared peccary (Pecari tajacu) using analyses based on mtDNA variation.

    PubMed

    Cooper, J D; Vitalis, R; Waser, P M; Gopurenko, D; Hellgren, E C; Gabor, T M; DeWoody, J A

    2010-01-01

    Recent advances in the statistical analysis of microsatellite data permit calculation of sex-specific dispersal rates through sex- and age-specific comparisons of genetic variation. This approach, developed for the analysis of data derived from co-dominant autosomal markers, should be applicable to a sex-specific marker such as mitochondrial DNA. To test this premise, we amplified a 449 bp control region DNA sequence from the mitochondrial genome of the collared peccary (Pecari tajacu), and estimated intra-class correlations among herds sampled from three Texas populations. Analyses on data partitioned by breeding group showed a clear signal of male-biased dispersal; sex-specific fixation indices associated with genetic variation among social groups within populations yielded values for females (F(GP)=0.91), which were significantly larger than values for males (F(GP)=0.24; P=0.0015). The same general pattern emerged when the analyses were conducted on age classes (albeit nonsignificantly), as well as categories of individuals that were predicted a posteriori to be dispersers (adult males) and philopatric (adult females and all immatures). By extending a previously published methodology based on biparentally inherited markers to matrilineally inherited haploid data, we calculated sex-specific rates of contemporary dispersal among social groups within populations (m(male symbol)=0.37). These results support the idea that mitochondrial DNA haplotype frequency data can be used to estimate sex-specific instantaneous dispersal rates in a social species.

  3. Structure-function relationships in human testis-determining factor SRY: an aromatic buttress underlies the specific DNA-bending surface of a high mobility group (HMG) box.

    PubMed

    Racca, Joseph D; Chen, Yen-Shan; Maloy, James D; Wickramasinghe, Nalinda; Phillips, Nelson B; Weiss, Michael A

    2014-11-21

    Human testis determination is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in SRY cause 46 XY gonadal dysgenesis with female somatic phenotype (Swyer syndrome) and confer a high risk of malignancy (gonadoblastoma). Such mutations cluster in the SRY high mobility group (HMG) box, a conserved motif of specific DNA binding and bending. To explore structure-function relationships, we constructed all possible substitutions at a site of clinical mutation (W70L). Our studies thus focused on a core aromatic residue (position 15 of the consensus HMG box) that is invariant among SRY-related HMG box transcription factors (the SOX family) and conserved as aromatic (Phe or Tyr) among other sequence-specific boxes. In a yeast one-hybrid system sensitive to specific SRY-DNA binding, the variant domains exhibited reduced (Phe and Tyr) or absent activity (the remaining 17 substitutions). Representative nonpolar variants with partial or absent activity (Tyr, Phe, Leu, and Ala in order of decreasing side-chain volume) were chosen for study in vitro and in mammalian cell culture. The clinical mutation (Leu) was found to markedly impair multiple biochemical and cellular activities as respectively probed through the following: (i) in vitro assays of specific DNA binding and protein stability, and (ii) cell culture-based assays of proteosomal degradation, nuclear import, enhancer DNA occupancy, and SRY-dependent transcriptional activation. Surprisingly, however, DNA bending is robust to this or the related Ala substitution that profoundly impairs box stability. Together, our findings demonstrate that the folding, trafficking, and gene-regulatory function of SRY requires an invariant aromatic "buttress" beneath its specific DNA-bending surface. PMID:25258310

  4. European Mitochondrial DNA Haplogroups and Metabolic Changes during Antiretroviral Therapy in AIDS Clinical Trials Group Study A5142

    PubMed Central

    Hulgan, Todd; Haubrich, Richard; Riddler, Sharon A.; Tebas, Pablo; Ritchie, Marylyn D.; McComsey, Grace A.; Haas, David W.; Canter, Jeffrey A.

    2010-01-01

    Background Mitochondrial DNA (mtDNA) influences metabolic diseases and perhaps antiretroviral therapy (ART) complications. We explored associations between European mtDNA haplogroups and metabolic changes among A5142 participants. Methods 757 ART-naïve subjects were randomized to one of three class-sparing ART regimens including efavirenz and/or lopinavir/ritonavir with or without nucleoside reverse transcriptase inhibitors (NRTIs). Non-randomized NRTIs included stavudine, tenofovir, or zidovudine, each with lamivudine. Fasting lipid profiles and whole-body dual-energy X-ray absorptiometry (DEXA) were performed. Nine European mtDNA haplogroups were determined for 231 self-identified non-Hispanic white subjects. Metabolic changes from baseline to 96 weeks were analyzed by haplogroup. Results Median age was 39 years, 9% were female, and 37%, 32%, and 30% were randomized to NRTI-containing regimens with either efavirenz or lopinavir/ritonavir, and an NRTI-sparing regimen respectively. Among NRTI-containing regimens, 51% included zidovudine, 28% tenofovir, and 21% stavudine. Compared with other haplogroups, mtDNA haplogroup I (N=10) had higher baseline non-HDL cholesterol (160 mg/dL [interquartile range 137–171] vs. 120 mg/dL [104–136]; p=0.005), a decrease in non-HDL cholesterol over 96 weeks (−14% [−20-+6] vs. +25% [+8-+51]; p<0.001), tended to have more baseline extremity fat, and had more extremity fat loss by DEXA (−13% [−31-+12] vs. +9% [−13-+26]; p=0.08) and lipoatrophy (50% vs. 20%; p=0.04). Haplogroup W (N=5; all randomized to NRTI-sparing regimens) had the greatest increase in extremity fat (+35.5% [+26.8 - +54.9]; P=0.02). Conclusions Lipids and extremity fat were associated with European mtDNA haplogroups in this HIV-infected population. These preliminary results suggest that mitochondrial genomics may influence metabolic parameters before and during ART. PMID:20871389

  5. The molecular evolution and structural organization of self-splicing group I introns at position 516 in nuclear SSU rDNA of myxomycetes.

    PubMed

    Haugen, Peik; Coucheron, Dag H; Rønning, Sissel B; Haugli, Kari; Johansen, Steinar

    2003-01-01

    Group I introns are relatively common within nuclear ribosomal DNA of eukaryotic microorganisms, especially in myxomycetes. Introns at position S516 in the small subunit ribosomal RNA gene are particularly common, but have a sporadic occurrence in myxomycetes. Fuligo septica, Badhamia gracilis, and Physarum flavicomum, all members of the family Physaraceae, contain related group IC1 introns at this site. The F. septica intron was studied at the molecular level and found to self-splice as naked RNA and to generate full-length intron RNA circles during incubation. Group I introns at position S516 appear to have a particularly widespread distribution among protists and fungi. Secondary structural analysis of more than 140 S516 group I introns available in the database revealed five different types of organization, including IC1 introns with and without His-Cys homing endonuclease genes, complex twin-ribozyme introns, IE introns, and degenerate group I-like introns. Both intron structural and phylogenetic analyses indicate a multiple origin of the S516 introns during evolution. The myxomycete introns are related to S516 introns in the more distantly related brown algae and Acanthamoeba species. Possible mechanisms of intron transfer both at the RNA- and DNA-levels are discussed in order to explain the observed widespread, but scattered, phylogenetic distribution.

  6. Complete structure of nuclear rDNA of the obligate plant parasite Plasmodiophora brassicae: intraspecific polymorphisms in the exon and group I intron of the large subunit rDNA.

    PubMed

    Niwa, Rieko; Kawahara, Ai; Murakami, Hiroharu; Tanaka, Shuhei; Ezawa, Tatsuhiro

    2011-07-01

    Plasmodiophora brassicae is a soil-borne obligate intracellular parasite in the phylum Cercozoa of the Rhizaria that causes clubroot disease of crucifer crops. To control the disease, understanding the distribution and infection routes of the pathogen is essential, and thus development of reliable molecular markers to discriminate geographic populations is required. In this study, the nuclear ribosomal RNA gene (rDNA) repeat unit of P. brassicae was determined, with particular emphasis on the structure of large subunit (LSU) rDNA, in which polymorphic regions were expected to be present. The complete rDNA complex was 9513bp long, which included the small subunit, 5.8S and LSU rDNAs as well as the internal transcribed spacer and intergenic spacer regions. Among eight field populations collected from throughout Honshu Island, Japan, a 1.1 kbp region of the LSU rDNA, including the divergent 8 domain, exhibited intraspecific polymorphisms that reflected geographic isolation of the populations. Two new group I introns were found in this region in six out of the eight populations, and the sequences also reflected their geographic isolation. The polymorphic region found in this study may have potential for the development of molecular markers for discrimination of field populations/isolates of this organism.

  7. Complete structure of nuclear rDNA of the obligate plant parasite Plasmodiophora brassicae: intraspecific polymorphisms in the exon and group I intron of the large subunit rDNA.

    PubMed

    Niwa, Rieko; Kawahara, Ai; Murakami, Hiroharu; Tanaka, Shuhei; Ezawa, Tatsuhiro

    2011-07-01

    Plasmodiophora brassicae is a soil-borne obligate intracellular parasite in the phylum Cercozoa of the Rhizaria that causes clubroot disease of crucifer crops. To control the disease, understanding the distribution and infection routes of the pathogen is essential, and thus development of reliable molecular markers to discriminate geographic populations is required. In this study, the nuclear ribosomal RNA gene (rDNA) repeat unit of P. brassicae was determined, with particular emphasis on the structure of large subunit (LSU) rDNA, in which polymorphic regions were expected to be present. The complete rDNA complex was 9513bp long, which included the small subunit, 5.8S and LSU rDNAs as well as the internal transcribed spacer and intergenic spacer regions. Among eight field populations collected from throughout Honshu Island, Japan, a 1.1 kbp region of the LSU rDNA, including the divergent 8 domain, exhibited intraspecific polymorphisms that reflected geographic isolation of the populations. Two new group I introns were found in this region in six out of the eight populations, and the sequences also reflected their geographic isolation. The polymorphic region found in this study may have potential for the development of molecular markers for discrimination of field populations/isolates of this organism. PMID:21497131

  8. The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair.

    PubMed

    Brosh, R M; Balajee, A S; Selzer, R R; Sunesen, M; Proietti De Santis, L; Bohr, V A

    1999-11-01

    Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. PMID:10564257

  9. Induction of cytokine production in cholesteatoma keratinocytes by extracellular high-mobility group box chromosomal protein 1 combined with DNA released by apoptotic cholesteatoma keratinocytes.

    PubMed

    Chi, Zhangcai; Wang, Zhengmin; Liang, Qiong; Zhu, Yaying; du, Qiang

    2015-02-01

    High-mobility group box chromosomal protein 1 (HMGB-1), a nuclear DNA binding protein, was recently rediscovered as a new proinflammatory cytokine. The purpose of this study was to determine HMGB-1 expression in vivo and to identify the effect of extracellular HMGB-1 in inflammatory process associated with bone destruction in cholesteatoma. We investigated the expression and location of HMGB-1 in the cholesteatoma and healthy skin using an immunofluorescence assay. We also detected apoptosis and DNA fragments in the cholesteatoma by TUNEL staining. HMGB-1 concentration in apoptotic supernatants from UV light-treated cells, culture supernatants and its translocation in cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells were measured by immunoblot analysis and immunofluorescence assay. Cultures of human cholesteatoma keratinocytes were exposed to CpG-DNA, HMGB-1, or CpG-DNA complexed to HMGB-1 for 24 h. Cytokines in the culture supernatant were measured by ELISA. In addition, levels of proinflammatory cytokines released by cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells with or without anti-HMGB-1 antibodies and supernatants from UV light-treated cells with DNase 1 were measured by enzyme-linked immunosorbent assay. The expression of HMGB-1 in cholesteatoma increased and it translocated both to the cytoplasm and extracellular space. Furthermore, the HMGB-1 concentration in supernatants increased significantly after addition of supernatants from UV light-treated cells. TNF-α and IL-1β can be induced by purified HMGB-1 combined with CpG-DNA in the cholesteatoma keratinocytes. In addition, supernatants of apoptotic cells containing HMGB-1-DNA were effective in inducing TNF-α and IL-1β secretion. This study suggested that persistent expression of extracellular HMGB-1 and DNA fragments in cholesteatoma leads to TNF-α and IL-1β production, causing bone resorption and destruction. Thus, we have

  10. DNA Barcoding of Rhodiola (Crassulaceae): A Case Study on a Group of Recently Diversified Medicinal Plants from the Qinghai-Tibetan Plateau

    PubMed Central

    Zhang, Jian-Qiang; Meng, Shi-Yong; Wen, Jun; Rao, Guang-Yuan

    2015-01-01

    DNA barcoding, the identification of species using one or a few short standardized DNA sequences, is an important complement to traditional taxonomy. However, there are particular challenges for barcoding plants, especially for species with complex evolutionary histories. We herein evaluated the utility of five candidate sequences — rbcL, matK, trnH-psbA, trnL-F and the internal transcribed spacer (ITS) — for barcoding Rhodiola species, a group of high-altitude plants frequently used as adaptogens, hemostatics and tonics in traditional Tibetan medicine. Rhodiola was suggested to have diversified rapidly recently. The genus is thus a good model for testing DNA barcoding strategies for recently diversified medicinal plants. This study analyzed 189 accessions, representing 47 of the 55 recognized Rhodiola species in the Flora of China treatment. Based on intraspecific and interspecific divergence and degree of monophyly statistics, ITS was the best single-locus barcode, resolving 66% of the Rhodiola species. The core combination rbcL+matK resolved only 40.4% of them. Unsurprisingly, the combined use of all five loci provided the highest discrimination power, resolving 80.9% of the species. However, this is weaker than the discrimination power generally reported in barcoding studies of other plant taxa. The observed complications may be due to the recent diversification, incomplete lineage sorting and reticulate evolution of the genus. These processes are common features of numerous plant groups in the high-altitude regions of the Qinghai-Tibetan Plateau. PMID:25774915

  11. Identification, molecular characterization, and evolution of group I introns at the expansion segment D11 of 28S rDNA in Rhizoctonia species.

    PubMed

    González, Dolores

    2013-09-01

    The nuclear ribosomal DNA of Rhizoctonia species is polymorphic in terms of the nucleotide composition and length. Insertions of 349-410 nucleotides in length with characteristics of group I introns were detected at a single insertion point at the expansion segment D11 of 28S rDNA in 12 out of 64 isolates. Eleven corresponded to Rhizoctonia solani (teleomorph: Thanatephorous) and one (AG-Q) to Rhizoctonia spp. (teleomorph: Ceratobasidium). Sequence data showed that all but AG-Q contained conserved DNA catalytic core regions (P, Q, R, and S) essential for selfsplicing. The predicted secondary structure revealed that base-paired helices corresponded to subgroup IC1. Isolates from same anastomosis group and even subgroups within R. solani were variable with regard to possession of introns. Phylogenetic analyses indicated that introns were vertically transmitted. Unfortunately, sequence data from the conserved region from all 64 isolates were not useful for delimiting species. Analyses with IC1 introns at same insertion point, of both Ascomycota and Basidiomycota indicated the possibility of horizontal transfer at this site. The present study uncovered new questions on evolutionary pattern of change of these introns within Rhizoctonia species.

  12. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

    PubMed

    Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M

    2013-07-01

    Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications. PMID:23697550

  13. Intermittent hair loss in a child with PIBI(D)S syndrome and trichothiodystrophy with defective DNA repair-xeroderma pigmentosum group D.

    PubMed

    Kleijer, W J; Beemer, F A; Boom, B W

    1994-08-15

    We describe a girl with photosensitivity (P), ichthyosis (I), brittle hair (B), impaired intelligence (I), possibly decreased fertility (D), and short stature (S). The clinical findings fit into the PIBI(D)S syndrome and trichothiodystrophy. A remarkable and probably unique observation for this disorder was the intermittent character of the scalp hair loss during infectious periods in this patient. Easy suntanning suggested photosensitivity and prompted DNA repair studies which demonstrated reduced UV-induced DNA repair synthesis. Subsequent studies have assigned this patient to xeroderma pigmentosum group D and suggested a specific deficiency of 6-4 photoproduct repair. An unaffected child was diagnosed in the next pregnancy of the mother. PMID:7802014

  14. RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability.

    PubMed

    Park, E; Guzder, S N; Koken, M H; Jaspers-Dekker, I; Weeda, G; Hoeijmakers, J H; Prakash, S; Prakash, L

    1992-12-01

    Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M(r) 95,356). The RAD25 (SSL2)- and XPBC-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UV sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys392 residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD25 (SSL2) ATPase/DNA helicase activity in viability. PMID:1333609

  15. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China*

    PubMed Central

    Wang, Hong-dan; Shen, Chun-mei; Liu, Wen-juan; Zhang, Yu-dang; Yang, Guang; Yan, Jiang-wei; Qin, Hai-xia; Zhu, Bo-feng

    2013-01-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capillary electrophoresis, the PCR products of the 21 STR loci were separated and genotyped. A total of 161 alleles were observed in the Russian ethnic minority group, and corresponding allelic frequencies ranged from 0.0044 to 0.5965. The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population’s genetic background, for individual identification, and for paternity testing in forensic practice. PMID:23733431

  16. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China.

    PubMed

    Wang, Hong-dan; Shen, Chun-mei; Liu, Wen-juan; Zhang, Yu-dang; Yang, Guang; Yan, Jiang-wei; Qin, Hai-xia; Zhu, Bo-feng

    2013-06-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capillary electrophoresis, the PCR products of the 21 STR loci were separated and genotyped. A total of 161 alleles were observed in the Russian ethnic minority group, and corresponding allelic frequencies ranged from 0.0044 to 0.5965. The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population's genetic background, for individual identification, and for paternity testing in forensic practice.

  17. Biological and DNA evidence of two dissimilar populations of the Rhipicephalus sanguineus tick group (Acari: Ixodidae) in South America.

    PubMed

    Szabó, Matias P J; Mangold, Atilio J; João, Carolina F; Bechara, Gervásio H; Guglielmone, Alberto A

    2005-06-10

    In this work, the biology, mitochondrial DNA and fertility of hybrids from two strains of Rhipicephalus sanguineus, from Brazil and Argentina, were compared. Engorged larvae, nymphs and adults from Argentina weighed more and the engorgement period of adult females was significantly longer than those of their Brazilian counterparts, whereas adult female tick yield rate was higher for the Brazilian strain. High intraspecific divergence of mitochondrial DNA was detected between R. sanguineus from Brazil and Argentina. On the other hand, a strong genetic relationship was detected between European and Argentinean R. sanguineus populations while the Brazilian population appeared to be related to the African Rhipicephalus turanicus. Adult hybrid females laid eggs, which were mostly unviable, whereas a mean of more than 1400 larvae hatched per egg mass from pure Brazilian and Argentinean strains. These results showed that differences between these strains are greater than previously assumed and that the biosystematic status of R. sanguineus ticks from South America should be re-evaluated. Wide variations, such as these might account for the reported worldwide differences in biology and vector capacity of this species.

  18. Karyotype diversity of four species of the incertae sedis group (Characidae) from different hydrographic basins: analysis of AgNORs, CMA3 and 18S rDNA.

    PubMed

    Mendes, M M; da Rosa, R; Giuliano-Caetano, L; Dias, A L

    2011-01-01

    A large number of genera in the tropical fish family Characidae are incertae sedis. Cytogenetic analysis was made of four of these species: Astyanax eigenmanniorum, Deuterodon stigmaturus, Hyphessobrycon luetkenii, and H. anisitsi, collected from various hydrographic basins: hydrographic system from Laguna dos Patos/RS, Tramandaí basin/RS and Tibagi River basin/PR. The first two species were collected in their type locality in the State of Rio Grande do Sul. The 2n = 48 karyotype was observed only in A. eigenmanniorum, while the other species had 2n = 50 chromosomes, with different karyotypic formulas. There was weak heterochromatin staining in the pericentromeric region of A. eigenmanniorum, D. stigmaturus and H. luetkenni chromosomes. In H. anisitsi, heterochromatin appeared to be more abundant and distributed in the pericentromeric and terminal regions of the chromosomes; three pairs showed more evident heterochromatic blocks. There were multiple Ag-NORs in all populations, visualized by FISH with an 18S rDNA probe. While D. stigmaturus and H. luetkenii had conserved AgNOR, CMA3 and 18S rDNA sites, the other two species showed intra- and interindividual variation at these sites. The karyotype variability was high, as is common in this group of fish. Different species arising from isolated hydrographic basins maintain an elevated level of karyotype differentiation, mainly with respect to chromosome structure, heterochromatin distribution and rDNA localization. This is the first report with cytogenetic data for D. stigmaturus and H. luetkenii. PMID:22179995

  19. Identification of Optimal Protocols for Sequencing Difficult Templates: Results of the 2008 ABRF DNA Sequencing Research Group Difficult Template Study 2008

    PubMed Central

    Kieleczawa, Jan; Adam, Debbie; Bintzler, Doug; Detwiler, Michelle; Needleman, David; Schweitzer, Peter; Singh, Sushmita; Steen, Robert; Zianni, Michael

    2009-01-01

    The 2008 ABRF DNA Sequencing Research Group (DSRG) difficult template sequencing study was designed to identify a general set of guidelines that would constitute the best approaches for sequencing difficult templates. This was a continuation of previous DSRG difficult template studies performed in 1996, 1997, and 2003. The distinguishing factors in the present study were the number of DNA templates used, the number of different types of difficult regions tested, and the inclusion of a follow-up phase of the study to identify optimal protocols for each type of difficult template. DNA templates with associated sequencing primers were distributed to participating laboratories and each laboratory returned their sequencing results along with descriptions of the experimental conditions used. The data were analyzed and the best protocols were identified for each difficult template. This information was subsequently distributed to the participating laboratories for a second round of sequencing to evaluate the general applicability of the optimized protocols. The average improvements in sequencing results were 11% overall, with a range of −25% to +43% using the optimized protocols. The full results from this study are presented here and they demonstrate that general experimental protocols and common additives can be used to improve the sequencing success for many difficult templates. PMID:19503623

  20. Synthesis and Structure of a New Copper(II) Coordination Polymer Alternately Bridged by Oxamido and Carboxylate Groups: Evaluation of DNA/BSA Binding and Cytotoxic Activities.

    PubMed

    Jin, Xiao-Ting; Zheng, Kang; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2015-08-01

    A new one-dimensional (1D) copper(II) coordination polymer {[Cu2 (dmaepox)(dabt)](NO3) · 0.5 H2 O}n , where H3 dmaepox and dabt denote N-benzoato-N'-(3-methylaminopropyl)oxamide and 2,2'-diamino-4,4'-bithiazole, respectively, was synthesized and characterized by single-crystal X-ray diffraction and other methods. The crystal structure analysis revealed that the two copper(II) ions are bridged alternately by cis-oxamido and carboxylato groups to form a 1-D coordination polymer with the corresponding Cu · · · Cu separations of 5.1946(19) and 5.038(2) Å. There is a three-dimensional supramolecular structure constructed by hydrogen bonding and π-π stacking interactions in the crystal. The reactivity towards herring sperm DNA (HS-DNA) and bovine serum albumin (BSA) indicated that the copper(II) polymer can interact with the DNA in the mode of intercalation, and bind to BSA responsible for quenching of tryptophan fluorescence by the static quenching mechanism. The in vitro cytotoxicity suggested that the copper(II) polymer exhibits cytotoxic effects against the selected tumor cell lines.

  1. Phylogenetic Analysis of Rhinosporidium seeberi’s 18S Small-Subunit Ribosomal DNA Groups This Pathogen among Members of the Protoctistan Mesomycetozoa Clade

    PubMed Central

    Herr, Roger A.; Ajello, Libero; Taylor, John W.; Arseculeratne, Sarath N.; Mendoza, Leonel

    1999-01-01

    For the past 100 years the phylogenetic affinities of Rhinosporidium seeberi have been controversial. Based on its morphological features, it has been classified as a protozoan or as a member of the kingdom Fungi. We have amplified and sequenced nearly a full-length 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence from R. seeberi. Using phylogenetic analysis, by parsimony and distance methods, of R. seeberi’s 18S SSU rDNA and that of other eukaryotes, we found that this enigmatic pathogen of humans and animals clusters with a novel group of fish parasites referred to as the DRIP clade (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium), near the animal-fungal divergence. Our phylogenetic analyses also indicate that R. seeberi is the sister taxon of the two Dermocystidium species used in this study. This molecular affinity is remarkable since members of the genus Dermocystidium form spherical structures in infected hosts, produce endospores, have not been cultured, and possess mitochondria with flat cristae. With the addition of R. seeberi to this clade, the acronym DRIP is no longer appropriate. We propose to name this monophyletic clade Mesomycetozoa to reflect the group’s phylogenetic association within the Eucarya. PMID:10449446

  2. DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizoctonia blight (sensu lato) is a common and serious disease of many turfgrass species. The most widespread causal agent, R. solani, consists of several genetically different subpopulations. Though hyphal anastomosis reactions have been used to group Rhizoctonia species, they are time consuming a...

  3. Patterns of inter- and intra-group genetic diversity in the Vlax Roma as revealed by Y chromosome and mitochondrial DNA lineages.

    PubMed

    Kalaydjieva, L; Calafell, F; Jobling, M A; Angelicheva, D; de Knijff, P; Rosser, Z H; Hurles, M E; Underhill, P; Tournev, I; Marushiakova, E; Popov, V

    2001-02-01

    Previous genetic studies, supported by linguistic and historical data, suggest that the European Roma, comprising a large number of socially divergent endogamous groups, may be a complex conglomerate of founder populations. The boundaries and characteristics of such founder populations and their relationship to the currently existing social stratification of the Roma have not been investigated. This study is an attempt to address the issues of common vs independent origins and the history of population fissioning in three Romani groups that are well defined and strictly endogamous relative to each other. According to linguistic classifications, these groups belong to the Vlax Roma, who account for a large proportion of the European Romani population. The analysis of mtDNA sequence variation has shown that a large proportion of maternal lineages are common to the three groups. The study of a set of Y chromosome markers of different mutability has revealed that over 70% of males belong to a single lineage that appears unique to the Roma and presents with closely related microsatellite haplotypes and MSY1 codes. The study unambiguously points to the common origins of the three Vlax groups and the recent nature of the population fissions, and provides preliminary evidence of limited genetic diversity in this young founder population.

  4. A diverse group of small circular ssDNA viral genomes in human and non-human primate stools

    PubMed Central

    Ng, Terry Fei Fan; Zhang, Wen; Sachsenröder, Jana; Kondov, Nikola O.; da Costa, Antonio Charlys; Vega, Everardo; Holtz, Lori R.; Wu, Guang; Wang, David; Stine, Colin O.; Antonio, Martin; Mulvaney, Usha S.; Muench, Marcus O.; Deng, Xutao; Ambert-Balay, Katia; Pothier, Pierre; Vinjé, Jan; Delwart, Eric

    2015-01-01

    Viral metagenomics sequencing of fecal samples from outbreaks of acute gastroenteritis from the US revealed the presence of small circular ssDNA viral genomes encoding a replication initiator protein (Rep). Viral genomes were ∼2.5 kb in length, with bi-directionally oriented Rep and capsid (Cap) encoding genes and a stem loop structure downstream of Rep. Several genomes showed evidence of recombination. By digital screening of an in-house virome database (1.04 billion reads) using BLAST, we identified closely related sequences from cases of unexplained diarrhea in France. Deep sequencing and PCR detected such genomes in 7 of 25 US (28 percent) and 14 of 21 French outbreaks (67 percent). One of eighty-five sporadic diarrhea cases in the Gambia was positive by PCR. Twenty-two complete genomes were characterized showing that viruses from patients in the same outbreaks were closely related suggesting common origins. Similar genomes were also characterized from the stools of captive chimpanzees, a gorilla, a black howler monkey, and a lemur that were more diverse than the human stool-associated genomes. The name smacovirus is proposed for this monophyletic viral clade. Possible tropism include mammalian enteric cells or ingested food components such as infected plants. No evidence of viral amplification was found in immunodeficient mice orally inoculated with smacovirus-positive stool supernatants. A role for smacoviruses in diarrhea, if any, remains to be demonstrated. PMID:27774288

  5. Elevated levels of STAT1 in Fanconi anemia group A lymphoblasts correlate with the cells’ sensitivity to DNA interstrand crosslinking drugs

    PubMed Central

    Prieto-Remón, Inés; Sánchez-Carrera, Dámaso; López-Duarte, Mónica; Richard, Carlos; Pipaón, Carlos

    2013-01-01

    Progressive bone marrow failure starting in the first decade of life is one of the main characteristics of Fanconi anemia. Along with the bone marrow failure, this pathology is characterized by congenital malformations, endocrine dysfunction and an extraordinary predisposition to develop cancer. The fact that hematopoietic progenitor cells from subjects with Fanconi anemia are sensitive to both DNA-interstrand crosslinking agents and inflammatory cytokines, which are aberrantly overproduced in these patients, has led to different explanations for the causes of the bone marrow failure. We analyzed STAT1 expression in lymphoblastoid cell lines derived from patients with Fanconi anemia group A and correlated this with aspects of the Fanconi anemia phenotype such as sensitivity to genotoxic agents or to inhibitory cytokines. We provide evidence of overexpression of STAT1 in FANCA-deficient cells which has both transcriptional and post-translational components, and is related to the constitutive activation of ERK in Fanconi anemia group A cells, since it can be reverted by treatment with U0126. STAT1 phosphorylation was not defective in the lymphoblasts, so these cells accumulated higher levels of active STAT1 in response to interferon gamma, probably in relation to their greater sensitivity to this cytokine. On the other hand, inhibition of STAT1 by genetic or chemical means reverted the hypersensitivity of Fanconi anemia group A lymphoblasts to DNA interstrand crosslinking agents. Our data provide an explanation for the mixed sensitivity of Fanconi anemia group A cells to both genotoxic stress and inflammatory cytokines and indicate new targets for the treatment of bone marrow failure in these patients. PMID:23585528

  6. MtDNA Haplogroup A10 Lineages in Bronze Age Samples Suggest That Ancient Autochthonous Human Groups Contributed to the Specificity of the Indigenous West Siberian Population

    PubMed Central

    Pilipenko, Aleksandr S.; Trapezov, Rostislav O.; Zhuravlev, Anton A.; Molodin, Vyacheslav I.; Romaschenko, Aida G.

    2015-01-01

    Background The craniometric specificity of the indigenous West Siberian human populations cannot be completely explained by the genetic interactions of the western and eastern Eurasian groups recorded in the archaeology of the area from the beginning of the 2nd millennium BC. Anthropologists have proposed another probable explanation: contribution to the genetic structure of West Siberian indigenous populations by ancient human groups, which separated from western and eastern Eurasian populations before the final formation of their phenotypic and genetic features and evolved independently in the region over a long period of time. This hypothesis remains untested. From the genetic point of view, it could be confirmed by the presence in the gene pool of indigenous populations of autochthonous components that evolved in the region over long time periods. The detection of such components, particularly in the mtDNA gene pool, is crucial for further clarification of early regional genetic history. Results and Conclusion We present the results of analysis of mtDNA samples (n = 10) belonging to the A10 haplogroup, from Bronze Age populations of West Siberian forest-steppe (V—I millennium BC), that were identified in a screening study of a large diachronic sample (n = 96). A10 lineages, which are very rare in modern Eurasian populations, were found in all the Bronze Age groups under study. Data on the A10 lineages’ phylogeny and phylogeography in ancient West Siberian and modern Eurasian populations suggest that A10 haplogroup underwent a long-term evolution in West Siberia or arose there autochthonously; thus, the presence of A10 lineages indicates the possible contribution of early autochthonous human groups to the genetic specificity of modern populations, in addition to contributions of later interactions of western and eastern Eurasian populations. PMID:25950581

  7. DNA evidence on the phylogenetic systematics of New World monkeys: support for the sister-grouping of Cebus and Saimiri from two unlinked nuclear genes.

    PubMed

    Harada, M L; Schneider, H; Schneider, M P; Sampaio, I; Czelusniak, J; Goodman, M

    1995-09-01

    Previous inferences from epsilon-globin gene sequences on cladistic relationships among the 16 extant genera of Ceboidea (the New World monkeys) were tested by strength of grouping and bootstrap values for the clades in the most parsimonious trees found: for this epsilon data set enlarged with additional Cebus and Saimiri orthologues; for another nuclear DNA sequence data set consisting of IRBP (interstitial retinol-binding protein gene) intron 1 orthologues; and for tandemly combined epsilon and IRBP sequences. Different ceboid species of the same genus always grouped strongly together as demonstrated by results on Cebus (capuchin monkeys), Saimiri (squirrel monkeys), Callicebus (titi monkeys), Aotus (night monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys). Other strong groupings that could be represented as monophyletic taxa in a cladistic classification were: Cebuella (pygmy marmoset) and Callithrix (marmoset) into subtribe Callitrichina; Callitrichina, Callimico (Goeldi's monkey), Leontopithecus (lion tamarin), and Saguinus (tamarin) into subfamily Callitrichinae; Callitrichinae, Aotus, Cebus, and Saimiri into family Cebidae; Cacajao (uakari monkey) and Chiropotes (saki) into subtribe Chiropotina; Chiropotina and Pithecia (bearded saki) into tribe Pitheciini; Pitheciini and Callicebus into subfamily Pitheciinae; Brachyteles (woolly spider monkey), Lagothrix (woolly monkey), and Ateles into tribe Atelini; and Atelini and Alouatta into subfamily Atelinae. In addition the epsilon and IRBP results congruently grouped (but at lesser strengths) Brachyteles and Lagothrix into subtribe Brachytelina within Atelini, and also Cebus and Saimiri into subfamily Cebinae within Cebidae. Because the IRBP results weakly grouped Pitheciinae with Cebidae, whereas the epsilon results weakly grouped Pitheciinae with Atelinae, the present evidence is best represented in an interim cladistic classification of ceboids by dividing the superfamily Ceboidea into three

  8. PolA1, a Putative DNA Polymerase I, Is Coexpressed with PerR and Contributes to Peroxide Stress Defenses of Group A Streptococcus

    PubMed Central

    Toukoki, Chadia

    2013-01-01

    The peroxide stress response regulator PerR coordinates the oxidative-stress defenses of group A Streptococcus (GAS). We now show that PerR is expressed from an operon encoding a putative DNA polymerase I (PolA1), among other GAS products. A polA1 deletion mutant exhibited wild-type growth but showed reduced capacity to repair DNA damage caused by UV light or ciprofloxacin. Mutant bacteria were hypersensitive to H2O2, compared with the wild type or a complemented mutant strain, and remained severely attenuated even after adaptation at sublethal H2O2 levels, whereas wild-type bacteria could adapt to withstand peroxide challenge under identical conditions. The hypersensitivity of the mutant was reversed when bacteria were grown in iron-depleted medium and challenged in the presence of a hydroxyl radical scavenger, results that indicated sensitivity to hydroxyl radicals generated by Fenton chemistry. The peroxide resistance of a perR polA1 double mutant following adaptation at sublethal H2O2 levels was decreased 9-fold relative to a perR single mutant, thus implicating PolA1 in PerR-mediated defenses against peroxide stress. Cultures of the polA1 mutant grown with or without prior H2O2 exposure yielded considerably lower numbers of rifampin-resistant mutants than cultures of the wild type or the complemented mutant strain, a finding consistent with PolA1 lacking proofreading activity. We conclude that PolA1 promotes genome sequence diversity while playing an essential role in oxidative DNA damage repair mechanisms of GAS, dual functions predicted to confer optimal adaptive capacity and fitness in the host. Together, our studies reveal a unique genetic and functional relationship between PerR and PolA1 in streptococci. PMID:23204468

  9. Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

    PubMed

    Yan, Y X; Schiestl, R H; Prakash, L

    1995-06-01

    The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

  10. Design, synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino, N-formamido, pyrrole- and imidazole-containing polyamides.

    PubMed

    Chavda, Sameer; Babu, Balaji; Patil, Pravin; Plaunt, Adam; Ferguson, Amanda; Lee, Megan; Tzou, Samuel; Sjoholm, Robert; Rice, Toni; Mackay, Hilary; Ramos, Joseph; Wang, Shuo; Lin, Shicai; Kiakos, Konstantinos; Wilson, W David; Hartley, John A; Lee, Moses

    2013-07-01

    Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K(eq)=2.4×10(8) M(-1)) and with comparable sequence selectivity to its cognate sequence 5'-ACGCGT-3' when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5'-ACGCGT-3' via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5'-ATGCAT-3' (K(eq)=7.4×10(6) M(-1)) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5'-AAATTT-3' (K(eq)=4.8×10(7) M(-1)), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5'-ATCGAT-3' as a stacked dimer and it has the lowest affinity among the diamino PAs tested (Keq <1×10(5) M(-1)). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the 'core rules' of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants

  11. Design, synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino, N-formamido, pyrrole- and imidazole-containing polyamides.

    PubMed

    Chavda, Sameer; Babu, Balaji; Patil, Pravin; Plaunt, Adam; Ferguson, Amanda; Lee, Megan; Tzou, Samuel; Sjoholm, Robert; Rice, Toni; Mackay, Hilary; Ramos, Joseph; Wang, Shuo; Lin, Shicai; Kiakos, Konstantinos; Wilson, W David; Hartley, John A; Lee, Moses

    2013-07-01

    Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K(eq)=2.4×10(8) M(-1)) and with comparable sequence selectivity to its cognate sequence 5'-ACGCGT-3' when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5'-ACGCGT-3' via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5'-ATGCAT-3' (K(eq)=7.4×10(6) M(-1)) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5'-AAATTT-3' (K(eq)=4.8×10(7) M(-1)), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5'-ATCGAT-3' as a stacked dimer and it has the lowest affinity among the diamino PAs tested (Keq <1×10(5) M(-1)). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the 'core rules' of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants

  12. Redox-mediated Mechanisms Regulate DNA Binding Activity of the G-group of Basic Region Leucine Zipper (bZIP) Transcription Factors in Arabidopsis*

    PubMed Central

    Shaikhali, Jehad; Norén, Louise; de Dios Barajas-López, Juan; Srivastava, Vaibhav; König, Janine; Sauer, Uwe H.; Wingsle, Gunnar; Dietz, Karl-Josef; Strand, Åsa

    2012-01-01

    Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys330 in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors. PMID:22718771

  13. The sequences of heat shock protein 40 (DnaJ) homologs provide evidence for a close evolutionary relationship between the Deinococcus-thermus group and cyanobacteria.

    PubMed

    Bustard, K; Gupta, R S

    1997-08-01

    The genes encoding for heat shock protein 40 (Hsp40 or DnaJ) homologs were cloned and sequenced from the archaebacterium Halobacterium cutirubrum and the eubacterium Deinococcus proteolyticus to add to sequences from the gene banks. These genes were identified downstream of the Hsp70 (or DnaK) genes in genomic fragments spanning this region and, as in other prokaryotic species, Hsp70-Hsp40 genes are likely part of the same operon. The Hsp40 homolog from D. proteolyticus was found to be lacking a central 204 base pair region present in H. cutirubrum that encodes for the four cysteine-rich domains of the repeat consensus sequence CxxCxGxG (where x is any amino acid), present in most Hsp40 homologs. The available sequences from various archaebacteria, eubacteria, and eukaryotes show that the same deletion is also present in the homologs from Thermus aquaticus and two cyanobacteria, but in no other species tested. This unique deletion and the clustering of homologs from the Deinococcus-Thermus group and cyanobacterial species in the Hsp40 phylogenetic trees suggest a close evolutionary relationship between these groups as was also shown recently for Hsp70 sequences (R.S. Gupta et al., J Bacteriol 179:345-357, 1997). Sequence comparisons indicate that the Hsp40 homologs are not as conserved as the Hsp70 sequences. Phylogenetic analysis provides no reliable information concerning evolutionary relationship between prokaryotes and eukaryotes and their usefulness in this regard is limited. However, in phylogenetic trees based on Hsp40 sequences, the two archaebacterial homologs showed a polyphyletic branching within Gram-positive bacteria, similar to that seen with Hsp70 sequences.

  14. Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis

    PubMed Central

    Lekang, Katrine; Langeland, Nina; Wiker, Harald G.

    2011-01-01

    The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing. PMID:21436365

  15. A study of genetic polymorphisms in mitochondrial DNA hypervariable regions I and II of the five major ethnic groups and Vedda population in Sri Lanka.

    PubMed

    Ranasinghe, Ruwandi; Tennekoon, Kamani H; Karunanayake, Eric H; Lembring, Maria; Allen, Marie

    2015-11-01

    Diversity of the hypervariable regions (HV) I and II of the mitochondrial genome was studied in maternally unrelated Sri Lankans (N=202) from six ethnic groups (i.e.: Sinhalese, Sri Lankan Tamil, Muslim, Malay, Indian Tamil and Vedda). DNA was extracted from blood and buccal swabs and HVI and HVII regions were PCR amplified and sequenced. Resulting sequences were aligned and edited between 16024-16365 and 73-340 regions and compared with revised Cambridge reference sequences (rCRS). One hundred and thirty-five unique haplotypes and 22 shared haplotypes were observed. A total of 145 polymorphic sites and 158 polymorphisms were observed. Hypervariable region I showed a higher polymorphic variation than hypervariable region II. Nucleotide diversities were quite low and similar for all ethnicities apart from a slightly higher value for Indian Tamils and a much lower value for the Vedda population compared to the other groups. When the total population was considered South Asian (Indian) haplogroups were predominant, but there were differences in the distribution of phylo-geographical haplogroups between ethnic groups. Sinhalese, Sri Lankan Tamil and Vedda populations had a considerable presence of West Eurasian haplogroups. About 2/3rd of the Vedda population comprised of macro-haplogroup N or its subclades R and U, whereas macro-haplogroup M was predominant in all other populations. The Vedda population clustered separately from other groups and Sri Lankan Tamils showed a closer genetic affiliation to Sinhalese than to Indian Tamils. Thus this study provides useful information for forensic analysis and anthropological studies of Sri Lankans.

  16. Genetic variation in DNA-repair pathways and response to radiochemotherapy in esophageal adenocarcinoma: a retrospective cohort study of the Eastern Cooperative Oncology Group

    PubMed Central

    2011-01-01

    Background Recent data in esophageal cancer suggests the variant allele of a single-nucleotide polymorphism (SNP) in XRCC1 may be associated with resistance to radiochemotherapy. However, this SNP has not been assessed in a histologically homogeneous clinical trial cohort that has been treated with a uniform approach. In addition, whether germline DNA may serve as a surrogate for tumor genotype at this locus is unknown in this disease. Our objective was to assess this SNP in relation to the pathologic complete response (pCR) rate in subjects with esophageal adenocarcinoma who received cisplatin-based preoperative radiochemotherapy in a multicenter clinical trial (Eastern Cooperative Oncology Group 1201). As a secondary aim, we investigated the rate of allelic imbalance between germline and tumor DNA. Methods Eighty-one eligible treatment-naïve subjects with newly diagnosed resectable esophageal adenocarcinoma received radiotherapy (45 Gy) concurrent with cisplatin-based chemotherapy, with planned subsequent surgical resection. The primary endpoint was pCR, defined as complete absence of tumor in the surgical specimen after radiochemotherapy. Using germline DNA from 60 subjects, we examined the base-excision repair SNP, XRCC1 Arg399Gln, and 4 other SNPs in nucleotide excision (XPD Lys751Gln and Asp312Asn, ERCC1 3' flank) and double-stranded break (XRCC2 5' flank) repair pathways, and correlated genotype with pCR rate. Paired tumor tissue was used to estimate the frequency of allelic imbalance at the XRCC1 SNP. Results The variant allele of the XRCC1 SNP (399Gln) was detected in 52% of subjects. Only 6% of subjects with the variant allele experienced a pCR, compared to 28% of subjects without the variant allele (odds ratio 5.37 for failing to achieve pCR, p = 0.062). Allelic imbalance at this locus was found in only 10% of informative subjects, suggesting that germline genotype may reflect tumor genotype at this locus. No significant association with pCR was noted

  17. Molecular cloning, sequence, and expression of a human GDP-L-fucose:. beta. -D-galactoside 2-. alpha. -L-fucosyltransferase cDNA that can form the H blood group antigen

    SciTech Connect

    Larsen, R.D.; Ernst, L.K.; Nair, R.P.; Lowe, J.B. )

    1990-09-01

    The authors have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:{beta}D-galactoside 2-{alpha}-L-fucosyltransferase. Although this fragment determined expression of an {alpha}(1,2)FT whose kinetic properties mirror those of the human H blood group {alpha}(1,2)FT, their precise nature remained undefined. They describe here the molecular cloning, sequence, and expression of a human of cDNA corresponding to these human genomic sequences. When expressed in COS-1 cells, the cDNA directs expression of cell surface H structures and a cognate {alpha}(1,2)FT activity with properties analogous to the human H blood group {alpha}(1,2)FT. The cDNA sequence predicts a 365-amino acid polypeptide characteristic of a type II transmembrane glycoprotein with a domain structure analogous to that of other glycosyltransferases but without significant primary sequence similarity to these or other known proteins. To directly demonstrate that the cDNA encodes an {alpha}(1,2)FT, the COOH-terminal domain predicted to be Golgi-resident was expressed in COS-1 cells as a catalytically active, secreted, and soluble protein A fusion peptide. Southern blot analysis showed that this cDNA identified DNA sequences syntenic to the human H locus on chromosome 19. These results strongly suggest that this cloned {alpha}(1,2)FT cDNA represents the product of the human H blood group locus.

  18. A novel trimeric Zn (II) complex based on 8-hydroxyquinoline with trifluoromethylbenzene group: Synthesis, crystal structure, photophysical properties and DNA binding

    NASA Astrophysics Data System (ADS)

    Huo, Yanping; Wang, Chunquan; Lu, Jiguo; Hu, Sheng; Li, Xiaoyang; Zhang, Li

    2015-10-01

    A novel 2-substituted-8-hydroxyquinoline ligand (E)-2-[2-(4-trifluoromethylphenyl)ethenyl]-8-hydroxyquinoline (3, HL) was synthesized and characterized by ESI-MS, NMR spectroscopy and elemental analysis. Using solvothermal method, a trimeric complex [Zn3L6] (4) was fabricated by self-assembly of Zn(II) ions with 3. X-ray structural analysis shows that 4 exhibits a trinuclear core, which was bridged and encapsulated by six 8-hydroxyquinolinate-based ligands. The supramolecular structure of 4 features a lamellar solid constructed by aromatic stacking interactions and nonclassical C-H···F hydrogen bonds derived from 4-trifluoromethylphenyl group of the 3. The coordination behavior of zinc salt and 3 in solution was performed by 1H NMR, UV-vis and Photoluminescence (PL). The experimental results show that the complex 4 emits yellow luminescence in the solid state. To investigate its properties further, we also studied the thermal stability, photophysical properties (fluorescent emission, lifetime) of complex 4, and the interactions between 4 and C60 or EtBr-DNA system.

  19. Virion-associated cofactor high-mobility group DNA-binding protein-1 facilitates transposition from the herpes simplex virus/Sleeping Beauty amplicon vector platform.

    PubMed

    de Silva, Suresh; Lotta, Louis T; Burris, Clark A; Bowers, William J

    2010-11-01

    The development of the integration-competent, herpes simplex virus/Sleeping Beauty (HSV/SB) amplicon vector platform has created a means to efficiently and stably deliver therapeutic transcription units (termed "transgenons") to neurons within the mammalian brain. Furthermore, an investigation into the transposition capacity of the HSV/SB vector system revealed that the amplicon genome provides an optimal substrate for the transposition of transgenons at least 12 kb in length [de Silva, S., Mastrangelo, M.A., Lotta, L.T., Jr., Burris, C.A., Federoff, H.J., and Bowers, W.J. ( 2010 ). Gene Ther. 17, 424-431]. These results prompted an investigation into the factors that may contribute toward efficient transposition from the HSV/SB amplicon. One of the cellular cofactors known to play a key role during SB-mediated transposition is the high-mobility group DNA-binding protein-1 (HMGB1). Our present investigation into the role of HMGB1 during amplicon-based transposition revealed that transposition is not strictly dependent on the presence of cellular HMGB1, contrary to what had been previously demonstrated with plasmid-based SB transposition. We have shown for the first time that during amplicon preparation, biologically active HMGB1 derived from the packaging cell line is copackaged into amplicon vector particles. As a result, HSV/SB amplicon virions arrive prearmed with HMGB1 protein at levels sufficient for facilitating SB-mediated transposition in the transduced mammalian cell. PMID:20568967

  20. Variations of SSU rDNA group I introns in different isolates of Cordyceps militaris and the loss of an intron during cross-mating.

    PubMed

    Lian, Tiantian; Yang, Tao; Sun, Junde; Guo, Suping; Yang, Huaijun; Dong, Caihong

    2014-08-01

    Cordyceps militaris, the type species of genus Cordyceps, is one of the most popular mushrooms and a nutraceutical in eastern Asia. It is considered a model organism for the study of Cordyceps species because it can complete its life cycle when cultured in vitro. In the present study, the occurrence and sequence variation of SSU rDNA group I introns, Cmi.S943 and Cmi.S1199, among different isolates of C. militaris were analyzed. Based on the secondary structure predictions, the Cmi.S943 intron has been placed in subgroup IC1, and the Cmi.S1199 intron has been placed in subgroup IE. No significant similarity between Cmi.S943 and Cmi.S1199 suggested different origins. Three genotypes, based on the frequency and distribution of introns, were described to discriminate the 57 surveyed C. militaris strains. It was found that the genotype was related to the stroma characteristics. The stromata of all of the genotype II strains, which possessed only Cmi.S943, could produce perithecium. In contrast, the stromata of all genotype III strains, which had both Cmi.S943 and Cmi.S1199, could not produce perithecium. Cmi.S1199 showed the lowest level of intra-specific variation among the tested strains. Group I introns can be lost during strain cross-mating. Therefore, we presumed that during cross-mating and recombination, intron loss could be driven by positive Darwinian selection due to the energetic cost of transcribing long introns.

  1. Variations of SSU rDNA group I introns in different isolates of Cordyceps militaris and the loss of an intron during cross-mating.

    PubMed

    Lian, Tiantian; Yang, Tao; Sun, Junde; Guo, Suping; Yang, Huaijun; Dong, Caihong

    2014-08-01

    Cordyceps militaris, the type species of genus Cordyceps, is one of the most popular mushrooms and a nutraceutical in eastern Asia. It is considered a model organism for the study of Cordyceps species because it can complete its life cycle when cultured in vitro. In the present study, the occurrence and sequence variation of SSU rDNA group I introns, Cmi.S943 and Cmi.S1199, among different isolates of C. militaris were analyzed. Based on the secondary structure predictions, the Cmi.S943 intron has been placed in subgroup IC1, and the Cmi.S1199 intron has been placed in subgroup IE. No significant similarity between Cmi.S943 and Cmi.S1199 suggested different origins. Three genotypes, based on the frequency and distribution of introns, were described to discriminate the 57 surveyed C. militaris strains. It was found that the genotype was related to the stroma characteristics. The stromata of all of the genotype II strains, which possessed only Cmi.S943, could produce perithecium. In contrast, the stromata of all genotype III strains, which had both Cmi.S943 and Cmi.S1199, could not produce perithecium. Cmi.S1199 showed the lowest level of intra-specific variation among the tested strains. Group I introns can be lost during strain cross-mating. Therefore, we presumed that during cross-mating and recombination, intron loss could be driven by positive Darwinian selection due to the energetic cost of transcribing long introns. PMID:24996897

  2. Phosphorus-nitrogen compounds: Part 28. Syntheses, structural characterizations, antimicrobial and cytotoxic activities, and DNA interactions of new phosphazenes bearing vanillinato and pendant ferrocenyl groups

    NASA Astrophysics Data System (ADS)

    Tümer, Yasemin; Asmafiliz, Nuran; Kılıç, Zeynel; Hökelek, Tuncer; Yasemin Koç, L.; Açık, Leyla; Yola, Mehmet Lütfi; Solak, Ali Osman; Öner, Yağmur; Dündar, Devrim; Yavuz, Makbule

    2013-10-01

    The gradually Cl replacement reactions of spirocyclic mono (1 and 2) and bisferrocenyl cyclotriphosphazenes (3-5) with the potassium salt of 4-hydroxy-3-methoxybenzaldehyde (potassium vanillinate) gave mono (1a-5a), geminal (gem-1b-5b), non-geminal (cis-1b, cis-5b and trans-2b-5b), tri (1c-5c) and tetra-substituted phosphazenes (1d-5d). Some phosphazenes have stereogenic P-center(s). The chirality of 4c was verified using chiral HPLC column. Electrochemical behaviors were influenced only by the number of ferrocene groups, but not the length of the amine chains and the substituent(s). The structures of the new phosphazenes were determined by FTIR, MS, 1H, 13C and 31P NMR, HSQC and HMBC spectral data. The solid-state structures of cis-1b and 4d were examined by single crystal X-ray diffraction techniques. The twelve phosphazene derivatives were screened for antimicrobial activity and the compounds 5a, cis-1b and 2c exhibited the highest antibacterial activity against G(+) and G(-) bacteria. In addition, it was found that overall gem-1b inhibited the growth of Mycobacterium tuberculosis. The compounds 1d, 2d and 4d were tested in HeLa cancer cell lines. Among these compounds, 2d had cytotoxic effect on HeLa cell in the first 48 h. Moreover, interactions between compounds 2a, gem-1b, gem-2b, cis-1b, 2c, 3c, 4c, 5c, 1d, 2d and 4d, and pBR322 plasmid DNA were investigated.

  3. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm.

  4. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  5. Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

    NASA Astrophysics Data System (ADS)

    Krupovic, Mart; Koonin, Eugene V.

    2014-06-01

    Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

  6. Reactions of 5-methylcytosine cation radicals in DNA and model systems: thermal deprotonation from the 5-methyl group vs. excited state deprotonation from sugar

    PubMed Central

    Adhikary, Amitava; Kumar, Anil; Palmer, Brian J.; Todd, Andrew D.; Heizer, Alicia N.; Sevilla, Michael D.

    2014-01-01

    Purpose To study the formation and subsequent reactions of the 5-methyl-2′-deoxycytidine cation radical (5-Me-2′-dC•+) in nucleosides and DNA-oligomers and compare to one electron oxidized thymidine. Materials and methods Employing electron spin resonance (ESR), cation radical formation and its reactions were investigated in 5-Me-2′-dC, thymidine (Thd) and their derivatives, in fully double stranded (ds) d[GC*GC*GC*GC*]2 and in the 5-Me-C/A mismatched, d[GGAC*AAGC:CCTAATCG], where C* = 5-Me-C. Results We report 5-Me-2′-dC•+ production by one-electron oxidation of 5-Me-2′-dC by Cl2•− via annealing in the dark at 155 K. Progressive annealing of 5-Me-2′-dC•+ at 155 K produces the allylic radical (C-CH2•). However, photoexcitation of 5-Me-2′-dC•+ by 405 nm laser or by photoflood lamp leads to only C3′• formation. Photoexcitation of N3-deprotonated thyminyl radical in Thd and its 5′-nucleotides leads to C3′• formation but not in 3′-TMP which resulted in the allylic radical (U-CH2•) and C5′• production. For excited 5-Me-2′,3′-ddC•+, absence of the 3′-OH group does not prevent C3′• formation. For d[GC*GC*GC*GC*]2 and d[GGAC*AAGC:CCTAATCG], intra-base paired proton transferred form of G cation radical (G(N1-H)•:C(+H+)) is found with no observable 5-Me-2′-dC•+ formation. Photoexcitation of (G(N1-H)•:C(+H+)) in d[GC*GC*GC*GC*]2 produced only C1′• and not the expected photoproducts from 5-Me-2′-dC•+. However, photoexcitation of (G(N1-H)•:C(+H+)) in d[GGAC*AAGC:CCTAATCG] led to C5′• and C1′• formation. Conclusions C-CH2• formation from 5-Me-2′-dC•+ occurs via ground state deprotonation from C5-methyl group on the base. In the excited 5-Me-2′-dC•+ and 5-Me-2′,3′-ddC•+, spin and charge localization at C3′ followed by deprotonation leads to C3′• formation. Thus, deprotonation from C3′ in the excited cation radical is kinetically controlled and sugar C-H bond energies are

  7. DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).

    PubMed

    Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia; Porrini, Marisa; Langie, Sabine A S; van Schooten, Frederik-Jan; Azqueta, Amaya; Collins, Andrew R; Jones, George D D; Kwok, Rachel W L; Phillips, David H; Sozeri, Osman; Allione, Alessandra; Matullo, Giuseppe; Möller, Lennart; Forchhammer, Lykke; Loft, Steffen; Møller, Peter

    2013-09-18

    The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.

  8. The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex.

    PubMed

    Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

    2013-07-01

    A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy=2,2-bipyridine, Hbcpip=2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5±1.4)×10(5) M(-1) in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived.

  9. The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex

    NASA Astrophysics Data System (ADS)

    Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

    2013-07-01

    A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy = 2,2-bipyridine, Hbcpip = 2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5 ± 1.4) × 105 M-1 in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived.

  10. A biosensing of Toxoplasma gondii DNA with CdTe/Fe3O4 dual functional quantum dot as reporter group

    NASA Astrophysics Data System (ADS)

    Liang, Chu; Xu, Shichao; Yang, Juan; Zhang, Jimei; Dai, Zhao; Sun, Bo; Sun, Shuqing; Feng, Tielin; Zi, Yan; Liu, Jingwei; Luo, Hao

    2009-07-01

    Toxoplasma gondii is an intestinal coccidium that parasitizes members of the cat family as definitive hosts and has a wide range of intermediate hosts. Infection is common in many warm-blooded animals, including humans, the early detection of Toxoplasma gondii was concerned in recent years. In the current research, we presented a fast, specific, and sensitive sensing probe to detect Toxoplasma gondii DNA based on mechanism of fluorescence energy transfer (FRET), and a magnetic-fluorescent CdTe/Fe3O4 core-shell quantum dots (mQDs) was utilized as energy donor, and a commercial quencher (BHQ-2) was used as energy acceptor, respectively. The CdTe/Fe3O4 mQDs were prepared by layer-by-layer (LBL) process at ambient temperature. The sensing probe was fabricated through labeling a stem-loop Toxoplasma gondii DNA oligonucleotide with mQDs at the 5' end and BHQ-2 at 3' end, respectively, and the resulting sensing probe can be simply isolated and purified from the reactant with a common magnet. Properties of mQDs and sensing probe were determined by transmission electron microscopy (TEM) and fluorescence spectrum (FS). The TEM data demonstrated that the size of mQDs was ~20nm. the FS data indicated fluorescence intensity (FI) was doubled after the complete complimentary target Toxoplasma gondii DNA was introduced comparing with the FI before addition of target Toxoplasma gondii DNA. Moreover, only weak FI change was observed when the target DNA with one-mismatch base pair was added, this result revealed the sensing probe has high sensitivity and specificity. The current sensing probe will has great potential applications in the life science and related research.

  11. Sperm DNA damage and its relation with leukocyte DNA damage.

    PubMed

    Babazadeh, Zahra; Razavi, Shahnaz; Tavalaee, Marziyeh; Deemeh, Mohammad Reza; Shahidi, Maryam; Nasr-Esfahani, Mohammad Hossein

    2010-01-01

    DNA fragmentation in human sperm has been related to endogenous and exogenous factors. Exogenous factors can also affect leukocyte DNA integrity. This study evaluated the relation between sperm DNA damage and leukocyte DNA integrity, as a predictor of exogenous factors. DNA damage in the sperm and leukocytes of 41 individuals undergoing ICSI were measured by Comet assay. In addition, sperm chromatin dispersion (SCD) was carried out on semen samples. A positive correlation was observed between the DNA integrity of sperm with leukocytes. When patients were divided into low and high DNA exposure groups, sperm DNA fragmentation was significantly different between the two groups. Cleavage rate and embryo quality showed significant correlation with leukocyte DNA integrity. The results showed that leukocyte DNA integrity could be used to identify individuals at high risk in order to reduce the extent of DNA damage in patients before ICSI in order to improve the subsequent outcome of this procedure.

  12. Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups

    PubMed Central

    Licursi, Valerio; Brito, Catarina; La Torre, Mattia; Alves, Paula M.; Simao, Daniel; Mottini, Carla; Salinas, Sara; Negri, Rodolfo; Tagliafico, Enrico; Kremer, Eric J.; Saggio, Isabella

    2015-01-01

    Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR. PMID:26207738

  13. Specific oligonucleotide probes for in situ detection of a major group of gram-positive bacteria with low DNA G + C content.

    PubMed

    Meier, H; Amann, R; Ludwig, W; Schleifer, K H

    1999-05-01

    Almost one thousand 16S rRNA sequences of Gram-positive bacteria with a low DNA G + C content from public databases were analyzed using the ARB software package. A signature region was identified between positions 354 and 371 (E. coli numbering) for the Bacillus sub-branch of the Gram-positive bacteria with a low DNA G + C content, the former orders Bacillales and Lactobacillales. Three oligonucleotide probes, namely LGC354A, LGC354B, and LGC354C, were designed to target this diagnostic site. Their fluorescent derivatives were suitable for whole cell detection by fluorescence in situ hybridization (FISH). Hybridization conditions were adjusted for differentiation of target and related non-target reference species. When applying FISH to whole bacterial cells in a sample of activated sludge from a communal wastewater treatment plant, members of the Bacillus sub-branch were detected at levels from 0.01% of cells in samples fixed with paraformaldehyde to over 8 percent in the same samples fixed with ethanol and treated with lysozyme. The problems of quantitative in situ analysis of Gram-positive bacteria with a low DNA G + C content in biofilm flocs are discussed and recommendations made. Members of the Bacillus sub-branch were detected in different abundances in activated sludge samples from different wastewater plants.

  14. DNA Nanotechnology-- Architectures Designed with DNA

    NASA Astrophysics Data System (ADS)

    Han, Dongran

    As the genetic information storage vehicle, deoxyribonucleic acid (DNA) molecules are essential to all known living organisms and many viruses. It is amazing that such a large amount of information about how life develops can be stored in these tiny molecules. Countless scientists, especially some biologists, are trying to decipher the genetic information stored in these captivating molecules. Meanwhile, another group of researchers, nanotechnologists in particular, have discovered that the unique and concise structural features of DNA together with its information coding ability can be utilized for nano-construction efforts. This idea culminated in the birth of the field of DNA nanotechnology which is the main topic of this dissertation. The ability of rationally designed DNA strands to self-assemble into arbitrary nanostructures without external direction is the basis of this field. A series of novel design principles for DNA nanotechnology are presented here, from topological DNA nanostructures to complex and curved DNA nanostructures, from pure DNA nanostructures to hybrid RNA/DNA nanostructures. As one of the most important and pioneering fields in controlling the assembly of materials (both DNA and other materials) at the nanoscale, DNA nanotechnology is developing at a dramatic speed and as more and more construction approaches are invented, exciting advances will emerge in ways that we may or may not predict.

  15. Circular DNA by "Bis-Click" Ligation: Template-Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides.

    PubMed

    Yang, Haozhe; Seela, Frank

    2016-01-22

    A highly effective and convenient "bis-click" strategy was developed for the template-independent circularization of single-stranded oligonucleotides by employing copper(I)-assisted azide-alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7-deaza-2'-deoxyadenosine and 2'-deoxyuridine residues with different side chains were used in solid-phase synthesis with phosphoramidite chemistry. The bis-click ligation of linear 9- to 36-mer oligonucleotides with 1,4-bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9-mers. Compared with linear duplexes, circular bis-click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis-click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides.

  16. Synthesis, spectral characterization, crystal structure and in vitro DNA/protein binding studies of phosphorous ylide palladacyclic complexes containing azide group.

    PubMed

    Karami, Kazem; Shirani-Sarmazeh, Zahra; Hosseini-Kharat, Mahboubeh; Lipkowski, Janusz; Saeidifar, Maryam

    2015-03-01

    The reaction between (4-nitrobenzoylmethylene)triphenylphosphorane Pd(II) complex [Pd{κ(2)(C,C)-C₆ H₄PPh₂C(H)CO(C₆ H₄NO₂-4)}(μ-Cl)]₂ and excess of NaN₃ resulted in the μ-N₃ bridged Pd(II) complex [Pd{κ(2)(C,C)-C₆H₄PPh₂C(H)CO(C₆ H₄NO₂-4)}(μ-N₃)]₂ (1), which underwent bridge cleavage reactions with monodentate ligands to afford the monomeric, neutral complexes [Pd{κ(2)(C,C)-C₆ H₄PPh₂C(H)CO(C₆ H₄NO₂-4)}N₃(L)] (L=Me₃Py (1a), PPh₃ (1b)). The complexes were identified and characterized by elemental analyses, infrared (IR), ((1))H, ((13))C{((1))H} and ((31))P{((1))H} NMR spectroscopy. The molecular structure of 1b was determined by single-crystal X-ray diffraction. The interactions of complexes with FS-DNA were investigated using UV absorption and fluorescence spectra. The results suggested that both complexes could interact with FS-DNA through the intercalation mode and follow the binding affinity order of 1a>1b. The reactivity toward protein BSA revealed that the quenching of BSA fluorescence by the two complexes are static quenching, and complex 1a exhibits a higher BSA-binding ability than the complex 1b.

  17. The katablepharids are a distant sister group of the Cryptophyta: A proposal for Katablepharidophyta divisio nova/ Kathablepharida phylum novum based on SSU rDNA and beta-tubulin phylogeny.

    PubMed

    Okamoto, Noriko; Inouye, Isao

    2005-08-01

    The katablepharids are a morphologically well-defined group of heterotrophic flagellates. Since their original description in 1939, they have been classified in the Cryptophyceae (Cryptophyta) based on their similar cell shape, flagellar orientation, and the presence of ejectisomes visible by light microscopy. However, electron microscopy suggests that the katablepharids are distinct from cryptomonads. A possible affinity with the Alveolata has been proposed which is mainly based on the resemblance of their feeding apparatus to the apical complex of the Apicomplexa or to the tentacles of the Ciliophora. In this study, we provide the first SSU rDNA and beta-tubulin molecular sequence data for two katablepharids: Katablepharis japonica sp. nov. and Leucocryptos marina. We reveal that the katablepharids are not closely related to the Alveolata; rather, phylogenetic reconstruction analyses of SSU rDNA and beta-tubulin suggest that the katablepharids are a distant sister group of the Cryptophyta. We therefore conclude that the katablepharids should be a group equivalent to the Cryptophyta and propose Katablepharidophyta divisio nova (ICBN)/Kathablepharida phylum novum (ICZN). PMID:16171184

  18. DNA-cell conjugates

    DOEpatents

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  19. DNA barcoding for plants.

    PubMed

    de Vere, Natasha; Rich, Tim C G; Trinder, Sarah A; Long, Charlotte

    2015-01-01

    DNA barcoding uses specific regions of DNA in order to identify species. Initiatives are taking place around the world to generate DNA barcodes for all groups of living organisms and to make these data publically available in order to help understand, conserve, and utilize the world's biodiversity. For land plants the core DNA barcode markers are two sections of coding regions within the chloroplast, part of the genes, rbcL and matK. In order to create high quality databases, each plant that is DNA barcoded needs to have a herbarium voucher that accompanies the rbcL and matK DNA sequences. The quality of the DNA sequences, the primers used, and trace files should also be accessible to users of the data. Multiple individuals should be DNA barcoded for each species in order to check for errors and allow for intraspecific variation. The world's herbaria provide a rich resource of already preserved and identified material and these can be used for DNA barcoding as well as by collecting fresh samples from the wild. These protocols describe the whole DNA barcoding process, from the collection of plant material from the wild or from the herbarium, how to extract and amplify the DNA, and how to check the quality of the data after sequencing.

  20. Using DNA-barcoding for sorting out protist species complexes: a case study of the Nebela tincta-collaris-bohemica group (Amoebozoa; Arcellinida, Hyalospheniidae).

    PubMed

    Kosakyan, Anush; Gomaa, Fatma; Mitchell, Edward A D; Heger, Thierry J; Lara, Enrique

    2013-05-01

    Species identification by means of morphology is often problematic in protists. Nebela tincta-collaris-bohemica (Arcellinida) is a species complex of small to medium-sized (ca.100 μm) testate amoebae common in peat bogs and forest soils. The taxonomic validity of characters used to define species within this group is debated and causes confusion in studies of biogeography, and applications in palaeoecology. We examined the relationship between morphological and genetic diversity within this species complex by combined analyses of light microscopy imaging and Cytochrome Oxidase Subunit 1(COI) sequences obtained from the same individual amoeba cells. Our goals were (1) to clarify the taxonomy and the phylogenetic relationships within this group, and (2) to evaluate if individual genotypes corresponded to specific morphotypes and the extent of phenotypic plasticity. We show here that small variations in test morphology that have been often overlooked by traditional taxonomy correspond to distinct haplotypes. We therefore revise the taxonomy of the group. We redefine Nebela tincta (Leidy) Kosakyan et Lara and N. collaris (Ehrenberg 1848) Kosakyan et Gomaa, change N. tincta var. rotunda Penard to N. rotunda (Penard 1890), describe three new species: N. guttata n. sp. Kosakyan et Lara, N. pechorensis n. sp. Kosakyan et Mitchell, and N. aliciae n. sp. Mitchell et Lara.

  1. Bipolar localization of the group II intron Ll.LtrB is maintained in Escherichia coli deficient in nucleoid condensation, chromosome partitioning and DNA replication.

    PubMed

    Beauregard, Arthur; Chalamcharla, Venkata R; Piazza, Carol Lyn; Belfort, Marlene; Coros, Colin J

    2006-11-01

    Group II introns are mobile genetic elements that invade their cognate intron-minus alleles via an RNA intermediate, in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. In Escherichia coli, retrotransposition of the lactococcal group II intron, Ll.LtrB, occurs preferentially within the Ori and Ter macrodomains of the E. coli chromosome. These macrodomains migrate towards the poles of the cell, where the intron-encoded protein, LtrA, localizes. Here we investigate whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron integration and LtrA distribution in E. coli. We thus examined these properties in the absence of the nucleoid-associated proteins H-NS, StpA and MukB, in variants of partitioning functions including the centromere-like sequence migS and the actin homologue MreB, as well as in the replication mutants DeltaoriC, seqA, tus and topoIV (ts). Although there were some dramatic fluctuations in retrotransposition levels in these hosts, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid-free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron-encoded protein at the poles of the cell.

  2. Immobilization of DNA in polyacrylamide gel for the manufacture of DNA and DNA-oligonucleotide microchips.

    SciTech Connect

    Proudnikov, D.; Timofeev, E.; Mirzabekov, A.; Center for Mechanistic Biology and Biotechnology; Engelhardt Inst. of Molecular Biology

    1998-05-15

    Activated DNA was immobilized in aldehyde-containing polyacrylamide gel for use in manufacturing the MAGIChip (microarrays of gel-immobilized compounds on a chip). First, abasic sites were generated in DNA by partial acidic depurination. Amino groups were then introduced into the abasic sites by reaction with ethylenediamine and reduction of the aldimine bonds formed. It was found that DNA could be fragmented at the site of amino group incorporation or preserved mostly unfragmented. In similar reactions, both amino-DNA and amino-oligonucleotides were attached through their amines to polyacrylamide gel derivatized with aldehyde groups. Single- and double-stranded DNA of 40 to 972 nucleotides or base pairs were immobilized on the gel pads to manufacture a DNA microchip. The microchip was hybridized with fluorescently labeled DNA-specific oligonucleotide probes. This procedure for immobilization of amino compounds was used to manufacture MAGIChips containing both DNA and oligonucleotides.

  3. The 1998-1999 collaborative exercises and proficiency testing program on DNA typing of the Spanish and Portuguese Working Group of the International Society for Forensic Genetics (GEP-ISFG).

    PubMed

    Gómez, J; Carracedo, A

    2000-10-01

    A total of 28 laboratories (labs) submitted results for the 1998 collaborative exercise and the proficiency testing program of the Spanish and Portuguese Working Group of the International Society for Forensic Genetics (GEP-ISFG) group. This number increased to 46 labs in 1999. Six bloodstains were submitted, each one with 200 microl soaked in cotton except the sample no. 6 submitted for DNA quantification which had 2 microl. One of the samples was a mixed stain. A paternity testing case and a criminal case in the 1998 trial (GEP'98) and two paternity testing cases in 1999 (GEP'99) were included and the statistical evaluation of the evidence was requested in both cases. In the GEP'99 trial, a theoretical paternity testing case was included. A total of 52 DNA genetic markers were used by the participants in the GEP'98 trial, which increased to 101 in GEP'99. Despite this increasing number of participating labs, results remained quite satisfactory. All the labs used PCR-based DNA polymorphisms with an increasing number of markers, obtaining good results. SLPs were used by a decreasing number of labs but the results indicated a good level of expertise despite the different protocols used. Good results were also obtained for mtDNA despite the difficulties presented by the samples due to the presence of length heteroplasmy in some samples in both trials. The detection of heteroplasmy should, however, be improved. Similar conclusions were reached for both, the paternity and the criminal case by all the labs. Common methodologies for the statistical evaluation of the paternity case were used and the paternity index and the probability of paternity (with an a priori value of 0.5) reported by most of the labs. Also, a great uniformity was found in the evaluation of the criminal case despite the lack of a specific hypothesis in the design of the exercise. Some errors in statistical programs or in calculations were detected in a theoretical paternity case included in the GEP

  4. Solution conformation of the (-)-trans-anti-5-methylchrysene-dG adduct opposite dC in a DNA duplex: DNA bending associated with wedging of the methyl group of 5-methylchrysene to the 3'-side of the modification site.

    PubMed

    Cosman, M; Xu, R; Hingerty, B E; Amin, S; Harvey, R G; Geacintov, N E; Broyde, S; Patel, D J

    1995-05-01

    This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(C1-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17-G18- A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG.dC 11-mer duplex]. This adduct is derived from the trans addition at C4 of (-)-anti-1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4-tetrahydro-5-met hylchrysen e [(-)-anti-5-MeCDE] to the N2 position of dG6 in this duplex sequence. The 5-methyl group is located adjacent to the MC(C4) binding site, with these groups juxtaposed in a sterically crowded bay region in the adduct duplex. The 5-methylchrysenyl and the nucleic acid exchangeable and nonexchangeable protons were assigned following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[MC]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the [MC]dG6.dC17 base pair and flanking dC5.dG18 and dC7.dG16 base pairs retain Watson-Crick alignments upon adduct formation. The aromatic chrysenyl ring is positioned in the minor groove of a right-handed B-DNA helix and stacks predominantly over the sugar of the dC17 residue across from it on the unmodified complementary strand. The chrysenyl ring points toward the 3'-end of the modified strand with its 5-methyl group inserting between the modified [MC]dG6.dC17 and dC7.dG16 base pairs. The adduct duplex bends by approximately 47 degrees as a result of the wedged insertion of the 5-methyl group from the minor groove face of the duplex. The solution structure of the (-)-trans-anti-[MC] dG.dC 11-mer duplex is compared with that of the corresponding (-)-trans-anti-[BP]dG.dC 11-mer [De los Santos et al. (1992

  5. Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron aI2.

    PubMed Central

    Moran, J V; Mecklenburg, K L; Sass, P; Belcher, S M; Mahnke, D; Lewin, A; Perlman, P

    1994-01-01

    Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X. Images PMID:8029012

  6. Noninvasive determination of fetal rh blood group, D antigen status by cell-free DNA analysis in maternal plasma: experience in a Brazilian population.

    PubMed

    Chinen, Paulo Alexandre; Nardozza, Luciano Marcondes Machado; Martinhago, Ciro Dresch; Camano, Luiz; Daher, Silvia; Pares, David Baptista da Silva; Minett, Thais; Araujo Júnior, Edward; Moron, Antonio Fernandes

    2010-11-01

    We evaluated the diagnostic accuracy of Rh blood group, D antigen (RHD) fetal genotyping, using real-time polymerase chain reaction in maternal blood samples, in a racially mixed population. We performed a prospective study conducted between January 2006 and December 2007, analyzing fetal RHD genotype in the plasma of 102 D- pregnant women by real-time polymerase chain reaction, targeting exons 7 and 10 of the RHD gene. Genotype results were compared with cord blood phenotype obtained after delivery or before the first intrauterine transfusion when necessary. Most of the participants (75.5%) were under 28 weeks of pregnancy, and 87.5% had at least one relative of black ancestry. By combining amplification of two exons, the accuracy of genotyping was 98%, sensitivity was 100%, and specificity was 92%. The positive likelihood ratio was 12.5, and the negative likelihood ratio was 0. The two false-positive cases were confirmed to be pseudogene RHD by real-time polymerase chain reaction. There were no differences between the patients with positive or negative Coombs test ( P = 0.479). Determination of fetal RHD status in maternal peripheral blood was highly sensitive in this racially mixed population and was not influenced by the presence of antierythrocyte antibodies.

  7. The Association of the Xeroderma Pigmentosum Group D DNA Helicase (XPD) with Transcription Factor IIH Is Regulated by the Cytosolic Iron-Sulfur Cluster Assembly Pathway.

    PubMed

    Vashisht, Ajay A; Yu, Clarissa C; Sharma, Tanu; Ro, Kevin; Wohlschlegel, James A

    2015-05-29

    Xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH (TFIIH) transcription complex and plays essential roles in transcription and nucleotide excision repair. Although iron-sulfur (Fe-S) cluster binding by XPD is required for activity, the process mediating Fe-S cluster assembly remains poorly understood. We recently identified a cytoplasmic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, CIAO1, and FAM96B that is required for the biogenesis of extramitochondrial Fe-S proteins including XPD. Here, we use XPD as a prototypical Fe-S protein to further characterize how Fe-S assembly is facilitated by the CIA targeting complex. Multiple lines of evidence indicate that this process occurs in a stepwise fashion in which XPD acquires a Fe-S cluster from the CIA targeting complex before assembling into TFIIH. First, XPD was found to associate in a mutually exclusive fashion with either TFIIH or the CIA targeting complex. Second, disrupting Fe-S cluster assembly on XPD by either 1) depleting cellular iron levels or 2) utilizing XPD mutants defective in either Fe-S cluster or CIA targeting complex binding blocks Fe-S cluster assembly and prevents XPD incorporation into TFIIH. Finally, subcellular fractionation studies indicate that the association of XPD with the CIA targeting complex occurs in the cytoplasm, whereas its association with TFIIH occurs largely in the nucleus where TFIIH functions. Together, these data establish a sequential assembly process for Fe-S assembly on XPD and highlight the existence of quality control mechanisms that prevent the incorporation of immature apoproteins into their cellular complexes.

  8. Solution confirmation of the (-)-trans-anti-5-Methylchrysene-dG adduct oppposite dC in a DNA duplex: DNA bending associated with wedging of the Methyl group of 5-Methylchrysene to the 3{prime}-side of the modification site

    SciTech Connect

    Cosman, M.; Patel, D.J.

    1995-05-09

    This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(Cl-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11){sm_bullet}d(G12-G13-T14-A15-G16-C17-G 18-A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG{sm_bullet}dC 11-mer duplex]. This adduct is derived from the trans addition at C{sup 4} of (-)-anti-1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4-tetrahydro-5-methylchrysene [(-)-anti-5-MeCDE] to the N{sup 2} position of dG6 in this duplex sequence. The 5-methyl group is located adjacent to the MC(C{sup 4}) binding site, with these groups juxtaposed in a sterically crowded bay region in the adduct duplex. The 5-methylchrysenyl and the nucleic acid exchangeable and nonexchangeable protons were assigned following analysis of two-dimensional NMR data sets in H{sub 2}O and D{sub 2}O buffer solution. The solution structure of the trans-anti-[MC]dG{sm_bullet}dC 11-mer duplex has been determined by incorporating DNA-DNA and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the [MC]dG6{sm_bullet}dC17 base pair and flanking dC5{sm_bullet}dG18 and dC7{sm_bullet}dG16 base pairs retain Watson-Crick alignments upon adduct formation. 61 refs., 9 figs., 2 tabs.

  9. Xeroderma pigmentosum group E and DDB2, a smaller subunit of damage-specific DNA binding protein: proposed classification of xeroderma pigmentosum, Cockayne syndrome, and ultraviolet-sensitive syndrome.

    PubMed

    Itoh, Toshiki

    2006-02-01

    Xeroderma pigmentosum is a rare photosensitive syndrome that comprises eight different genetic diseases (A to G; variant (V)). Although genotype-phenotype correlations have been evaluated in most XP groups, the relationship between the E subgroup of xeroderma pigmentosum (XP-E) and damage-specific DNA binding protein (DDB) still remained a mystery. Recent studies have provided new insight for XP-E and the role(s) of DDB2, a smaller subunit of DDB. Reclassification studies have confirmed that mutations in DDB2 give rise to XP-E. The mouse model of XP-E demonstrated that DDB2 was well conserved between mouse and human and was critical in controlling proper cell-survival through regulating the tumor suppressor p53-mediated responses after ultraviolet (UV)-irradiation: i.e. defective DDB2 causes the resistance to cell-killing by UV-irradiation due to decreased p53-mediated apoptosis. These phenotypes are unique to XP-E because other XP groups show normal (XP-V) or hypersensitivity (XP-A, B, C, D, F, and G) to UV-irradiation. Thus XP-E is defined as a skin cancer prone disease with unique resistance to UV-irradiation. PMID:16325378

  10. A high-mobility group box 1 that binds to DNA, enhances pro-inflammatory activity, and acts as an anti-infection molecule in black rockfish, Sebastes schlegelii.

    PubMed

    Xin-Peng, Zhao; Yong-Hua, Hu; Yong, Liu; Jing-Jing, Wang; Guang-Hua, Wang; Ren-Jie, Wang; Min, Zhang

    2016-09-01

    High-mobility group box (HMGB) 1 is a chromosomal protein that plays critical roles in DNA transcription, replication and repair. In addition, HMGB1 functions as a pro-inflammatory molecule in many vertebrates and invertebrates. In teleosts, very limited studies of HMGB1 have been reported. In this study, we identified a HMGB1 homologue (SsHMGB1) from black rockfish (Sebastes schlegelii) and analyzed its structure, expression and biological function. The open reading frame of SsHMGB1 is 621 bp, with a 5'-untranslated region (UTR) of 62 bp and a 3'-UTR of 645 bp. SsHMGB1 contains two typical HMG boxes and an acidic C-terminal tail. The deduced amino acid sequence of SsHMGB1 shares the highest overall identity (89.4%) with the HMGB1 of Anoplopoma fimbria. The expression of SsHMGB1 occurred in multiple tissues and was highest in the brain. Moreover, the mRNA level of SsHMGB1 in head kidney (HK) macrophages could be induced by Listonella anguillarum in a time-dependent manner. Recombinant SsHMGB1 purified from Escherichia coli (i) bound DNA fragments in a dose-dependent manner; and (ii) induced the expression of cytokines in HK macrophages, including a significant increase in TNF-α activity and enhanced mRNA level of TNF13B and IL-1 β, which are known to be involved in antibacterial defense; moreover, (iii) significantly improved the macrophage bactericidal activity together with reduced pathogen dissemination and replication of bacteria in fish kidney. These results indicated that SsHMGB1 is a novel HMGB1 that possesses apparent immunoregulatory properties and is likely to be involved in fighting bacterial infection. PMID:27492120

  11. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  12. DNA Banking

    SciTech Connect

    Reilly, P.R. )

    1992-11-01

    The author is involved in the ethical, legal, and social issues of banking of DNA and data from DNA analysis. In his attempt to determine the extent of DNA banking in the U.S., the author surveyed some commercial companies performing DNA banking services. This article summarizes the results of that survey, with special emphasis on the procedures the companies use to protect the privacy of individuals. 4 refs.

  13. DNA-DNA hybridization study of strains of Chryseobacterium, Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc, IIe, IIh and IIi, mostly from human clinical sources, and proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov.

    PubMed

    Holmes, B; Steigerwalt, A G; Nicholson, A C

    2013-12-01

    The taxonomic classification of 182 phenotypically similar isolates was evaluated using DNA-DNA hybridization and 16S rRNA gene sequence analysis. These bacterial isolates were mainly derived from clinical sources; all were Gram-negative non-fermenters and most were indole-producing. Phenotypically, they resembled species from the genera Chryseobacterium, Elizabethkingia or Empedobacter or belonged to CDC groups IIc, IIe, IIh and IIi. Based on these analyses, four novel species are described: Chryseobacterium bernardetii sp. nov. (type strain NCTC 13530(T) = CCUG 60564(T) = CDC G229(T)), Chryseobacterium carnis sp. nov. (type strain NCTC 13525(T) = CCUG 60559(T) = CDC G81(T)), Chryseobacterium lactis sp. nov. (type strain NCTC 11390(T) = CCUG 60566(T) = CDC KC1864(T)) and Chryseobacterium nakagawai sp. nov. (type strain NCTC 13529(T) = CCUG 60563(T) = CDC G41(T)). The new combination Chryseobacterium taklimakanense comb. nov. (type strain NCTC 13490(T) = X-65(T) = CCTCC AB 208154(T) = NRRL B-51322(T)) is also proposed to accommodate the reclassified Planobacterium taklimakanense.

  14. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  15. Molecular and Cellular Analysis of the DNA Repair Defect in a Patient in Xeroderma Pigmentosum Complementation Group D Who Has the Clinical Features of Xeroderma Pigmentosum and Cockayne Syndrome

    PubMed Central

    Broughton, B. C.; Thompson, A. F.; Harcourt, S. A.; Vermeulen, W.; Hoeijmakers, J. H. J.; Botta, E.; Stefanini, M.; King, M. D.; Weber, C. A.; Cole, J.; Arlett, C. F.; Lehmann, A. R.

    1995-01-01

    Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30%–40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly→arg change at amino acid 675 in the allele inherited from the patient's mother and a −1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in patients with trichothiodystrophy. ImagesFigure 3Figure 5 PMID:7825573

  16. DNA mini-barcodes.

    PubMed

    Hajibabaei, Mehrdad; McKenna, Charly

    2012-01-01

    Conventional DNA barcoding uses an approximately 650 bp DNA barcode of the mitochondrial gene COI for species identification in animal groups. Similar size fragments from chloroplast genes have been proposed as barcode markers for plants. While PCR amplification and sequencing of a 650 bp fragment is consistent in freshly collected and well-preserved specimens, it is difficult to obtain a full-length barcode in older museum specimens and samples which have been preserved in formalin or similar DNA-unfriendly preservatives. A comparable issue may prevent effective DNA-based authentication and testing in processed biological materials, such as food products, pharmaceuticals, and nutraceuticals. In these cases, shorter DNA sequences-mini-barcodes-have been robustly recovered and shown to be effective in identifying majority of specimens to a species level. Furthermore, short DNA regions can be utilized via high-throughput sequencing platforms providing an inexpensive and comprehensive means of large-scale species identification. These properties of mini-barcodes, coupled with the availability of standardized and universal primers make mini-barcodes a feasible option for DNA barcode analysis in museum samples and applied diagnostic and environmental biodiversity analysis.

  17. Cinnamate-based DNA photolithography.

    PubMed

    Feng, Lang; Romulus, Joy; Li, Minfeng; Sha, Ruojie; Royer, John; Wu, Kun-Ta; Xu, Qin; Seeman, Nadrian C; Weck, Marcus; Chaikin, Paul

    2013-08-01

    As demonstrated by means of DNA nanoconstructs, as well as DNA functionalization of nanoparticles and micrometre-scale colloids, complex self-assembly processes require components to associate with particular partners in a programmable fashion. In many cases the reversibility of the interactions between complementary DNA sequences is an advantage. However, permanently bonding some or all of the complementary pairs may allow for flexibility in design and construction. Here, we show that the substitution of a cinnamate group for a pair of complementary bases provides an efficient, addressable, ultraviolet light-based method to bond complementary DNA covalently. To show the potential of this approach, we wrote micrometre-scale patterns on a surface using ultraviolet light and demonstrated the reversible attachment of conjugated DNA and DNA-coated colloids. Our strategy enables both functional DNA photolithography and multistep, specific binding in self-assembly processes.

  18. Cinnamate-based DNA photolithography

    PubMed Central

    Romulus, Joy; Li, Minfeng; Sha, Ruojie; Royer, John; Wu, Kun-Ta; Xu, Qin

    2013-01-01

    As demonstrated by means of DNA nanoconstructs[1], as well as DNA functionalization of nanoparticles[2-4] and micrometre-scale colloids[5-8], complex self-assembly processes require components to associate with particular partners in a programmable fashion. In many cases the reversibility of the interactions between complementary DNA sequences is an advantage[9]. However, permanently bonding some or all of the complementary pairs may allow for flexibility in design and construction[10]. Here, we show that the substitution of a pair of complementary bases by a cinnamate group provides an efficient, addressable, UV light-based method to covalently bond complementary DNA. To show the potential of this approach, we wrote micrometre-scale patterns on a surface via UV light and demonstrate the reversible attachment of conjugated DNA and DNA-coated colloids. Our strategy enables both functional DNA photolithography and multi-step, specific binding in self-assembly processes. PMID:23685865

  19. Chilean Pitavia more closely related to Oceania and Old World Rutaceae than to Neotropical groups: evidence from two cpDNA non-coding regions, with a new subfamilial classification of the family

    PubMed Central

    Groppo, Milton; Kallunki, Jacquelyn A.; Pirani, José Rubens; Antonelli, Alexandre

    2012-01-01

    Abstract The position of the plant genus Pitavia within an infrafamilial phylogeny of Rutaceae (rue, or orange family) was investigated with the use of two non-coding regions from cpDNA, the trnL-trnF region and the rps16 intron. The only species of the genus, Pitavia punctata Molina, is restricted to the temperate forests of the Coastal Cordillera of Central-Southern Chile and threatened by loss of habitat. The genus traditionally has been treated as part of tribe Zanthoxyleae (subfamily Rutoideae) where it constitutes the monogeneric tribe Pitaviinae. This tribe and genus are characterized by fruits of 1 to 4 fleshy drupelets, unlike the dehiscent fruits typical of the subfamily. Fifty-five taxa of Rutaceae, representing 53 genera (nearly one-third of those in the family) and all subfamilies, tribes, and almost all subtribes of the family were included. Parsimony and Bayesian inference were used to infer the phylogeny; six taxa of Meliaceae, Sapindaceae, and Simaroubaceae, all members of Sapindales, were also used as out-groups. Results from both analyses were congruent and showed Pitavia as sister to Flindersia and Lunasia, both genera with species scattered through Australia, Philippines, Moluccas, New Guinea and the Malayan region, and phylogenetically far from other Neotropical Rutaceae, such as the Galipeinae (Galipeeae, Rutoideae) and Pteleinae (Toddalieae, former Toddalioideae). Additionally, a new circumscription of the subfamilies of Rutaceae is presented and discussed. Only two subfamilies (both monophyletic) are recognized: Cneoroideae (including Dictyolomatoideae, Spathelioideae, Cneoraceae, and Ptaeroxylaceae) and Rutoideae (including not only traditional Rutoideae but also Aurantioideae, Flindersioideae, and Toddalioideae). As a consequence, Aurantioideae (Citrus and allies) is reduced to tribal rank as Aurantieae. PMID:23717188

  20. Patterning nanocrystals using DNA

    SciTech Connect

    Williams, Shara Carol

    2003-09-01

    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices to a length greater than

  1. DNA nanomachines.

    PubMed

    Bath, Jonathan; Turberfield, Andrew J

    2007-05-01

    We are learning to build synthetic molecular machinery from DNA. This research is inspired by biological systems in which individual molecules act, singly and in concert, as specialized machines: our ambition is to create new technologies to perform tasks that are currently beyond our reach. DNA nanomachines are made by self-assembly, using techniques that rely on the sequence-specific interactions that bind complementary oligonucleotides together in a double helix. They can be activated by interactions with specific signalling molecules or by changes in their environment. Devices that change state in response to an external trigger might be used for molecular sensing, intelligent drug delivery or programmable chemical synthesis. Biological molecular motors that carry cargoes within cells have inspired the construction of rudimentary DNA walkers that run along self-assembled tracks. It has even proved possible to create DNA motors that move autonomously, obtaining energy by catalysing the reaction of DNA or RNA fuels.

  2. Recombinant DNA means and method

    SciTech Connect

    Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.

    1987-05-19

    This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.

  3. Training Groups, Encounter Groups, Sensitivity Groups and Group Psychotherapy

    PubMed Central

    Gottschalk, Louis A.; Pattison, E. Mansell; Schafer, Donald W.

    1971-01-01

    Descriptions and comparison of group therapies and the new group procedures (training groups and sensitivity groups—an outgrowth of the so-called Laboratory Movement methods of the mid-1930's) have been provided for the better understanding of non-psychiatric physicians. A group leader must have proper training and must help his group in its search for its avowed goals, whether he is a group therapist, a sensitivity trainer, or anyone else interested in utilizing group processes. Those goals are either the therapeutic benefit of the individual, as defined in group psychotherapy, or a better understanding of how one functions in groups, as in T-groups or the other group processes in the area of sensitive living. All group situations contain powerful tools which must be handled with proper respect. When so handled by experienced leaders, the individuals involved can achieve their goals in these group experiences. PMID:18730582

  4. Group Counseling

    ERIC Educational Resources Information Center

    Mahler, Clarence A.

    1971-01-01

    This article reviews the major concerns of group counseling and differentiates among group guidance, group counseling, and group therapy. It also evaluates the research status of group counseling and presents implications for the future of this approach. Comment by Carl E. Thoresen follows. (Author)

  5. [DNA computing].

    PubMed

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  6. Deciphering the Positional Influence of the Hydroxyl Group in the Cinnamoyl Part of 3-Hydroxy Flavonoids for Structural Modification and Their Interaction with the Protonated and B Form of Calf Thymus DNA Using Spectroscopic and Molecular Modeling Studies.

    PubMed

    Pradhan, Ankur Bikash; Haque, Lucy; Bhuiya, Sutanwi; Ganguly, Aniruddha; Das, Suman

    2015-06-11

    Studies on the interaction of naturally occurring flavonoids with different polymorphic forms of nucleic acid are helpful for understanding the molecular aspects of binding mode and providing direction for the use and design of new efficient therapeutic agents. However, much less information is available on the interactions of these compounds with different polymorphic forms of DNA at the molecular level. In this report we investigated the interaction of two widely abundant dietary flavonoids quercetin (Q) and morin (M) with calf thymus (CT) DNA. Spectrophotometric, spectropolarimetric, viscosity measurement, and molecular docking simulation methods are used as tools to delineate the binding mode and probable location of the flavonoids and their effects on the stability and conformation of DNA. It is observed that in the presence of the protonated form of DNA the dual fluorescence of Q and M resulting from the excited-state intramolecular proton transfer (ESIPT) is modified significantly. Structural analysis showed Q and M binds weakly to the B form (groove binding) compared to the protonated form of CT DNA (electrostatic interaction). In both cases, Q binds strongly to both forms of DNA compared to M.

  7. Dancing DNA.

    ERIC Educational Resources Information Center

    Pennisi, Elizabeth

    1991-01-01

    An imaging technique that uses fluorescent dyes and allows scientists to track DNA as it moves through gels or in solution is described. The importance, opportunities, and implications of this technique are discussed. (KR)

  8. DNA Dynamics.

    ERIC Educational Resources Information Center

    Warren, Michael D.

    1997-01-01

    Explains a method to enable students to understand DNA and protein synthesis using model-building and role-playing. Acquaints students with the triplet code and transcription. Includes copies of the charts used in this technique. (DDR)

  9. DNA Adductomics

    PubMed Central

    2015-01-01

    Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the 32P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC–MSn), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC–MSn instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology. PMID:24437709

  10. What Is Mitochondrial DNA?

    MedlinePlus

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  11. Group X

    SciTech Connect

    Fields, Susannah

    2007-08-16

    This project is currently under contract for research through the Department of Homeland Security until 2011. The group I was responsible for studying has to remain confidential so as not to affect the current project. All dates, reference links and authors, and other distinguishing characteristics of the original group have been removed from this report. All references to the name of this group or the individual splinter groups has been changed to 'Group X'. I have been collecting texts from a variety of sources intended for the use of recruiting and radicalizing members for Group X splinter groups for the purpose of researching the motivation and intent of leaders of those groups and their influence over the likelihood of group radicalization. This work included visiting many Group X websites to find information on splinter group leaders and finding their statements to new and old members. This proved difficult because the splinter groups of Group X are united in beliefs, but differ in public opinion. They are eager to tear each other down, prove their superiority, and yet remain anonymous. After a few weeks of intense searching, a list of eight recruiting texts and eight radicalizing texts from a variety of Group X leaders were compiled.

  12. Group Flow and Group Genius

    ERIC Educational Resources Information Center

    Sawyer, Keith

    2015-01-01

    Keith Sawyer views the spontaneous collaboration of group creativity and improvisation actions as "group flow," which organizations can use to function at optimum levels. Sawyer establishes ideal conditions for group flow: group goals, close listening, complete concentration, being in control, blending egos, equal participation, knowing…

  13. Ancient DNA.

    PubMed

    Willerslev, Eske; Cooper, Alan

    2005-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets.

  14. Ancient DNA

    PubMed Central

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets. PMID:15875564

  15. DNA vaccines

    NASA Astrophysics Data System (ADS)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  16. DNA topoisomerases.

    PubMed

    Wang, J C

    1996-01-01

    The various problems of disentangling DNA strands or duplexes in a cell are all rooted in the double-helical structure of DNA. Three distinct subfamilies of enzymes, known as the DNA topoisomerases, have evolved to solve these problems. This review focuses on work in the past decade on the mechanisms and cellular functions of these enzymes. Newly discovered members and recent biochemical and structural results are reviewed, and mechanistic implications of these results are summarized. The primary cellular functions of these enzymes, including their roles in replication, transcription, chromosome condensation, and the maintenance of genome stability, are then discussed. The review ends with a summary of the regulation of the cellular levels of these enzymes and a discussion of their association with other cellular proteins.

  17. Isopermutation group

    SciTech Connect

    Muktibodh, A. S.

    2015-03-10

    The concept of ‘Isotopy’ as formulated by Ruggero Maria Santilli [1, 2, 3] plays a vital role in the development of Iso mathematics. Santilli defined iso-fields of characteristic zero. In this paper we extend this definition to define Iso-Galois fields [4] which are essentially of non-zero characteristic. Isotopically isomorphic realizations of a group define isopermutation group which gives a clear cut distinction between automorphic groups and isotopic groups.

  18. Hot Groups.

    ERIC Educational Resources Information Center

    Vail, Kathleen

    1996-01-01

    Collaborators sparked by creative ideas and obsessed by a common task may not realize they're part of a "hot group"--a term coined by business professors Harold J. Leavitt and Jean Lipman-Blumen. Spawned by group decision making and employee empowerment, hot groups can flourish in education settings. They're typically small, short lived, and goal…

  19. Linking two DNA duplexes with a rigid linker for DNA nanotechnology

    PubMed Central

    Tashiro, Ryu; Iwamoto, Masahiro; Morinaga, Hironobu; Emura, Tomoko; Hidaka, Kumi; Endo, Masayuki; Sugiyama, Hiroshi

    2015-01-01

    DNA has recently emerged as a promising material for the construction of nanosized architectures. Chemically modified DNA has been suggested to be an important component of such architectural building blocks. We have designed and synthesized a novel H-shaped DNA oligonucleotide dimer that is cross-linked with a structurally rigid linker composed of phenylene and ethynylene groups. A rotatable DNA unit was constructed through the self-assembly of this H-shaped DNA component and two complementary DNA oligonucleotides. In addition to the rotatable unit, a locked DNA unit containing two H-shaped DNA components was also constructed. As an example of an extended locked structure, a hexagonal DNA origami dimer and oligomer were constructed by using H-shaped DNA as linkers. PMID:26130712

  20. DNA Looping, Supercoiling and Tension

    NASA Astrophysics Data System (ADS)

    Finzi, Laura

    2007-11-01

    In complex organisms, activation or repression of gene expression by proteins bound to enhancer or silencer elements located several kilobases away from the promoter is a well recognized phenomenon. However, a mechanistic understanding of any of these multiprotein interactions is still incomplete. Part of the difficulty in characterizing long-range interactions is the complexity of the regulatory systems and also an underestimation of the effect of DNA supercoiling and tension. Supercoiling is expected to promote interactions between DNA sites because it winds the DNA into compact plectonemes in which distant DNA segments more frequently draw close. The idea that DNA is also under various levels of tension is becoming more widely accepted. Forces that stretch the double helix in vivo are the electrostatic repulsion among the negatively charged phosphate groups along the DNA backbone, the action of motor enzymes perhaps acting upon a topologically constrained sequence of DNA or chromosome segregation during cell mitosis following DNA replication. Presently, little is known about the tension acting on DNA in vivo, but characterization of how physiological regulatory processes, such as loop formation, depend on DNA tension in vitro will indicate the stretching force regimes likely to exist in vivo. In this light, the well studied CI protein of bacteriophage l, which was recently found to cause a of 3.8 kbp loop in DNA, is an ideal system in which to characterize long-range gene regulation. The large size of the loop lends itself to single-molecule techniques, which allow characterization of the dynamics of CI-mediated l DNA looping under controlled levels of supercoiling and tension. Such experiments are being used to discover the principles of long-range interactions in l and in more complex systems.

  1. Galaxy groups

    SciTech Connect

    Brent Tully, R.

    2015-02-01

    Galaxy groups can be characterized by the radius of decoupling from cosmic expansion, the radius of the caustic of second turnaround, and the velocity dispersion of galaxies within this latter radius. These parameters can be a challenge to measure, especially for small groups with few members. In this study, results are gathered pertaining to particularly well-studied groups over four decades in group mass. Scaling relations anticipated from theory are demonstrated and coefficients of the relationships are specified. There is an update of the relationship between light and mass for groups, confirming that groups with mass of a few times 10{sup 12}M{sub ⊙} are the most lit up while groups with more and less mass are darker. It is demonstrated that there is an interesting one-to-one correlation between the number of dwarf satellites in a group and the group mass. There is the suggestion that small variations in the slope of the luminosity function in groups are caused by the degree of depletion of intermediate luminosity systems rather than variations in the number per unit mass of dwarfs. Finally, returning to the characteristic radii of groups, the ratio of first to second turnaround depends on the dark matter and dark energy content of the universe and a crude estimate can be made from the current observations of Ω{sub matter}∼0.15 in a flat topology, with a 68% probability of being less than 0.44.

  2. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  3. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  4. Forensic DNA typing in China.

    PubMed

    Hou, Y P

    2009-04-01

    In the field of forensic genetics, essential developmental impulses come from the advances of the molecular biology and human genome projects. This paper overviews existing technologies for forensic genetics in China and gives a perspective of forensic DNA analysis. In China, work has been done in the development of blood group serology of the conventional markers. Forensic scientists in China also contributed to the progress of DNA analysis by the validation of numerous test methods and by optimization of these methods. During these years, forensic DNA analysis in China has experienced tremendous progress towards development of robust, efficient and precise protocols, including the development of short tandem repeat analysis, mitochondrial DNA and Y-chromosome analysis. Forensic scientists are constantly looking for new methods to further improve DNA typing. Therefore, this paper also focuses on emerging new technologies in China, which represent an interest for forensic genetics.

  5. GROUP INEQUALITY

    PubMed Central

    Bowles, Samuel; Loury, Glenn C.; Sethi, Rajiv

    2014-01-01

    We explore the combined effect of segregation in social networks, peer effects, and the relative size of a historically disadvantaged group on the incentives to invest in market-rewarded skills and the dynamics of inequality between social groups. We identify conditions under which group inequality will persist in the absence of differences in ability, credit constraints, or labor market discrimination. Under these conditions, group inequality may be amplified even if initial group differences are negligible. Increases in social integration may destabilize an unequal state and make group equality possible, but the distributional and human capital effects of this depend on the demographic composition of the population. When the size of the initially disadvantaged group is sufficiently small, integration can lower the long-run costs of human capital investment in both groups and result in an increase the aggregate skill share. In contrast, when the initially disadvantaged group is large, integration can induce a fall in the aggregate skill share as the costs of human capital investment rise in both groups. We consider applications to concrete cases and policy implications. PMID:25554727

  6. Modified DNA aptamers against sweet agent aspartame.

    PubMed

    Saitoh, Hiroshi; Nakamura, Akiko; Kuwahara, Masayasu; Ozaki, Hiroaki; Sawai, Hiroaki

    2002-01-01

    We obtained a modified DNA aptamer against sweetener, aspartame, by in vitro selection method. The modified DNA was prepared from dATP, dGTP, dCTP and a modified dTTP bearing a terminal amino group at C-5 position in place of thymidine by PCR using a hyper thermophilic DNA polymerase, KOD Dash DNA polymerase. The synthetic 102-mer DNA with a 60-mer random region was used as an initial template for the PCR. The PCR-amplified modified DNA library was applied to an aspartame-agarose column, and then the bound modified DNA was eluted from the column for the affinity chromatography selection. Repeating the procedure, we selected the modified DNA aptamer against aspartame.

  7. PNA beacons for duplex DNA.

    PubMed

    Kuhn, H; Demidov, V V; Gildea, B D; Fiandaca, M J; Coull, J C; Frank-Kamenetskii, M D

    2001-08-01

    We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics.

  8. DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks

    PubMed Central

    Gan, Rui; Wu, Xiaolin; He, Wei; Liu, Zhenhua; Wu, Shuangju; Chen, Chao; Chen, Si; Xiang, Qianrong; Deng, Zixin; Liang, Dequan; Chen, Shi; Wang, Lianrong

    2014-01-01

    The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system. PMID:25319634

  9. Differential requirement for the ATPase domain of the Cockayne syndrome group B gene in the processing of UV-induced DNA damage and 8-oxoguanine lesions in human cells.

    PubMed

    Selzer, Rebecca R; Nyaga, Simon; Tuo, Jingsheng; May, Alfred; Muftuoglu, Meltem; Christiansen, Mette; Citterio, Elisabetta; Brosh, Robert M; Bohr, Vilhelm A

    2002-02-01

    Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by UV sensitivity, developmental abnormalities and premature aging. The cellular and molecular phenotypes of CS include increased sensitivity to oxidative and UV-induced DNA lesions. The CSB protein is thought to play a pivotal role in transcription-coupled repair and CS-B cells are defective in the repair of the transcribed strand of active genes, both after exposure to UV and in the presence of oxidative DNA lesions. A previous study has indicated that a conserved helicase ATPase motif II residue is essential for the function of the CSB protein in responding to UV-induced DNA damage in a hamster cell line. Due to the limitations in studying a complex human disorder in another species, this study introduced the site-directed mutation of the ATPase motif II in the human CSB gene in an isogenic human cell line. The CSB mutant allele was tested for genetic complementation of UV-sensitive phenotypes in the human CS-B cell line CS1AN.S3.G2. In addition, the incision of an 8-oxoguanine lesion by extracts of the CS-B cell lines stably transfected with the wild-type or ATPase mutant CSB gene has been investigated. The ATPase motif II point mutation (E646Q) abolished the function of the CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery and apoptosis. Interestingly, whole-cell extract prepared from these mutant cells retained wild-type incision activity on an oligonucleotide containing a single 8-oxoguanine lesion, whereas the absence of the CSB gene altogether resulted in reduced incision activity relative to wild-type. These results suggest damage-specific functional requirements for CSB in the repair of UV-induced and oxidative lesions in human cells. The transfection of the mutant or wild-type CSB gene into the CS1AN.S3.G2 cells did not alter the expression of the subset of genes examined by cDNA array analysis. PMID:11809892

  10. Group Grammar

    ERIC Educational Resources Information Center

    Adams, Karen

    2015-01-01

    In this article Karen Adams demonstrates how to incorporate group grammar techniques into a classroom activity. In the activity, students practice using the target grammar to do something they naturally enjoy: learning about each other.

  11. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  12. Group dynamics.

    PubMed

    Scandiffio, A L

    1990-12-01

    Group dynamics play a significant role within any organization, culture, or unit. The important thing to remember with any of these structures is that they are made up of people--people with different ideas, motivations, background, and sometimes different agendas. Most groups, formal or informal, look for a leader in an effort to maintain cohesiveness of the unit. At times, that cultural bond must be developed; once developed, it must be nurtured. There are also times that one of the group no longer finds the culture comfortable and begins to act out behaviorally. It is these times that become trying for the leader as she or he attempts to remain objective when that which was once in the building phase of group cohesiveness starts to fall apart. At all times, the manager must continue to view the employee creating the disturbance as an integral part of the group. It is at this time that it is beneficial to perceive the employee exhibiting problem behaviors as a special employee, as one who needs the benefit of your experience and skills, as one who is still part of the group. It is also during this time that the manager should focus upon her or his own views in the area of power, communication, and the corporate culture of the unit that one has established before attempting to understand another's point of view. Once we understand our own motivation and accept ourselves, it is then that we may move on to offer assistance to another. Once we understand our insecurities recognizing staff dysfunction as a symptom of system dysfunction will not be so threatening to the concept of the manager that we perceive ourselves to be. It takes a secure person to admit that she or he favors staff before deciding to do something to change things. The important thing to know is that it can be done. The favored staff can find a new way of relating to others, the special employee can find new modes of behavior (and even find self-esteem in the process), the group can find new ways

  13. Comparison of three DNA extraction methods for recovery of soil protist DNA.

    PubMed

    Santos, Susana S; Nielsen, Tue Kjærgaard; Hansen, Lars H; Winding, Anne

    2015-08-01

    The use of molecular methods to investigate protist communities in soil is in rapid development this decade. Molecular analysis of soil protist communities is usually dependant on direct genomic DNA extraction from soil and inefficient or differential DNA extraction of protist DNA can lead to bias in downstream community analysis. Three commonly used soil DNA extraction methods have been tested on soil samples from three European Long-Term Observatories (LTOs) with different land-use and three protist cultures belonging to different phylogenetic groups in different growth stages. The methods tested were: ISOm-11063 (a version of the ISO-11063 method modified to include a FastPrep ®-24 mechanical lysis step), GnS-GII (developed by the GenoSol platform to extract soil DNA in large-scale soil surveys) and a commercial DNA extraction kit - Power Lyzer™ PowerSoil® DNA Isolation Kit (MoBio). DNA yield and quality were evaluated along with DNA suitability for amplification of 18S rDNA fragments by PCR. On soil samples, ISOm-11063 yields significantly higher DNA for two of the three soil samples, however, MoBio extraction favors DNA quality. This method was also more effective to recover copies of 18S rDNA numbers from all soil types. In addition and despite the lower yields, higher DNA quality was observed with DNA extracted from protist cultures with the MoBio method. Likewise, a bead-beating step shows to be a good solution for DNA extraction of soil protists, since the recovery of DNA from protist cultures and from the different soil samples with the ISOm method proved to be efficient in recovering PCR-amplifiable DNA. This study showed that soil DNA extraction methods provide biased results towards the cyst stages of protist organism.

  14. Optical DNA

    NASA Astrophysics Data System (ADS)

    Vijaywargi, Deepak; Lewis, Dave; Kirovski, Darko

    A certificate of authenticity (COA) is an inexpensive physical object with a random and unique structure S which is hard to near-exactly replicate. An inexpensive device should be able to scan object’s physical “fingerprint,” a set of features that represents S. In this paper, we explore one set of requirements that optical media such as DVDs should satisfy, to be considered as COAs. As manufacturing of such media produces inevitable errors, we use the locations and count of these errors as a “fingerprint” for each optical disc: its optical DNA. The “fingerprint” is signed using publisher’s private-key and the resulting signature is stored onto the optical medium using a post-production process. Standard DVD players with altered firmware that includes publisher’s public-key, should be able to verify the authenticity of DVDs protected with optical DNA. Our key finding is that for the proposed protocol, only DVDs with exceptional wear-and-tear characteristics would result in an inexpensive and viable anti-counterfeiting technology.

  15. Group Learning.

    ERIC Educational Resources Information Center

    Black, Susan

    1992-01-01

    Research suggests that cooperative learning works best when students are first taught group-processing skills, such as leadership, decision making, communication, trust building, and conflict management. Inadequate teacher training and boring assignments can torpedo cooperative learning efforts. Administrators should reassure teachers with…

  16. Alkali lability of bacteriophage phi W-14 DNA.

    PubMed

    Lewis, H A; Miller, R C; Stone, J C; Warren, R A

    1975-12-01

    The molecular weight of bacteriophage phi W-14 DNA, determined by velocity sedimentation in neutral sucrose gradients, was 92 +/- 6 X 10(6). The DNA showed marked fragmentation in alkaline sucrose gradients. This fragmentation was not a consequence of preexisting single-strand interruptions in the DNA, since thermal denaturation of DNA yielded intact single strands. The alpha-putrescinylthymine groups in phi W-14 DNA appeared to be labile; some, or parts of some, of these groups were cleaved from the DNA in alkali. PMID:1202241

  17. DNA denaturation in the rodlike polyelectrolyte model

    NASA Astrophysics Data System (ADS)

    Passos, C. B.; Kuhn, P. S.; Barbosa, M. C.

    2014-11-01

    The denaturation of the DNA is analyzed using an analytic model. The DNA molecules are described in the Primitive Model of Polyelectrolytes (PMP), where the polyelectrolyte molecules are cylinders with charged sites. We show that the DNA stabilization arises as the result of the competition between the electrostatic repulsion of the phosphate groups and the attractive forces of the H-bonds. We also show that the addition of salt in the system screens the electrostatic interactions and favors the double strand configuration.

  18. Electronic transport in methylated fragments of DNA

    SciTech Connect

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L. Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

    2015-11-16

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  19. Electronic transport in methylated fragments of DNA

    NASA Astrophysics Data System (ADS)

    de Almeida, M. L.; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; de Moura, F. A. B. F.; Lyra, M. L.

    2015-11-01

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  20. Nanomechanical molecular devices made of DNA origami.

    PubMed

    Kuzuya, Akinori; Ohya, Yuichi

    2014-06-17

    CONSPECTUS: Eight years have passed since the striking debut of the DNA origami technique ( Rothemund, P. W. K. Nature 2006 , 440 , 297 - 302 ), in which long single-stranded DNA is folded into a designed nanostructure, in either 2D or 3D, with the aid of many short staple strands. The number of proposals for new design principles for DNA origami structures seems to have already reached a peak. It is apparent that DNA origami study is now entering the second phase of creating practical applications. The development of functional nanomechanical molecular devices using the DNA origami technique is one such application attracting significant interest from researchers in the field. Nanomechanical DNA origami devices, which maintain the characteristics of DNA origami structures, have various advantages over conventional DNA nanomachines. Comparatively high assembly yield, relatively large size visible via atomic force microscopy (AFM) or transmission electron microscopy (TEM), and the capability to assemble multiple functional groups with precision using multiple staple strands are some of the advantages of the DNA origami technique for constructing sophisticated molecular devices. This Account describes the recent developments of such nanomechanical DNA origami devices and reviews the emerging target of DNA origami studies. First, simple "dynamic" DNA origami structures with transformation capability, such as DNA origami boxes and a DNA origami hatch with structure control, are briefly summarized. More elaborate nanomechanical DNA origami devices are then reviewed. The first example describes DNA origami pinching devices that can be used as "single-molecule" beacons to detect a variety of biorelated molecules, from metal ions at the size of a few tens of atomic mass number units to relatively gigantic proteins with a molecular mass greater than a hundred kilodaltons, all on a single platform. Clamshell-like DNA nanorobots equipped with logic gates can discriminate

  1. DNA sequencing using fluorescence background electroblotting membrane

    DOEpatents

    Caldwell, K.D.; Chu, T.J.; Pitt, W.G.

    1992-05-12

    A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through amino groups contained on the surface. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to the target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membranes may be reprobed numerous times. No Drawings

  2. DNA sequencing using fluorescence background electroblotting membrane

    DOEpatents

    Caldwell, Karin D.; Chu, Tun-Jen; Pitt, William G.

    1992-01-01

    A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.

  3. Local Group

    NASA Astrophysics Data System (ADS)

    Mateo, M.; Murdin, P.

    2000-11-01

    Not long after EDWIN HUBBLE established that galaxies are `island universes' similar to our home galaxy, the MILKY WAY, he realized that a few of these external galaxies are considerably closer to us than any others. In 1936 he first coined the term `Local Group' in his famous book The Realm of the Nebulae to identify our nearest galactic neighbors. More than 60 yr later, the galaxies of the Loca...

  4. Underrepresented groups

    NASA Technical Reports Server (NTRS)

    Peters, David A.

    1990-01-01

    The problem with the shortage of under represented groups in science and engineering is absolutely crucial, especially considering that U.S. will experience a shortage of 560,000 science and engineering personnel by the year 2010. Most studies by the National Science Foundation also concluded that projected shortages cannot be alleviated without significant increases in the involvement of Blacks, Hispanics, Native Americans, handicapped persons, and women.

  5. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  6. [Mitochondrial DNA diversity in Kazym Khanty].

    PubMed

    Naumova, O Iu; Khaiat, S Sh; Rychkov, S Iu

    2009-06-01

    New data on mitochondrial DNA polymorphism in the representatives of Kazym territorial group of Northern Khanty are presented. MtDNA diversity observed in Kazym Khanty was compared with that in Khanty from Shuryshkarskii raion of Yamalo-Nenets Autonomous Okrug.

  7. Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

    SciTech Connect

    Verebová, Valéria; Adamcik, Jozef; Danko, Patrik; Podhradský, Dušan; Miškovský, Pavol; Staničová, Jana

    2014-01-31

    Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

  8. DNA ligase I, the replicative DNA ligase

    PubMed Central

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2013-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication. PMID:22918593

  9. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  10. The centipede genus Eupolybothrus Verhoeff, 1907 (Chilopoda: Lithobiomorpha: Lithobiidae) in North Africa, a cybertaxonomic revision, with a key to all species in the genus and the first use of DNA barcoding for the group.

    PubMed

    Stoev, Pavel; Akkari, Nesrine; Zapparoli, Marzio; Porco, David; Enghoff, Henrik; Edgecombe, Gregory D; Georgiev, Teodor; Penev, Lyubomir

    2010-06-30

    The centipede genus Eupolybothrus Verhoeff, 1907 in North Africa is revised. A new cavernicolous species, Eupolybothruskahfi Stoev & Akkari, sp. n., is described from a cave in Jebel Zaghouan, northeast Tunisia. Morphologically, it is most closely related to Eupolybothrusnudicornis (Gervais, 1837) from North Africa and Southwest Europe but can be readily distinguished by the long antennae and leg-pair 15, a conical dorso-median protuberance emerging from the posterior part of prefemur 15, and the shape of the male first genital sternite. Molecular sequence data from the cytochrome c oxidase I gene (mtDNA-5' COI-barcoding fragment) exhibit 19.19% divergence between Eupolybothruskahfi and Eupolybothrusnudicornis, an interspecific value comparable to those observed among four other species of Eupolybothrus which, combined with a low intraspecific divergence (0.3-1.14%), supports the morphological diagnosis of Eupolybothruskahfi as a separate species. This is the first troglomorphic myriapod to be found in Tunisia, and the second troglomorph lithobiomorph centipede known from North Africa. Eupolybothrusnudicornis is redescribed based on abundant material from Tunisia and its post-embryonic development, distribution and habitat preferences recorded. Eupolybothruscloudsley-thompsoni Turk, 1955, a nominal species based on Tunisian type material, is placed in synonymy with Eupolybothrusnudicornis. To comply with the latest technological developments in publishing of biological information, the paper implements new approaches in cybertaxonomy, such as fine granularity XML tagging validated against the NLM DTD TaxPub for PubMedCentral and dissemination in XML to various aggregators (GBIF, EOL, Wikipedia), vizualisation of all taxa mentioned in the text via the dynamically created Pensoft Taxon Profile (PTP) page, data publishing, georeferencing of all localities via Google Earth, and ZooBank, GenBank and MorphBank registration of datasets. An interactive key to all valid

  11. Permanent or reversible conjugation of 2′-O- or 5′-O-aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences

    PubMed Central

    Cieślak, Jacek; Grajkowski, Andrzej; Ausín, Cristina; Gapeev, Alexei; Beaucage, Serge L.

    2012-01-01

    2′-O-Aminooxymethyl ribonucleosides are prepared from their 3′,5′-disilylated 2′-O-phthalimidooxymethyl derivatives by treatment with NH4F in MeOH. The reaction of these novel ribonucleosides with 1-pyrenecarboxaldehyde results in the efficient formation of stable and yet reversible ribonucleoside 2′-conjugates in yields of 69–82%. Indeed, exposure of these conjugates to 0.5 M tetra-n-butylammonium fluoride (TBAF) in THF results in the cleavage of their iminoether functions to give the native ribonucleosides along with the innocuous nitrile side product. Conversely, the reaction of 5-cholesten-3-one or dansyl chloride with 2′-O-aminooxymethyl uridine provides permanent uridine 2′-conjugates, which are left essentially intact upon treatment with TBAF. Alternatively, 5′-O-aminooxymethyl thymidine is prepared by hydrazinolysis of its 3′-O-levulinyl-5′-O-phthalimidooxymethyl precursor. Pyrenylation of 5′-O-aminooxymethyl thymidine and the sensitivity of the 5′-conjugate to TBAF further exemplify the usefulness of this nucleoside for modifying DNA sequences either permanently or reversibly. Although the versatility and uniqueness of 2′-O-aminooxymethyl ribonucleosides in the preparation of modified RNA sequences is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2′-conjugate into an RNA sequence, the conjugation of 2′-O-aminooxymethyl ribonucleosides with aldehydes, including those generated from their acetals, provides reversible 2′-O-protected ribonucleosides for potential applications in the solid-phase synthesis of native RNA sequences. The synthesis of a chimeric polyuridylic acid is presented as an exemplary model. PMID:22067450

  12. DNA Damage and Pulmonary Hypertension

    PubMed Central

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  13. Synthesis of DNA

    DOEpatents

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  14. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  15. Cardiovascular group

    NASA Technical Reports Server (NTRS)

    Blomqvist, Gunnar

    1989-01-01

    As a starting point, the group defined a primary goal of maintaining in flight a level of systemic oxygen transport capacity comparable to each individual's preflight upright baseline. The goal of maintaining capacity at preflight levels would seem to be a reasonable objective for several different reasons, including the maintenance of good health in general and the preservation of sufficient cardiovascular reserve capacity to meet operational demands. It is also important not to introduce confounding variables in whatever other physiological studies are being performed. A change in the level of fitness is likely to be a significant confounding variable in the study of many organ systems. The principal component of the in-flight cardiovascular exercise program should be large-muscle activity such as treadmill exercise. It is desirable that at least one session per week be monitored to assure maintenance of proper functional levels and to provide guidance for any adjustments of the exercise prescription. Appropriate measurements include evaluation of the heart-rate/workload or the heart-rate/oxygen-uptake relationship. Respiratory gas analysis is helpful by providing better opportunities to document relative workload levels from analysis of the interrelationships among VO2, VCO2, and ventilation. The committee felt that there is no clear evidence that any particular in-flight exercise regimen is protective against orthostatic hypotension during the early readaptation phase. Some group members suggested that maintenance of the lower body muscle mass and muscle tone may be helpful. There is also evidence that late in-flight interventions to reexpand blood volume to preflight levels are helpful in preventing or minimizing postflight orthostatic hypotension.

  16. Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination.

    PubMed

    Sutton, M D; Walker, G C

    2001-07-17

    Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.

  17. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  18. DNA systematics. Volume II

    SciTech Connect

    Dutta, S.K.

    1986-01-01

    This book discusses the following topics: PLANTS: PLANT DNA: Contents and Systematics. Repeated DNA Sequences and Polyploidy in Cereal Crops. Homology of Nonrepeated DNA Sequences in Phylogeny of Fungal Species. Chloropast DNA and Phylogenetic Relationships. rDNA: Evolution Over a Billion Years. 23S rRNA-derived Small Ribosomal RNAs: Their Structure and Evolution with Reference to Plant Phylogeny. Molecular Analysis of Plant DNA Genomes: Conserved and Diverged DNA Sequences. A Critical Review of Some Terminologies Used for Additional DNA in Plant Chromosomes and Index.

  19. Energy transport in crystalline DNA composites

    SciTech Connect

    Xu, Zaoli; Xu, Shen; Tang, Xiaoduan; Wang, Xinwei

    2014-01-15

    This work reports on the synthesis of crystalline DNA-composited films and microfibers, and details the study of thermal energy transport in them. The transient electro-thermal technique is used to characterize the thermal transport in DNA composite microfibers, and the photothermal technique is used to explore the thermal transport in the thickness direction of DNA films. Compared with microfibers, the DNA films are found to have a higher thermal transport capacity, largely due to the carefully controlled crystallization process in film synthesis. In high NaCl concentration solutions, the bond of the Na{sup +} ion and phosphate group aligns the DNA molecules with the NaCl crystal structure during crystallization. This results in significant enhancement of thermal transport in the DNA films with aligned structure.

  20. DNA/RNA Detection Using DNA-Templated Few-Atom Silver Nanoclusters

    PubMed Central

    Obliosca, Judy M.; Liu, Cong; Batson, Robert Austin; Babin, Mark C.; Werner, James H.; Yeh, Hsin-Chih

    2013-01-01

    DNA-templated few-atom silver nanoclusters (DNA/Ag NCs) are a new class of organic/inorganic composite nanomaterials whose fluorescence emission can be tuned throughout the visible and near-IR range by simply programming the template sequences. Compared to organic dyes, DNA/Ag NCs can be brighter and more photostable. Compared to quantum dots, DNA/Ag NCs are smaller, less prone to blinking on long timescales, and do not have a toxic core. The preparation of DNA/Ag NCs is simple and there is no need to remove excess precursors as these precursors are non-fluorescent. Our recent discovery of the fluorogenic and color switching properties of DNA/Ag NCs have led to the invention of new molecular probes, termed NanoCluster Beacons (NCBs), for DNA detection, with the capability to differentiate single-nucleotide polymorphisms by emission colors. NCBs are inexpensive, easy to prepare, and compatible with commercial DNA synthesizers. Many other groups have also explored and taken advantage of the environment sensitivities of DNA/Ag NCs in creating new tools for DNA/RNA detection and single-nucleotide polymorphism identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs for developing DNA/RNA sensors. PMID:25586126

  1. Group evaporation

    NASA Technical Reports Server (NTRS)

    Shen, Hayley H.

    1991-01-01

    Liquid fuel combustion process is greatly affected by the rate of droplet evaporation. The heat and mass exchanges between gas and liquid couple the dynamics of both phases in all aspects: mass, momentum, and energy. Correct prediction of the evaporation rate is therefore a key issue in engineering design of liquid combustion devices. Current analytical tools for characterizing the behavior of these devices are based on results from a single isolated droplet. Numerous experimental studies have challenged the applicability of these results in a dense spray. To account for the droplets' interaction in a dense spray, a number of theories have been developed in the past decade. Herein, two tasks are examined. One was to study how to implement the existing theoretical results, and the other was to explore the possibility of experimental verifications. The current theoretical results of group evaporation are given for a monodispersed cluster subject to adiabatic conditions. The time evolution of the fluid mechanic and thermodynamic behavior in this cluster is derived. The results given are not in the form of a subscale model for CFD codes.

  2. DNA vaccines: a simple DNA sensing matter?

    PubMed

    Coban, Cevayir; Kobiyama, Kouji; Jounai, Nao; Tozuka, Miyuki; Ishii, Ken J

    2013-10-01

    Since the introduction of DNA vaccines two decades ago, this attractive strategy has been hampered by its low immunogenicity in humans. Studies conducted to improve the immunogenicity of DNA vaccines have shown that understanding the mechanism of action of DNA vaccines might be the key to successfully improving their immunogenicity. Our current understanding is that DNA vaccines induce innate and adaptive immune responses in two ways: (1) encoded protein (or polypeptide) antigen(s) by the DNA plasmid can be expressed in stromal cells (i.e., muscle cells) as well as DCs, where these antigens are processed and presented to naïve CD4 or CD8 T cells either by direct or cross presentation, respectively; and (2) the transfected DNA plasmid itself may bind to an un-identified cytosolic DNA sensor and activate the TBK1-STING pathway and the production of type I interferons (IFNs) which function as an adjuvant. Recent studies investigating double-stranded cytosolic DNA sensor(s) have highlighted new mechanisms in which cytosolic DNA may release secondary metabolites, which are in turn recognized by a novel DNA sensing machinery. Here, we discuss these new metabolites and the possibilities of translating this knowledge into improved immunogenicity for DNA vaccines.

  3. Glucocorticoid receptor transformation and DNA binding

    SciTech Connect

    Tienrungroj, W.

    1986-01-01

    The overall goal is to probe the mechanism whereby glucocorticoid receptors are transformed from a non-DNA-binding form to their active DNA-binding form. The author has examined the effect of an endogenous inhibitor purified from rat liver cytosol on receptor binding to DNA. The inhibitor binds to transformed receptors in whole cytosol and prevent their binding to DNA. He also examined the role of sulfhydryl groups in determining the DNA binding activity of the transformed receptor and in determining the transformation process. Treatment of rat liver cytosol containing temperature-transformed, (/sup 3/H)dexamethasone-bound receptors at 0/sup 0/C with the sulfhydryl modifying reagent methyl methanethiosulfonate inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol. In addition, he has examined the relationship between receptor phosphorylation and DNA binding. Untransformed receptor complexes purified from cytosol prepared from mouse L cells grown in medium containing (/sup 32/P)orthophosphate contain two components, a 100 k-Da and a 90-kDa subunit, both of which are phosphoproteins. On transformation, the receptor dissociates from the 90-kDa protein. Transformation of the complex under cell free conditions does not result in a dephosphorylation of the 100-kDa steroid-binding protein. Transformed receptor that has been bound to DNA and purified by monoclonal antibody is still in a phosphorylated form. These results suggest that dephosphorylation is not required for receptor binding to DNA.

  4. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  5. Investigation of perfluorooctanoic acid induced DNA damage using electrogenerated chemiluminescence associated with charge transfer in DNA.

    PubMed

    Lu, Liping; Guo, Linqing; Li, Meng; Kang, Tianfang; Cheng, Shuiyuan; Miao, Wujian

    2016-10-01

    An electrogenerated chemiluminescence (ECL)-DNA sensor was designed and fabricated for the investigation of DNA damage by a potential environmental pollutant, perfluorooctanoic acid (PFOA). The ECL-DNA sensor consisted of a Au electrode that had a self-assembled monolayer of 15 base-pair double-stranded (ds) DNA oligonucleotides with covalently attached semiconductor CdSe quantum dots (QDs) at the distal end of the DNA. Characterization of the ECL-DNA sensor was conducted with X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy (EIS), ECL, and cyclic voltammetry before and after the exposure of the sensor to PFOA. Consistent data revealed that the dsDNA on Au was severely damaged upon the incubation of the electrode in PFOA, causing significant increase in charge (or electron) transfer (CT) resistance within DNA strands. Consequently, the cathodic coreactant ECL responses of the Au/dsDNA-QDs electrode in the presence of K2S2O8 were markedly decreased. The strong interaction between DNA and PFOA via the hydrophobic interaction, especially the formation of F···H hydrogen bonds by insertion of the difluoro-methylene group of PFOA into the DNA base pairs, was believed to be responsible for the dissociation or loosening of dsDNA structure, which inhibited the CT through DNA. A linear relationship between the ECL signal of the sensor and the logarithmical concentration of PFOA displayed a dynamic range of 1.00 × 10(-14)-1.00 × 10(-4) M, with a limit of detection of 1.00 × 10(-15) M at a signal-to-noise ratio of 3. Graphical Abstract Illustration of ECL detection of PFOA on a Au/dsDNA-QDs ECL-DNA sensor.

  6. Investigation of perfluorooctanoic acid induced DNA damage using electrogenerated chemiluminescence associated with charge transfer in DNA.

    PubMed

    Lu, Liping; Guo, Linqing; Li, Meng; Kang, Tianfang; Cheng, Shuiyuan; Miao, Wujian

    2016-10-01

    An electrogenerated chemiluminescence (ECL)-DNA sensor was designed and fabricated for the investigation of DNA damage by a potential environmental pollutant, perfluorooctanoic acid (PFOA). The ECL-DNA sensor consisted of a Au electrode that had a self-assembled monolayer of 15 base-pair double-stranded (ds) DNA oligonucleotides with covalently attached semiconductor CdSe quantum dots (QDs) at the distal end of the DNA. Characterization of the ECL-DNA sensor was conducted with X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy (EIS), ECL, and cyclic voltammetry before and after the exposure of the sensor to PFOA. Consistent data revealed that the dsDNA on Au was severely damaged upon the incubation of the electrode in PFOA, causing significant increase in charge (or electron) transfer (CT) resistance within DNA strands. Consequently, the cathodic coreactant ECL responses of the Au/dsDNA-QDs electrode in the presence of K2S2O8 were markedly decreased. The strong interaction between DNA and PFOA via the hydrophobic interaction, especially the formation of F···H hydrogen bonds by insertion of the difluoro-methylene group of PFOA into the DNA base pairs, was believed to be responsible for the dissociation or loosening of dsDNA structure, which inhibited the CT through DNA. A linear relationship between the ECL signal of the sensor and the logarithmical concentration of PFOA displayed a dynamic range of 1.00 × 10(-14)-1.00 × 10(-4) M, with a limit of detection of 1.00 × 10(-15) M at a signal-to-noise ratio of 3. Graphical Abstract Illustration of ECL detection of PFOA on a Au/dsDNA-QDs ECL-DNA sensor. PMID:27108285

  7. Photochemical modulation of DNA conformation by organic dications.

    PubMed

    Zinchenko, Anatoly A; Tanahashi, Mao; Murata, Shizuaki

    2012-01-01

    A group of azobenzene derivatives containing two quaternary ammonium groups with various intercharge distances between them was synthesised and used to control photochemically the conformation of genomic DNA by switching the distance between cationic ammonium groups in the dications. It was found that isomerisation of either dication from the trans form to cis resulted in an increase in the dication's efficiency for DNA compaction; this is associated with a decrease in intercharge distance between ammonium groups and leads to a better match of the binder's cationic groups to adjacent phosphate groups of DNA. Ammonium dications have several important advantages over the photosensitive surfactant type of diazobenzene reported earlier: they can be used at significantly lower (>100-fold) concentrations than photosensitive surfactants, and DNA conformation control can be performed over a broader concentration range of dications. The influence of intercharge distance in photosensitive dications on photo-induced DNA binding discrimination is discussed, and the molecular mechanism is proposed. PMID:22109974

  8. DNA Damage, DNA Repair, Aging, and Neurodegeneration.

    PubMed

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L; Bohr, Vilhelm A

    2015-09-18

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span.

  9. Correlated motions in DNA

    SciTech Connect

    Sundaralingam, M.; Westhof, E.

    1980-10-01

    The furanose ring of nucleic acids plays a key role in detrmining the conformations of nucleic acids because it shares a common bond C3'-C4'(psi') with the sugar-phosphate backbone. This structural feature enables the transmission of conformational changes between the side-chain base and the backbone through conformational correlations between the base and sugar. Thermally-induced local fluctuations of P can be transmitted along the backbone through psi', particularly when the sugar is in the C2'-endo domain. The sugar pucker-dependent flexibility of DNA is further exemplified by studies that have shown that due to steric interactions, absence of the 2'-OH group in deoxyribose tends to increase the conformational flexibility about the internucleotide phosphodiester (..omega.., ..omega..') especially when the sugar assumes the C2'-endo pucker.

  10. Tumorigenic DNA viruses

    SciTech Connect

    Klein, G.

    1989-01-01

    The eighth volume of Advances in Viral Oncology focuses on the three major DNA virus groups with a postulated or proven tumorigenic potential: papillomaviruses, animal hepatitis viruses, and the Epstein-Bar virus. In the opening chapters, the contributors analyze the evidence that papillomaviruses and animal hepatitis viruses are involved in tumorigenesis and describe the mechanisms that trigger virus-host cell interactions. A detailed section on the Epstein-Barr virus (EBV) - comprising more than half the book - examines the transcription and mRNA processing patterns of the virus genome; the mechanisms by which EBV infects lymphoid and epithelial cells; the immunological aspects of the virus; the actions of EBV in hosts with Acquired Immune Deficiency Syndrome; and the involvement of EBV in the etiology of Burkitt's lymphoma.

  11. Retroviral Integrase Structure and DNA Recombination Mechanism

    PubMed Central

    Engelman, Alan; Cherepanov, Peter

    2015-01-01

    SUMMARY Due to the importance of human immunodeficiency virus type 1 (HIV-1) integrase as a drug target, the biochemistry and structural aspects of retroviral DNA integration have been the focus of intensive research during the past three decades. The retroviral integrase enzyme acts on the linear double-stranded viral DNA product of reverse transcription. Integrase cleaves specific phosphodiester bonds near the viral DNA ends during the 3′ processing reaction. The enzyme then uses the resulting viral DNA 3′-OH groups during strand transfer to cut chromosomal target DNA, which simultaneously joins both viral DNA ends to target DNA 5′-phosphates. Both reactions proceed via direct transesterification of scissile phosphodiester bonds by attacking nucleophiles: a water molecule for 3′ processing, and the viral DNA 3′-OH for strand transfer. X-ray crystal structures of prototype foamy virus integrase-DNA complexes revealed the architectures of the key nucleoprotein complexes that form sequentially during the integration process and explained the roles of active site metal ions in catalysis. X-ray crystallography furthermore elucidated the mechanism of action of HIV-1 integrase strand transfer inhibitors, which are currently used to treat AIDS patients, and provided valuable insights into the mechanisms of viral drug resistance. PMID:25705574

  12. DNA from plant mitochondria.

    PubMed

    Suyama, Y; Bonner, W D

    1966-03-01

    DNA WAS ISOLATED FROM A MITOCHONDRIAL FRACTION OF EACH OF THE FOLLOWING PLANT MATERIALS: Mung bean (Phaseolus aureus) etiolated hypocotyl; turnip (Brassica rapa) root; sweet potato (Ipomoea batatas) root; and onion (Allium cepa) bulb. It was found that all of these mitochondrial fractions contained DNA, the densities of which were identical (rho=1.706 g.cm(-3)). An additional DNA (rho=1.695) band found in the mitochondrial fraction of Brassica rapa, was identical to DNA separately isolated from the chloroplast-rich fraction. The origin of the second DNA from Allium mitochondrial fraction was not identified.Contrary to the identity of the mitochondrial DNA, DNA from nuclear fractions differed not only with each other but from the corresponding mitochondrial DNA.DNA from Phaseolus and Brassica mitochondria showed the hyperchromicity characteristic of double stranded, native DNA upon heating; Tm's in 0.0195 Na(+) were the same; 72.0 degrees . The amount of DNA within the mitochondrion of Phaseolus was estimated to be 5.0 x 10(-10) mug; this estimate was made by isolating the mitochondrial DNA concomitantly with the known amount of added (15)N(2)H B. subtilis DNA (rho=1.740). Approximately the same amount of DNA was present in the mitochondrion of Brassica or Ipomoea.

  13. Study of the DNA damage checkpoint using Xenopus egg extracts.

    PubMed

    Willis, Jeremy; DeStephanis, Darla; Patel, Yogin; Gowda, Vrushab; Yan, Shan

    2012-01-01

    On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin. MMS is an alkylating agent that inhibits DNA replication and activates the ATR

  14. LCAT DNA shearing.

    PubMed

    Okabe, Yuka; Lee, Abraham P

    2014-04-01

    We present a novel method to fragment DNA by using lateral cavity acoustic transducers (LCATs). DNA solution is placed within a microfluidic device containing LCATs. The LCATs cause microstreaming, which fragments DNA within the solution without any need for purification or downstream processing. The LCAT-based DNA fragmentation method offers an easy-to-use, low-cost, low-energy way to fragment DNA that is amenable to integration on microfluidic platforms to further automate DNA processing. Furthermore, the LCAT microdevice requires less than 10 µL of sample, and no external equipment is needed besides a piezoelectric transducer. PMID:23850863

  15. Reconfiguration of DNA methylation in aging.

    PubMed

    Zampieri, Michele; Ciccarone, Fabio; Calabrese, Roberta; Franceschi, Claudio; Bürkle, Alexander; Caiafa, Paola

    2015-11-01

    A complex interplay between multiple biological effects shapes the aging process. The advent of genome-wide quantitative approaches in the epigenetic field has highlighted the effective impact of epigenetic deregulation, particularly of DNA methylation, on aging. Age-associated alterations in DNA methylation are commonly grouped in the phenomenon known as "epigenetic drift" which is characterized by gradual extensive demethylation of genome and hypermethylation of a number of promoter-associated CpG islands. Surprisingly, specific DNA regions show directional epigenetic changes in aged individuals suggesting the importance of these events for the aging process. However, the epigenetic information obtained until now in aging needs a re-consideration due to the recent discovery of 5-hydroxymethylcytosine, a new DNA epigenetic mark present on genome. A recapitulation of the factors involved in the regulation of DNA methylation and the changes occurring in aging will be described in this review also considering the data available on 5 hmC.

  16. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  17. Direct electrical detection of DNA synthesis

    PubMed Central

    Pourmand, Nader; Karhanek, Miloslav; Persson, Henrik H. J.; Webb, Chris D.; Lee, Thomas H.; Zahradníková, Alexandra; Davis, Ronald W.

    2006-01-01

    Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3′-hydroxyl group of the DNA molecule during phosphodiester bond formation. PMID:16614066

  18. Parvovirus infection-induced DNA damage response

    PubMed Central

    Luo, Yong; Qiu, Jianming

    2014-01-01

    Parvoviruses are a group of small DNA viruses with ssDNA genomes flanked by two inverted terminal structures. Due to a limited genetic resource they require host cellular factors and sometimes a helper virus for efficient viral replication. Recent studies have shown that parvoviruses interact with the DNA damage machinery, which has a significant impact on the life cycle of the virus as well as the fate of infected cells. In addition, due to special DNA structures of the viral genomes, parvoviruses are useful tools for the study of the molecular mechanisms underlying viral infection-induced DNA damage response (DDR). This review aims to summarize recent advances in parvovirus-induced DDR, with a focus on the diverse DDR pathways triggered by different parvoviruses and the consequences of DDR on the viral life cycle as well as the fate of infected cells. PMID:25429305

  19. [Uracil-DNA glycosylases].

    PubMed

    Pytel, Dariusz; Słupianek, Artur; Ksiazek, Dominika; Skórski, Tomasz; Błasiak, Janusz

    2008-01-01

    Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

  20. Chemical method for introducing haptens on to DNA probes

    SciTech Connect

    Keller, G.H.; Cumming, C.U.; Huang, D.P.; Manak, M.M.; Ting, R.

    1988-05-01

    The authors developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. They have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 x 10/sup -17/ mol (1 x 10/sup 6/ copies) of HBV DNA in total DNA from human serum.

  1. Structural Organization of DNA.

    ERIC Educational Resources Information Center

    Banfalvi, Gaspar

    1986-01-01

    Explains the structural organization of DNA by providing information on the primary, secondary, tertiary, and higher organization levels of the molecule. Also includes illustrations and descriptions of sign-inversion and rotating models for supercoiling of DNA. (ML)

  2. DNA tagged microparticles

    DOEpatents

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  3. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  4. Repair synthesis step involving ERCC1-XPF participates in DNA repair of the Top1-DNA damage complex.

    PubMed

    Takahata, Chiaki; Masuda, Yuji; Takedachi, Arato; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2015-08-01

    Topoisomerase 1 (Top1) is the intercellular target of camptothecins (CPTs). CPT blocks DNA religation in the Top1-DNA complex and induces Top1-attached nick DNA lesions. In this study, we demonstrate that excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease and replication protein A (RPA) participate in the repair of Top1-attached nick DNA lesions together with other nucleotide excision repair (NER) factors. ERCC1-XPF shows nuclease activity in the presence of RPA on a 3'-phosphotyrosyl bond nick-containing DNA (Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. In addition, ERCC1-XPF and RPA form a DNA/protein complex on the nick DNA substrate in vitro, and co-localize in CPT-treated cells in vivo. Moreover, the DNA repair synthesis of Tyr-nick DNA lesions occurred in the presence of NER factors, including ERCC1-XPF, RPA, DNA polymerase delta, flap endonuclease 1 and DNA ligase 1. Therefore, some of the NER repair machinery might be an alternative repair pathway for Top1-attached nick DNA lesions. Clinically, these data provide insights into the potential of ERCC1 as a biomarker during CPT regimens.

  5. Effect of DNA type on response of DNA biosensor for carcinogens

    NASA Astrophysics Data System (ADS)

    Sani, Nor Diyana bt. Md.; Heng, Lee Yook; Surif, Salmijah; Lazim, Azwani Mat

    2013-11-01

    Carcinogens are cancer causing chemicals that can bind to DNA and cause damage to the DNA. These chemicals are available everywhere including in water, air, soil and food. Therefore, a sensor that can detect the presence of these chemicals will be a very useful tool. Since carcinogens bind to DNA, DNA can be used as the biological element in a biosensor. This study has utilized different types of DNA in a biosensor for carcinogen detection. The DNAs include double stranded calf thymus DNA, single stranded calf thymus DNA and guanine rich single stranded DNA. The modified SPE was exposed to a carcinogen followed by interaction with methylene blue which acts as the electroactive indicator. The SPE was then analysed using differential pulse voltammetry (DPV). Optimization studies were conducted for MB concentration and accumulation time, DNA concentration, as well as effect of buffer concentration, buffer pH and ionic strength. The performance of the biosensor was tested on a group 1 carcinogen, formaldehyde. The results indicated that the usage of guanine rich single stranded DNA also gives higher response as carcinogens prefer to bind with guanine compared to other bases.

  6. Crossing borders: the DNA of physics

    NASA Astrophysics Data System (ADS)

    Beijerinck, H. C. W.

    2015-01-01

    In cell culture, the physical environment plays an important role: "Everything is everywhere, but the environment selects"[1]. The education of physicists can be viewed within this framework. The Petri dish for the reproduction of physicists is a university research group. The full professor is its DNA. The selection process of new professors - new DNA - is a determining step in creating the right culture. [1] M.W. Beijerinck and L.G.M. Baas Becking, en.wikipedia.org.

  7. Oxidation of DNA: damage to nucleobases.

    PubMed

    Kanvah, Sriram; Joseph, Joshy; Schuster, Gary B; Barnett, Robert N; Cleveland, Charles L; Landman, Uzi

    2010-02-16

    All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and

  8. Extracellular DNA: the tip of root defenses?

    PubMed

    Hawes, Martha C; Curlango-Rivera, Gilberto; Wen, Fushi; White, Gerard J; Vanetten, Hans D; Xiong, Zhongguo

    2011-06-01

    This review discusses how extracellular DNA (exDNA) might function in plant defense, and at what level(s) of innate immunity this process might operate. A new role for extracellular factors in mammalian defense has been described in a series of studies. These studies reveal that cells including neutrophils, eosinophils, and mast cells produce 'extracellular traps' (ETs) consisting of histone-linked exDNA. When pathogens are attracted to such ETs, they are trapped and killed. When the exDNA component of ETs is degraded, trapping is impaired and resistance against invasion is reduced. Conversely, mutation of microbial genes encoding exDNases that degrade exDNA results in loss of virulence. This discovery that exDNases are virulence factors opens new avenues for disease control. In plants, exDNA is required for defense of the root tip. Innate immunity-related proteins are among a group of >100 proteins secreted from the root cap and root border cell populations. Direct tests revealed that exDNA also is rapidly synthesized and exported from the root tip. When this exDNA is degraded by the endonuclease DNase 1, root tip resistance to fungal infection is lost; when the polymeric structure is degraded more slowly, by the exonuclease BAL31, loss of resistance to fungal infection is delayed accordingly. The results suggest that root border cells may function in a manner analogous to that which occurs in mammalian cells.

  9. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

    PubMed Central

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-01-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

  10. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

    PubMed

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

  11. DNA nanoarchitectonics: assembled DNA at interfaces.

    PubMed

    Howorka, Stefan

    2013-06-18

    DNA is a powerful biomaterial for creating rationally designed and functionally enhanced nanostructures. DNA nanoarchitectures positioned at substrate interfaces can offer unique advantages leading to improved surface properties relevant to biosensing, nanotechnology, materials science, and cell biology. This Perspective highlights the benefits and challenges of using assembled DNA as a nanoscale building block for interfacial layers and surveys their applications in three areas: homogeneous dense surface coatings, bottom-up nanopatterning, and 3D nanoparticle lattices. Possible future research developments are discussed at the end of the Perspective.

  12. Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA-protein interaction

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Yasuda, Hachiro; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2009-04-01

    We carried out an individual DNA manipulation using an optical trapping for a microbead. This manipulation system is based on a fluorescent microscopy equipped with an IR laser. Both ends of linear DNA molecule were labeled with a biotin and a thiol group, respectively. Then the biotinylated end was attached to a microbead, and the other was immobilized on a thiol-linkable glass surface. We controlled the form of an individual DNA molecule by moving the focal point of IR laser, which trapped the microbead. In addition, we applied single-molecule approach to analyze DNA hydrolysis. We also used microchannel for single-molecule observation of DNA hydrolysis. The shortening of DNA in length caused by enzymatic hydrolysis was observed in real-time. The single-molecule DNA manipulation should contribute to elucidate detailed mechanisms of DNA-protein interactions.

  13. The Many Sides of DNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores the meaning of DNA. Discusses histories of DNA, literature on DNA, the contributions of Max Delbruck and Barbara McClintock, life, views of control, current research, and the language of DNA. Contains 24 references. (JRH)

  14. DNA Sequencing apparatus

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  15. Racemic DNA crystallography.

    PubMed

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

    2014-12-22

    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA.

  16. Functionalizing Designer DNA Crystals

    NASA Astrophysics Data System (ADS)

    Chandrasekaran, Arun Richard

    nucleotides is usually pH dependent (pH < 6) four different TFOs were examined: TFO-1 was unmodified while TFOs 2-4 contained additional stabilizing analogues capable of extending triplex formation to pH 7. In addition, each of the TFOs contained a Cy5 dye at the 5'-end of the oligonucleotide to aid in characterization of TFO binding - crystals were obtained with all four variations of TFOs. Formation of DNA triplex in the motif was characterized by an electrophoretic mobility shift assay (EMSA), UV melting studies and FRET. Crystals containing TFO-1 (unmodified) and TFO-2 (with 2'-amino ethoxy modification) were isolated and flash-frozen in liquid nitrogen for X-ray data collection at beam line NSLS-X25. X-ray data was also collected for crystals of the 3-turn triangle without any TFO bound to it. Difference maps were done between the crystals with TFO against the one without to identify any additional electron density corresponding to the third strand in the triplex binding region. The data from the crystal containing TFO-2 was used to further analyze if the additional density can match the expected position of the TFO on the triangle motif. Since the additional density did not correspond to the entire binding region, 2Fo-Fc, 3Fo-2Fc and 4Fo-3Fc maps were done to check for missing pieces of the electron density. From the resulting 2Fo-Fc map, the asymmetric unit from the 3-turn triangle (31-bp duplex model based on previous structure 3UBI) was inserted into the density as a reference. However, the electron density corresponding to the TFO was still not continuous throughout the 13-nt triplex binding region and allowed only a partial fit of the TFO. The third nucleotide in positions 1, 3, 4, 6, 7 were fit into the density in the major groove of the underlying duplex with proper triplex configuration. The third chapter describes the triplex approach to position a functional group (the UV cross-linking agent psoralen) within a pre-formed DNA motif. Triplex formation and

  17. DNA structure and function.

    PubMed

    Travers, Andrew; Muskhelishvili, Georgi

    2015-06-01

    The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase.

  18. Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond

    PubMed Central

    Davidson, David; Amrein, Lilian; Panasci, Lawrence; Aloyz, Raquel

    2012-01-01

    design will not only assist in identifying novel functional moieties to replace the metabolically labile morpholino group but will also facilitate the design of molecules to target the DNA-PKcs/Ku80 interface or one of the autophosphorylation sites. PMID:23386830

  19. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  20. DNA barcoding amphibians and reptiles.

    PubMed

    Vences, Miguel; Nagy, Zoltán T; Sonet, Gontran; Verheyen, Erik

    2012-01-01

    Only a few major research programs are currently targeting COI barcoding of amphibians and reptiles (including chelonians and crocodiles), two major groups of tetrapods. Amphibian and reptile species are typically old, strongly divergent, and contain deep conspecific lineages which might lead to problems in species assignment with incomplete reference databases. As far as known, there is no single pair of COI primers that will guarantee a sufficient rate of success across all amphibian and reptile taxa, or within major subclades of amphibians and reptiles, which means that the PCR amplification strategy needs to be adjusted depending on the specific research question. In general, many more amphibian and reptile taxa have been sequenced for 16S rDNA, which for some purposes may be a suitable complementary marker, at least until a more comprehensive COI reference database becomes available. DNA barcoding has successfully been used to identify amphibian larval stages (tadpoles) in species-rich tropical assemblages. Tissue sampling, DNA extraction, and amplification of COI is straightforward in amphibians and reptiles. Single primer pairs are likely to have a failure rate between 5 and 50% if taxa of a wide taxonomic range are targeted; in such cases the use of primer cocktails or subsequent hierarchical usage of different primer pairs is necessary. If the target group is taxonomically limited, many studies have followed a strategy of designing specific primers which then allow an easy and reliable amplification of all samples.

  1. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA

  2. Group Work Publication-1991.

    ERIC Educational Resources Information Center

    Zimpfer, David G.

    1992-01-01

    Lists 21 new publications in group work, of which 9 are reviewed. Those discussed include publications on group counseling and psychotherapy, structured groups, support groups, psychodrama, and social group work. (Author/NB)

  3. Group Cohesion in Experiential Growth Groups

    ERIC Educational Resources Information Center

    Steen, Sam; Vasserman-Stokes, Elaina; Vannatta, Rachel

    2014-01-01

    This article explores the effect of web-based journaling on changes in group cohesion within experiential growth groups. Master's students were divided into 2 groups. Both used a web-based platform to journal after each session; however, only 1 of the groups was able to read each other's journals. Quantitative data collected before and…

  4. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    NASA Astrophysics Data System (ADS)

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

  5. Adsorption of DNA onto anionic lipid surfaces.

    PubMed

    Martín-Molina, Alberto; Luque-Caballero, Germán; Faraudo, Jordi; Quesada-Pérez, Manuel; Maldonado-Valderrama, Julia

    2014-04-01

    Currently self-assembled DNA delivery systems composed of DNA multivalent cations and anionic lipids are considered to be promising tools for gene therapy. These systems become an alternative to traditional cationic lipid-DNA complexes because of their low cytotoxicity lipids. However, currently these nonviral gene delivery methods exhibit low transfection efficiencies. This feature is in large part due to the poorly understood DNA complexation mechanisms at the molecular level. It is well-known that the adsorption of DNA onto like charged lipid surfaces requires the presence of multivalent cations that act as bridges between DNA and anionic lipids. Unfortunately, the molecular mechanisms behind such adsorption phenomenon still remain unclear. Accordingly a historical background of experimental evidence related to adsorption and complexation of DNA onto anionic lipid surfaces mediated by different multivalent cations is firstly reviewed. Next, recent experiments aimed to characterise the interfacial adsorption of DNA onto a model anionic phospholipid monolayer mediated by Ca(2+) (including AFM images) are discussed. Afterwards, modelling studies of DNA adsorption onto charged surfaces are summarised before presenting preliminary results obtained from both CG and all-atomic MD computer simulations. Our results allow us to establish the optimal conditions for cation-mediated adsorption of DNA onto negatively charged surfaces. Moreover, atomistic simulations provide an excellent framework to understand the interaction between DNA and anionic lipids in the presence of divalent cations. Accordingly,our simulation results in conjunction go beyond the macroscopic picture in which DNA is stuck to anionic membranes by using multivalent cations that form glue layers between them. Structural aspects of the DNA adsorption and molecular binding between the different charged groups from DNA and lipids in the presence of divalent cations are reported in the last part of the study

  6. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  7. DNA profiles from fingermarks.

    PubMed

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success.

  8. Electrocatalysis in DNA Sensors.

    PubMed

    Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

    2014-12-14

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  9. Electrocatalysis in DNA Sensors

    PubMed Central

    Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  10. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    PubMed Central

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J.; Kaur, Baljeet; Sarwar, Naveed; Seckl, Michael J.; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA) (from 9% to 53% of total cfDNA) in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis. PMID:26981554

  11. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  12. DNA-based machines.

    PubMed

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications.

  13. DNA-based machines.

    PubMed

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications. PMID:24647836

  14. DNA nanomachines and nanostructures involving quadruplexes.

    PubMed

    Alberti, Patrizia; Bourdoncle, Anne; Saccà, Barbara; Lacroix, Laurent; Mergny, Jean-Louis

    2006-09-21

    DNA is an attractive component for molecular recognition, because of its self-assembly properties. Its three-dimensional structure can differ markedly from the classical double helix. For example, DNA or RNA strands carrying guanine or cytosine stretches associate into four-stranded structures called G-quadruplexes or i-DNA, respectively. Since 2002, several groups have described nanomachines that take advantage of this structural polymorphism. We first introduce the unusual structures that are involved in these devices (i.e., i-DNA and G-quadruplexes) and then describe the opening and closing steps that allow cycling. A quadruplex-duplex molecular machine is then presented in detail, together with the rules that govern its formation, its opening/closing kinetics and the various technical and physico-chemical parameters that play a role in the efficiency of this device. Finally, we review the few examples of nanostructures that involve quadruplexes.

  15. All that is gold does not glitter? Age, taxonomy, and ancient plant DNA quality

    PubMed Central

    Choi, JinHee; Lee, HyeJi

    2015-01-01

    More than 600 herbarium samples from four distantly related groups of flowering plants were used for DNA extraction and subsequent measurements of DNA purity and concentration. We did not find any significant relation between DNA purity and the age of the sample. However, DNA yields were different between plant groups studied. We believe that there there should be no reservations about “old” samples if the goal is to extract more DNA of better purity. We argue that the older herbarium samples are the mine for the future DNA studies, and have the value not less than the “fresh” specimens. PMID:26244108

  16. Getting Ready for the Dance: FANCJ Irons Out DNA Wrinkles

    PubMed Central

    Bharti, Sanjay Kumar; Awate, Sanket; Banerjee, Taraswi; Brosh, Robert M.

    2016-01-01

    Mounting evidence indicates that alternate DNA structures, which deviate from normal double helical DNA, form in vivo and influence cellular processes such as replication and transcription. However, our understanding of how the cellular machinery deals with unusual DNA structures such as G-quadruplexes (G4), triplexes, or hairpins is only beginning to emerge. New advances in the field implicate a direct role of the Fanconi Anemia Group J (FANCJ) helicase, which is linked to a hereditary chromosomal instability disorder and important for cancer suppression, in replication past unusual DNA obstacles. This work sets the stage for significant progress in dissecting the molecular mechanisms whereby replication perturbation by abnormal DNA structures leads to genomic instability. In this review, we focus on FANCJ and its role to enable efficient DNA replication when the fork encounters vastly abundant naturally occurring DNA obstacles, which may have implications for targeting rapidly dividing cancer cells. PMID:27376332

  17. Multiprotein DNA Looping

    NASA Astrophysics Data System (ADS)

    Vilar, Jose M. G.; Saiz, Leonor

    2006-06-01

    DNA looping plays a fundamental role in a wide variety of biological processes, providing the backbone for long range interactions on DNA. Here we develop the first model for DNA looping by an arbitrarily large number of proteins and solve it analytically in the case of identical binding. We uncover a switchlike transition between looped and unlooped phases and identify the key parameters that control this transition. Our results establish the basis for the quantitative understanding of fundamental cellular processes like DNA recombination, gene silencing, and telomere maintenance.

  18. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  19. DNA origami nanopores.

    PubMed

    Bell, Nicholas A W; Engst, Christian R; Ablay, Marc; Divitini, Giorgio; Ducati, Caterina; Liedl, Tim; Keyser, Ulrich F

    2012-01-11

    We demonstrate the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA origami structures into solid-state nanopores. In our experiments, single artificial nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state nanopores with diameters around 15 nm. We show that these hybrid nanopores can be employed for the detection of λ-DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly.

  20. DNA polymerase profiling.

    PubMed

    Summerer, Daniel

    2008-01-01

    We report a simple homogeneous fluorescence assay for quantification of DNA polymerase function in high throughput. The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon extension of the template strand. A resulting distance alteration is reported by fluorescence resonance energy transfer between two dyes introduced into the molecular beacon stem. We describe real-time reaction profiling of two model DNA polymerases. We demonstrate kinetic characterization, rapid optimization of reaction conditions, and inhibitor profiling using the presented assay. Furthermore, to supersede purification steps in screening procedures of DNA polymerase mutant libraries, detection of enzymatic activity in bacterial expression lysates is described.

  1. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  2. Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

    PubMed

    Deng, M Q; Cliver, D O

    1999-08-01

    A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

  3. Genome Sequence of Bacillus cereus Group Phage SalinJah.

    PubMed

    Erill, Ivan; Caruso, Steven M

    2016-01-01

    The double-stranded DNA (dsDNA) Myoviridae Bacillus cereus group bacteriophage SalinJah was isolated from soil collected in Gyeonggi-do, South Korea. SalinJah, a cluster C phage with a broad host range, suggests the need to create a new subcluster with SalinJah and Helga as founding members.

  4. Genome Sequence of Bacillus cereus Group Phage SalinJah

    PubMed Central

    2016-01-01

    The double-stranded DNA (dsDNA) Myoviridae Bacillus cereus group bacteriophage SalinJah was isolated from soil collected in Gyeonggi-do, South Korea. SalinJah, a cluster C phage with a broad host range, suggests the need to create a new subcluster with SalinJah and Helga as founding members. PMID:27688335

  5. Genome Sequence of Bacillus cereus Group Phage SalinJah.

    PubMed

    Erill, Ivan; Caruso, Steven M

    2016-01-01

    The double-stranded DNA (dsDNA) Myoviridae Bacillus cereus group bacteriophage SalinJah was isolated from soil collected in Gyeonggi-do, South Korea. SalinJah, a cluster C phage with a broad host range, suggests the need to create a new subcluster with SalinJah and Helga as founding members. PMID:27688335

  6. DNA-Based Methods in the Immunohematology Reference Laboratory

    PubMed Central

    Denomme, Gregory A

    2010-01-01

    Although hemagglutination serves the immunohematology reference laboratory well, when used alone, it has limited capability to resolve complex problems. This overview discusses how molecular approaches can be used in the immunohematology reference laboratory. In order to apply molecular approaches to immunohematology, knowledge of genes, DNA-based methods, and the molecular bases of blood groups are required. When applied correctly, DNA-based methods can predict blood groups to resolve ABO/Rh discrepancies, identify variant alleles, and screen donors for antigen-negative units. DNA-based testing in immunohematology is a valuable tool used to resolve blood group incompatibilities and to support patients in their transfusion needs. PMID:21257350

  7. DNA-based methods in the immunohematology reference laboratory.

    PubMed

    Reid, Marion E; Denomme, Gregory A

    2011-02-01

    Although hemagglutination serves the immunohematology reference laboratory well, when used alone, it has limited capability to resolve complex problems. This overview discusses how molecular approaches can be used in the immunohematology reference laboratory. In order to apply molecular approaches to immunohematology, knowledge of genes, DNA-based methods, and the molecular bases of blood groups are required. When applied correctly, DNA-based methods can predict blood groups to resolve ABO/Rh discrepancies, identify variant alleles, and screen donors for antigen-negative units. DNA-based testing in immunohematology is a valuable tool used to resolve blood group incompatibilities and to support patients in their transfusion needs.

  8. Gene silencing and sex determination by programmed DNA elimination in parasitic nematodes.

    PubMed

    Streit, Adrian; Wang, Jianbin; Kang, Yuanyuan; Davis, Richard E

    2016-08-01

    Maintenance of genome integrity is essential. However, programmed DNA elimination removes specific DNA sequences from the genome during development. DNA elimination occurs in unicellular ciliates and diverse metazoa ranging from nematodes to vertebrates. Two distinct groups of nematodes use DNA elimination to silence germline-expressed genes in the soma (ascarids) or for sex determination (Strongyloides spp.). Data suggest that DNA elimination likely evolved independently in these nematodes. Recent studies indicate that differential CENP-A deposition within chromosomes defines which sequences are retained and lost during Ascaris DNA elimination. Additional studies are needed to determine the distribution, functions, and mechanisms of DNA elimination in nematodes. PMID:27315434

  9. Investigation of human papillomavirus DNA in colorectal carcinomas and adenomas.

    PubMed

    Yavuzer, Dilek; Karadayi, Nimet; Salepci, Taflan; Baloglu, Huseyin; Dabak, Resat; Bayramicli, Oya Uygur

    2011-03-01

    Human papillomavirus (HPV) has been considered to be an etiological agent for anogenital cancers, such as cervical cancer and possibly a subset of cancers of the aerodigestive tract. The aim of the study was to evaluate the presence of human papillomavirus DNA in colorectal carcinomas and adenomas. Formalin-fixed and paraffin-embedded archival tissue samples were used for DNA extraction. One hundred and six colorectal carcinomas and 62 adenomas were screened by nested polymerase chain reaction (PCR) for HPV DNA with a control group of 49 cervical tissues with invasive cervical carcinoma and cervical intraepithelial neoplasia (CIN). In the study group, we did not find HPV DNA positivity in any of all the colorectal carcinomas and adenomas. In the control group with cervical lesions, 34 out of 49 (69.4%) samples were positive for the HPV DNA. These results indicated that there was no correlation between HPV infection and colorectal carcinomas and adenomas. PMID:20082157

  10. Standard atomic volumes in double-stranded DNA and packing in protein--DNA interfaces.

    PubMed

    Nadassy, K; Tomás-Oliveira, I; Alberts, I; Janin, J; Wodak, S J

    2001-08-15

    Standard volumes for atoms in double-stranded B-DNA are derived using high resolution crystal structures from the Nucleic Acid Database (NDB) and compared with corresponding values derived from crystal structures of small organic compounds in the Cambridge Structural Database (CSD). Two different methods are used to compute these volumes: the classical Voronoi method, which does not depend on the size of atoms, and the related Radical Planes method which does. Results show that atomic groups buried in the interior of double-stranded DNA are, on average, more tightly packed than in related small molecules in the CSD. The packing efficiency of DNA atoms at the interfaces of 25 high resolution protein-DNA complexes is determined by computing the ratios between the volumes of interfacial DNA atoms and the corresponding standard volumes. These ratios are found to be close to unity, indicating that the DNA atoms at protein-DNA interfaces are as closely packed as in crystals of B-DNA. Analogous volume ratios, computed for buried protein atoms, are also near unity, confirming our earlier conclusions that the packing efficiency of these atoms is similar to that in the protein interior. In addition, we examine the number, volume and solvent occupation of cavities located at the protein-DNA interfaces and compared them with those in the protein interior. Cavities are found to be ubiquitous in the interfaces as well as inside the protein moieties. The frequency of solvent occupation of cavities is however higher in the interfaces, indicating that those are more hydrated than protein interiors. Lastly, we compare our results with those obtained using two different measures of shape complementarity of the analysed interfaces, and find that the correlation between our volume ratios and these measures, as well as between the measures themselves, is weak. Our results indicate that a tightly packed environment made up of DNA, protein and solvent atoms plays a significant role in

  11. Interagency mechanical operations group numerical systems group

    SciTech Connect

    1997-09-01

    This report consists of the minutes of the May 20-21, 1971 meeting of the Interagency Mechanical Operations Group (IMOG) Numerical Systems Group. This group looks at issues related to numerical control in the machining industry. Items discussed related to the use of CAD and CAM, EIA standards, data links, and numerical control.

  12. Group Dynamic Processes in Email Groups

    ERIC Educational Resources Information Center

    Alpay, Esat

    2005-01-01

    Discussion is given on the relevance of group dynamic processes in promoting decision-making in email discussion groups. General theories on social facilitation and social loafing are considered in the context of email groups, as well as the applicability of psychodynamic and interaction-based models. It is argued that such theories may indeed…

  13. Facilitating Reminiscence Groups: Perceptions of Group Leaders

    ERIC Educational Resources Information Center

    Christensen, Teresa M.; Hulse-Killacky, Diana; Salgado, Roy A.; Thornton, Mark D.; Miller, Jason L.

    2006-01-01

    This article presents the results of a two-year qualitative investigation in which group leaders provided their perceptions of the process of facilitating reminiscence groups with elderly persons in a residential care facility. Group Culture emerged as the dominant construct. Findings from this study can serve guide leaders who are interested in…

  14. Many Ways to Loop DNA

    PubMed Central

    Griffith, Jack D.

    2013-01-01

    In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered. PMID:24005675

  15. DNA repair in Chromobacterium violaceum.

    PubMed

    Duarte, Fábio Teixeira; Carvalho, Fabíola Marques de; Bezerra e Silva, Uaska; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella; Batistuzzo de Medeiros, Silvia Regina

    2004-03-31

    Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.

  16. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

    PubMed Central

    Vernon, Matthew M.; Dean, David A.; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  17. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons.

    PubMed

    Vernon, Matthew M; Dean, David A; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  18. Quantitative analysis of cell-free DNA in ovarian cancer

    PubMed Central

    SHAO, XUEFENG; He, YAN; JI, MIN; CHEN, XIAOFANG; QI, JING; SHI, WEI; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ovarian tumor cases, and 19 healthy, non-cancerous ovaries. bDNA techniques were used to detect serum cf-DNA concentrations. All data were analyzed using SPSS version 18.0. The cf-DNA levels were significantly increased in the ovarian cancer group compared with those of the benign ovarian tumor group and healthy ovarian group (P<0.01). Furthermore, cf-DNA levels were significantly increased in stage III and IV ovarian cancer compared with those of stages I and II (P<0.01). In addition, cf-DNA levels were significantly increased on the first day post-surgery (P<0.01), and subsequently demonstrated a gradual decrease. In the ovarian cancer group, the area under the receiver operating characteristic curve of cf-DNA and the sensitivity were 0.917 and 88.9%, respectively, which was higher than those of cancer antigen 125 (0.724, 75%) and human epididymis protein 4 (0.743, 80.6%). There was a correlation between the levels of serum cf-DNA and the occurrence and development of ovarian cancer in the patients evaluated. bDNA techniques possessed higher sensitivity and specificity than other methods for the detection of serum cf-DNA in patients exhibiting ovarian cancer, and bDNA techniques are more useful for detecting cf-DNA than other factors. Thus, the present study demonstrated the potential value for the use of bDNA as an adjuvant diagnostic method for ovarian cancer. PMID:26788153

  19. Routine DNA testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

  20. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  1. Translesion DNA synthesis

    PubMed Central

    Vaisman, Alexandra; McDonald, John P.; Woodgate, Roger

    2014-01-01

    All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell’s replicase. Under these situations, cells are forced to choose between recombination-mediated “damage avoidance” pathways, or use a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions, but also downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases. PMID:26442823

  2. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530

  3. Characterization of muntjac DNA

    SciTech Connect

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  4. Curating DNA specimens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA data are used in a variety of ethnobiological disciplines including archaeology, conservation, ecology, medicinal plants and natural products research, taxonomy and systematics, crop evolution and domestication, and genetic diversity. It frequently is convenient to store and share DNA among coop...

  5. Modeling DNA Replication Intermediates

    SciTech Connect

    Broyde, S.; Roy, D.; Shapiro, R.

    1997-06-01

    While there is now available a great deal of information on double stranded DNA from X-ray crystallography, high resolution NMR and computer modeling, very little is known about structures that are representative of the DNA core of replication intermediates. DNA replication occurs at a single strand/double strand junction and bulged out intermediates near the junction can lead to frameshift mutations. The single stranded domains are particularly challenging. Our interest is focused on strategies for modeling the DNA of these types of replication intermediates. Modeling such structures presents special problems in addressing the multiple minimum problem and in treating the electrostatic component of the force field. We are testing a number of search strategies for locating low energy structures of these types and we are also investigating two different distance dependent dielectric functions in the coulombic term of the force field. We are studying both unmodified DNA and DNA damaged by aromatic amines, carcinogens present in the environment in tobacco smoke, barbecued meats and automobile exhaust. The nature of the structure adopted by the carcinogen modified DNA at the replication fork plays a key role in determining whether the carcinogen will cause a mutation during replication that can initiate the carcinogenic process. In the present work results are presented for unmodified DNA.

  6. Human Mitochondrial DNA Replication

    PubMed Central

    Holt, Ian J.; Reyes, Aurelio

    2012-01-01

    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  7. Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos

    PubMed Central

    Zhang, Sheng; Chen, Xin; Wang, Fang; An, Xinglan; Tang, Bo; Zhang, Xueming; Sun, Liguang; Li, Ziyi

    2016-01-01

    DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner. PMID:27456302

  8. Premeltons in DNA.

    PubMed

    Sobell, Henry M

    2016-03-01

    Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible--and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5' and 3' ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions--all readily testable--as will be described in this review.

  9. Premeltons in DNA.

    PubMed

    Sobell, Henry M

    2016-03-01

    Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible--and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5' and 3' ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions--all readily testable--as will be described in this review

  10. Double-stranded DNA stereoselectively promotes aggregation of amyloid-like fibrils and generates peptide/DNA matrices.

    PubMed

    Yamada, Masanori; Hara, Sachiko; Yamada, Tetsuya; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi

    2014-11-01

    An amyloidogenic LAM-L peptide (AASIKVAVSADR, all-L configuration) derived from laminin promoted cell adhesion, neurite outgrowth, and angiogenesis. Here, we prepared novel matrices using double-stranded DNA and the LAM-L peptide. Double-stranded DNA promoted aggregation of amyloid-like fibrils and generated a LAM-L/DNA matrix through electrostatic interactions between the phosphate groups of DNA and the amino groups of LAM-L. This formation of peptide/DNA matrix depends on the Ile-Lys-Val-Ala-Val (IKVAV) sequence in the peptide, since LAM-RM peptide (AASVVIAKSADR), which is scrambled peptide of LAM-L, did not form a matrix with DNA. Further, LAM-D (all-D configuration of LAM-L), which forms amyloid-like fibrils and promotes similar biological activities as LAM-L, did not form amyloid-like fibrils with DNA, suggesting that DNA selectively interacts with the L-configured peptide. Moreover, the LAM-L/DNA matrices showed stronger cell attachment activity compared with LAM-L alone, suggesting the LAM-L/DNA matrices have potential for use as a novel biomaterial in tissue engineering.

  11. DNA Barcodes for Nearctic Auchenorrhyncha (Insecta: Hemiptera)

    PubMed Central

    Foottit, Robert G.; Maw, Eric; Hebert, P. D. N.

    2014-01-01

    Background Many studies have shown the suitability of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene as a DNA barcode for the identification of species in a wide range of animal groups. We examined 471 species in 147 genera of Hemiptera: Auchenorrhyncha drawn from specimens in the Canadian National Collection of Insects to assess the effectiveness of DNA barcoding in this group. Methodology/Principal Findings Analysis of the COI gene revealed less than 2% intra-specific divergence in 93% of the taxa examined, while minimum interspecific distances exceeded 2% in 70% of congeneric species pairs. Although most species are characterized by a distinct sequence cluster, sequences for members of many groups of closely related species either shared sequences or showed close similarity, with 25% of species separated from their nearest neighbor by less than 1%. Conclusions/Significance This study, although preliminary, provides DNA barcodes for about 8% of the species of this hemipteran suborder found in North America north of Mexico. Barcodes can enable the identification of many species of Auchenorrhyncha, but members of some species groups cannot be discriminated. Future use of DNA barcodes in regulatory, pest management, and environmental applications will be possible as the barcode library for Auchenorrhyncha expands to include more species and broader geographic coverage. PMID:25004106

  12. Parametric resonance in DNA.

    PubMed

    Lacitignola, Deborah; Saccomandi, Giuseppe

    2014-03-01

    We consider a simple mesoscopic model of DNA in which the binding of the RNA polymerase enzyme molecule to the promoter sequence of the DNA is included through a substrate energy term modeling the enzymatic interaction with the DNA strands. We focus on the differential system for solitary waves and derive conditions--in terms of the model parameters--for the occurrence of the parametric resonance phenomenon. We find that what truly matters for parametric resonance is not the ratio between the strength of the stacking and the inter-strand forces but the ratio between the substrate and the inter-strands. On the basis of these results, the standard objection that longitudinal motion is negligible because of the second order seems to fail, suggesting that all the studies involving the longitudinal degree of freedom in DNA should be reconsidered when the interaction of the RNA polymerase with the DNA macromolecule is not neglected. PMID:24510728

  13. Advances in DNA photonics

    NASA Astrophysics Data System (ADS)

    Heckman, Emily M.; Aga, Roberto S.; Fehrman Cory, Emily M.; Ouchen, Fahima; Lesko, Alyssa; Telek, Brian; Lombardi, Jack; Bartsch, Carrie M.; Grote, James G.

    2012-10-01

    In this paper we present our current research in exploring a DNA biopolymer for photonics applications. A new processing technique has been adopted that employs a modified soxhlet-dialysis (SD) rinsing technique to completely remove excess ionic contaminants from the DNA biopolymer, resulting in a material with greater mechanical stability and enhanced performance reproducibility. This newly processed material has been shown to be an excellent material for cladding layers in poled polymer electro-optic (EO) waveguide modulator applications. Thin film poling results are reported for materials using the DNA biopolymer as a cladding layer, as are results for beam steering devices also using the DNA biopolymer. Finally, progress on fabrication of a Mach Zehnder EO modulator with DNA biopolymer claddings using nanoimprint lithography techniques is reported.

  14. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

    PubMed Central

    Hayner, Jaclyn N.; Douma, Lauren G.; Bloom, Linda B.

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. PMID:25159615

  15. Structural features of DNA interaction with caffeine and theophylline

    NASA Astrophysics Data System (ADS)

    Nafisi, Shohreh; Manouchehri, Firouzeh; Tajmir-Riahi, Heidar-Ali; Varavipour, Maryam

    2008-03-01

    Caffeine and theophylline are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these xanthine derivatives with individual DNA at molecular level. The aim of this study was to examine the stability and structural features of calf-thymus DNA complexes with caffeine and theophylline in aqueous solution, using constant DNA concentration (6.25 mM) and various caffeine or theophylline/DNA(P) ratios of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. FTIR, UV-visible spectroscopic methods were used to determine the ligand external binding modes, the binding constant and the stability of caffeine, theophylline-DNA complexes in aqueous solution. Spectroscopic evidence showed that the complexation of caffeine and theophylline with DNA occurred via G-C and A-T and PO 2 group with overall binding constants of K(caffeine-DNA) = 9.7 × 10 3 M -1 and K(theophylline-DNA) = 1.7 × 10 4 M -1. The affinity of ligand-DNA binding is in the order of theophylline > caffeine. A partial B to A-DNA transition occurs upon caffeine and theophylline complexation.

  16. A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody.

    PubMed

    Yoo, S K; Yoon, M; Park, U J; Han, H S; Kim, J H; Hwang, H J

    1999-09-30

    High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA. PMID:10551259

  17. DNA supercoiling and its role in DNA decatenation and unknotting

    PubMed Central

    Witz, Guillaume; Stasiak, Andrzej

    2010-01-01

    Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supercoiled which protects it from thermal denaturation. While the role of DNA supercoiling connected to the control of DNA stability, is thoroughly researched and subject of many reviews, a less known role of DNA supercoiling emerges and consists of aiding DNA topoisomerases in DNA decatenation and unknotting. Although DNA catenanes are natural intermediates in the process of DNA replication of circular DNA molecules, it is necessary that they become very efficiently decatenated, as otherwise the segregation of freshly replicated DNA molecules would be blocked. DNA knots arise as by-products of topoisomerase-mediated intramolecular passages that are needed to facilitate general DNA metabolism, including DNA replication, transcription or recombination. The formed knots are, however, very harmful for cells if not removed efficiently. Here, we overview the role of DNA supercoiling in DNA unknotting and decatenation. PMID:20026582

  18. A DNA structural atlas for Escherichia coli.

    PubMed

    Pedersen, A G; Jensen, L J; Brunak, S; Staerfeldt, H H; Ussery, D W

    2000-06-16

    location of promoters at the apex of plectonemically supercoiled DNA. We have also analyzed the structural similarities between groups of genes by clustering all RNA and protein-encoding genes in E. coli, based on the average structural parameters. We find that most ribosomal genes (protein-encoding as well as rRNA genes) cluster together, and we suggest that DNA structure may play a role in the transcription of these highly expressed genes.

  19. Immune response to firefly luciferase as a naked DNA.

    PubMed

    Jeon, Yong Hyun; Choi, Yun; Kang, Joo Hyun; Kim, Chul Woo; Jeong, Jae Min; Lee, Dong Soo; Chung, June-Key

    2007-05-01

    Firefly luciferase (Fluc) has been widely used as a reporter gene. The aim of this study was to investigate immune response to luciferase protein after an intradermal injection of pcDNA3.1-Fluc in immunocompetent BALB/c mice. We observed bioluminescence at injection sites from one to seven days post-injection when pcDNA3.1-Fluc was intradermally injected into ear-pinnae. To observe induced immune response, the percentages of CD8+IFNgamma+ cells in the draining lymphoid cells of immunocompetent BALB/c mice immunized by pcDNA3.1-Fluc were measured. And the tumor growths of CT26/Fluc in pcDNA3.1-Fluc group were monitored by observing bioluminescent signals and measuring tumor mass, and these were compared with those of the pcDNA3.1 group in immunocompetent BALB/c mice and immunodeficient Nu/Nu mice. In the immunocompetent BALB/c mice, percentages of CD8+IFNgamma+ cells in the pcDNA3.1-Fluc group were higher than those in the pcDNA3.1 group. Ten days after tumor inoculation, tumor growth inhibition was found in the pcDNA3.1-Fluc group, but not in the pcDNA3.1 group in the immunocompetent BALB/c mice. No significant difference in tumor growth inhibition was observed when CT26/Fluc was injected into immunodeficient Nu/Nu mice. In terms of cytokine profiles of draining lymphoid cells of immunized mice, IFNgamma protein levels in the pcDNA3.1-Fluc group were higher than in pcDNA3.1 group animals among the immunocompetent BALB/c mice. In conclusion, Fluc induced a Th1 immune response to Fluc protein delivered by injecting pcDNA3.1-Fluc into immunocompetent BALB/c mice. We suggest that immune response to the Fluc gene is cautionary in preclinical or clinical trials involving the Fluc gene, and that the immunologic potential of firefly luciferase as a naked DNA may be useful in cancer immunotherapy.

  20. 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)

  1. Studying DNA in the Classroom.

    ERIC Educational Resources Information Center

    Zarins, Silja

    1993-01-01

    Outlines a workshop for teachers that illustrates a method of extracting DNA and provides instructions on how to do some simple work with DNA without sophisticated and expensive equipment. Provides details on viscosity studies and breaking DNA molecules. (DDR)

  2. One-electron oxidation of DNA: mechanism and consequences.

    PubMed

    Schuster, Gary B

    2009-01-01

    All living organisms store the information necessary to maintain life in their DNA. Any process that damages DNA and causes loss or corruption of that information threatens the viability of the organism. One-electron oxidation is such a process. Loss of an electron from DNA generates a radical cation that is located primarily on its nucleobases. The radical cation migrates reversibly through duplex DNA by hopping until it is eventually trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In its normal aqueous solutions, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counter ions (typically Na(+)) to the phosphate groups play an important role in facilitating both the migration of the radical cation and in its eventual reaction with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA that is comprised of the four common DNA nucleobases, reaction occurs most commonly at a guanine and results in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step primarily by a tandem process. The general outcomes revealed in the one-electron oxidation of DNA oligomers in solution appear to be generally valid also for more complex DNA structures and for the cellular DNA of living organisms.

  3. Developmental validation of the ParaDNA(®) Intelligence System-A novel approach to DNA profiling.

    PubMed

    Blackman, Stephen; Dawnay, Nick; Ball, Glyn; Stafford-Allen, Beccy; Tribble, Nicholas; Rendell, Paul; Neary, Kelsey; Hanson, Erin K; Ballantyne, Jack; Kallifatidis, Beatrice; Mendel, Julian; Mills, DeEtta K; Wells, Simon

    2015-07-01

    DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA(®) Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75min. The test uses direct PCR with fluorescent HyBeacon(®) detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI). PMID:25980941

  4. Developmental validation of the ParaDNA(®) Intelligence System-A novel approach to DNA profiling.

    PubMed

    Blackman, Stephen; Dawnay, Nick; Ball, Glyn; Stafford-Allen, Beccy; Tribble, Nicholas; Rendell, Paul; Neary, Kelsey; Hanson, Erin K; Ballantyne, Jack; Kallifatidis, Beatrice; Mendel, Julian; Mills, DeEtta K; Wells, Simon

    2015-07-01

    DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA(®) Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75min. The test uses direct PCR with fluorescent HyBeacon(®) detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI).

  5. Hierarchical-Multiplex DNA Patterns Mediated by Polymer Brush Nanocone Arrays That Possess Potential Application for Specific DNA Sensing.

    PubMed

    Liu, Wendong; Liu, Xueyao; Ge, Peng; Fang, Liping; Xiang, Siyuan; Zhao, Xiaohuan; Shen, Huaizhong; Yang, Bai

    2015-11-11

    This paper provides a facile and cost-efficient method to prepare single-strand DNA (ssDNA) nanocone arrays and hierarchical DNA patterns that were mediated by poly(2-hydroxyethyl methacrylate) (PHEMA) brush. The PHEMA brush nanocone arrays with different morphology and period were fabricated via colloidal lithography. The hierarchical structure was prepared through the combination of colloidal lithography and traditional photolithography. The DNA patterns were easily achieved via grafting the amino group modified ssDNA onto the side chain of polymer brush, and the anchored DNA maintained their reactivity. The as-prepared ssDNA nanocone arrays can be applied for target DNA sensing with the detection limit reaching 1.65 nM. Besides, with the help of introducing microfluidic ideology, the hierarchical-multiplex DNA patterns on the same substrate could be easily achieved with each kind of pattern possessing one kind of ssDNA, which are promising surfaces for the preparation of rapid, visible, and multiplex DNA sensors.

  6. Functionalization and self-assembly of DNA bidimensional arrays.

    PubMed

    Garibotti, Alejandra V; Pérez-Rentero, Sónia; Eritja, Ramon

    2011-01-01

    Oligonucleotides carrying amino, thiol groups, as well as fluorescein, c-myc peptide sequence and nanogold at internal positions were prepared and used for the assembly of bidimensional DNA arrays. PMID:22016615

  7. Quantitive DNA Fiber Mapping

    SciTech Connect

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  8. DNA vaccines against influenza.

    PubMed

    Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka

    2014-01-01

    Genetic vaccine technology has been considerably developed within the last two decades. This cost effective and promising strategy can be applied for therapy of cancers and for curing allergy, chronic and infectious diseases, such as a seasonal and pandemic influenza. Despite numerous advantages, several limitations of this technology reduce its performance and can retard its commercial exploitation in humans and its veterinary applications. Inefficient delivery of the DNA vaccine into cells of immunized individuals results in low intracellular supply of suitable expression cassettes encoding an antigen, in its low expression level and, in turn, in reduced immune responses against the antigen. Improvement of DNA delivery into the host cells might significantly increase effectiveness of the DNA vaccine. A vast array of innovative methods and various experimental strategies have been applied in order to enhance the effectiveness of DNA vaccines. They include various strategies improving DNA delivery as well as expression and immunogenic potential of the proteins encoded by the DNA vaccines. Researchers focusing on DNA vaccines against influenza have applied many of these strategies. Recent examples of the most successful modern approaches are discussed in this review.

  9. HIV DNA Integration

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2012-01-01

    Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV integration is strongly favored in active transcription units, which may promote efficient viral gene expression after integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified. PMID:22762018

  10. Reversible DNA compaction.

    PubMed

    González-Pérez, Alfredo

    2014-01-01

    In this review we summarize and discuss the different methods we can use to achieve reversible DNA compaction in vitro. Reversible DNA compaction is a natural process that occurs in living cells and viruses. As a result these process long sequences of DNA can be concentrated in a small volume (compacted) to be decompacted only when the information carried by the DNA is needed. In the current work we review the main artificial compacting agents looking at their suitability for decompaction. The different approaches used for decompaction are strongly influenced by the nature of the compacting agent that determines the mechanism of compaction. We focus our discussion on two main artificial compacting agents: multivalent cations and cationic surfactants that are the best known compacting agents. The reversibility of the process can be achieved by adding chemicals like divalent cations, alcohols, anionic surfactants, cyclodextrins or by changing the chemical nature of the compacting agents via pH modifications, light induced conformation changes or by redox-reactions. We stress the relevance of electrostatic interactions and self-assembly as a main approach in order to tune up the DNA conformation in order to create an on-off switch allowing a transition between coil and compact states. The recent advances to control DNA conformation in vitro, by means of molecular self-assembly, result in a better understanding of the fundamental aspects involved in the DNA behavior in vivo and serve of invaluable inspiration for the development of potential biomedical applications. PMID:24444152

  11. DNA-PK assay

    DOEpatents

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  12. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

    PubMed Central

    Maresca, Alessandra; Zaffagnini, Mirko; Caporali, Leonardo; Carelli, Valerio; Zanna, Claudia

    2015-01-01

    Autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN) and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E) are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5)-methyltransferase 1 (DNMT1). DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy, and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA) suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however, mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is regulated and

  13. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  14. A search for specificity in DNA-drug interactions.

    PubMed

    Cruciani, G; Goodford, P J

    1994-06-01

    The GRID force field and a principal component analysis have been used in order to predict the interactions of small chemical groups with all 64 different triplet sequences of B-DNA. Factors that favor binding to guanine-cytosine base pairs have been identified, and a dictionary of ligand groups and their locations is presented as a guide to the design of specific DNA ligands. PMID:7918250

  15. A search for specificity in DNA-drug interactions.

    PubMed

    Cruciani, G; Goodford, P J

    1994-06-01

    The GRID force field and a principal component analysis have been used in order to predict the interactions of small chemical groups with all 64 different triplet sequences of B-DNA. Factors that favor binding to guanine-cytosine base pairs have been identified, and a dictionary of ligand groups and their locations is presented as a guide to the design of specific DNA ligands.

  16. DNA Mismatch Repair

    PubMed Central

    MARINUS, M. G.

    2014-01-01

    DNA mismatch repair functions to correct replication errors in newly synthesized DNA and to prevent recombination between related, but not identical (homeologous), DNA sequences. The mechanism of mismatch repair is best understood in Escherichia coli and is the main focus of this review. The early genetic studies of mismatch repair are described as a basis for the subsequent biochemical characterization of the system. The effects of mismatch repair on homologous and homeologous recombination are described. The relationship of mismatch repair to cell toxicity induced by various drugs is included. The VSP (Very Short Patch) repair system is described in detail. PMID:26442827

  17. Focus: DNA probes

    SciTech Connect

    Not Available

    1986-11-01

    Progress in the development of DNA probes for the identification and quantitation of specific genetic sequences in biological samples is reviewed. Current research efforts in the development of DNA probes for the diagnosis of a wide variety of bacterial, viral, and other infectious diseases, such as herpes simplex and cytomegalovirus, and inherited genetic diseases such as cystic fibrosis and sickle cell anemia are discussed. Progress in development of DNA probe assays for cancer diagnosis, detection of Salmonella food poisoning, tissue typing (detection of histocompatibility antigens), mutagen screening, and animal diseases, among other applications is included.

  18. DNA damage induction of ribonucleotide reductase.

    PubMed

    Elledge, S J; Davis, R W

    1989-11-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. PMID:2513480

  19. Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

    NASA Technical Reports Server (NTRS)

    Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

    1982-01-01

    The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

  20. The interaction of amino acids, peptides, and proteins with DNA.

    PubMed

    Solovyev, Andrey Y; Tarnovskaya, Svetlana I; Chernova, Irina A; Shataeva, Larisa K; Skorik, Yury A

    2015-01-01

    Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby strengthening the DNA helix.

  1. Group Work: How to Use Groups Effectively

    ERIC Educational Resources Information Center

    Burke, Alison

    2011-01-01

    Many students cringe and groan when told that they will need to work in a group. However, group work has been found to be good for students and good for teachers. Employers want college graduates to have developed teamwork skills. Additionally, students who participate in collaborative learning get better grades, are more satisfied with their…

  2. Structure of large dsDNA viruses

    PubMed Central

    Klose, Thomas; Rossmann, Michael G.

    2015-01-01

    Nucleocytoplasmic large dsDNA viruses (NCLDVs) encompass an ever-increasing group of large eukaryotic viruses, infecting a wide variety of organisms. The set of core genes shared by all these viruses includes a major capsid protein with a double jelly-roll fold forming an icosahedral capsid, which surrounds a double layer membrane that contains the viral genome. Furthermore, some of these viruses, such as the members of the Mimiviridae and Phycodnaviridae have a unique vertex that is used during infection to transport DNA into the host. PMID:25003382

  3. DNA Damage among Wood Workers Assessed with the Comet Assay.

    PubMed

    Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B

    2016-01-01

    Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers' exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products.

  4. DNA Damage among Wood Workers Assessed with the Comet Assay

    PubMed Central

    Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B.

    2016-01-01

    Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers’ exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

  5. DNA Damage among Wood Workers Assessed with the Comet Assay.

    PubMed

    Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B

    2016-01-01

    Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers' exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

  6. Microscopic and spectroscopic analysis of chitosan-DNA conjugates.

    PubMed

    Agudelo, D; Kreplak, L; Tajmir-Riahi, H A

    2016-02-10

    Conjugations of DNA with chitosans 15 kD (ch-15), 100 kD (ch-100) and 200 kD (ch-200) were investigated in aqueous solution at pH 5.5-6.5. Multiple spectroscopic methods and atomic force microscopy (AFM) were used to locate the chitosan binding sites and the effect of polymer conjugation on DNA compaction and particle formation. Structural analysis showed that chitosan-DNA conjugation is mainly via electrostatic interactions through polymer cationic charged NH2 and negatively charged backbone phosphate groups. As polymer size increases major DNA compaction and particle formation occurs. At high chitosan concentration major DNA structural changes observed indicating a partial B to A-DNA conformational transition.

  7. African Mitochondrial DNA Subhaplogroups and Peripheral Neuropathy during Antiretroviral Therapy

    PubMed Central

    Canter, Jeffrey A.; Robbins, Gregory K.; Selph, Doug; Clifford, David B.; Kallianpur, Asha R.; Shafer, Robert; Levy, Shawn; Murdock, Deborah G.; Ritchie, Marylyn D.; Haas, David W.; Hulgan, Todd

    2010-01-01

    Susceptibility to peripheral neuropathy during antiretroviral therapy with nucleoside reverse transcriptase inhibitors (NRTIs) was previously associated with a European mitochondrial DNA (mtDNA) haplogroup among non-Hispanic white persons. To determine if NRTI-associated peripheral neuropathy was related to mtDNA variation in non-Hispanic black persons, we sequenced mtDNA of participants from AIDS Clinical Trials Group study 384. Of 156 non-Hispanic blacks with genomic data, 51 (33%) developed peripheral neuropathy. In a multivariate model, African mtDNA subhaplogroup L1c was an independent predictor of peripheral neuropathy (OR=3.7, 95% CI 1.1-12.0). An African mtDNA subhaplogroup is for the first time implicated in susceptibility to NRTI-associated toxicity. PMID:20402593

  8. Archaeal DNA polymerases in biotechnology.

    PubMed

    Zhang, Likui; Kang, Manyu; Xu, Jiajun; Huang, Yanchao

    2015-08-01

    DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols.

  9. MSUD Family Support Group

    MedlinePlus

    ... Group The MSUD Family Support Group is a non-profit 501 (c)(3) organization for those with MSUD ... Family Support Group is a 501(c)(3) non-profit organization with no paid staff. Funds are needed ...

  10. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

    SciTech Connect

    Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-05-17

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  11. Retroviral DNA Integration

    PubMed Central

    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  12. DNA vaccines: a review.

    PubMed

    Liu, M A

    2003-04-01

    The DNA vaccines are simple rings of DNA containing a gene encoding an antigen, and a promoter/terminator to make the gene express in mammalian cells. They are a promising new approach for generating all types of desired immunity: cytolytic T lymphocytes (CTL), T helper cells and antibodies, whilst being a technology that has the potential for global usage in terms of manufacturing ease, broad population administration and safety. This review gives an overview of the mechanisms, preclinical and clinical efficacy of DNA vaccines, and point out the limitations of the first generation of such vaccines, and some of the promising second-generation developments. This technology is also being utilized in the field of proteomics as a tool to elucidate the function of genes. The breadth of applications for DNA vaccines thus ranges from prophylactic vaccines to immunotherapy for infectious diseases, cancer, and autoimmune and allergic diseases. PMID:12653868

  13. Multiplex analysis of DNA

    DOEpatents

    Church, George M.; Kieffer-Higgins, Stephen

    1992-01-01

    This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.

  14. DNA damage and carcinogenesis

    SciTech Connect

    Stelow, R B

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10/sup 4/ fold.

  15. Close encounters with DNA

    PubMed Central

    Maffeo, C.; Yoo, J.; Comer, J.; Wells, D. B.; Luan, B.; Aksimentiev, A.

    2014-01-01

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena and we review the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field. PMID:25238560

  16. Forensic DNA and bioinformatics.

    PubMed

    Bianchi, Lucia; Liò, Pietro

    2007-03-01

    The field of forensic science is increasingly based on biomolecular data and many European countries are establishing forensic databases to store DNA profiles of crime scenes of known offenders and apply DNA testing. The field is boosted by statistical and technological advances such as DNA microarray sequencing, TFT biosensors, machine learning algorithms, in particular Bayesian networks, which provide an effective way of evidence organization and inference. The aim of this article is to discuss the state of art potentialities of bioinformatics in forensic DNA science. We also discuss how bioinformatics will address issues related to privacy rights such as those raised from large scale integration of crime, public health and population genetic susceptibility-to-diseases databases.

  17. Making DNA Fingerprints.

    ERIC Educational Resources Information Center

    Nunley, Kathie F.

    1996-01-01

    Presents an activity to simulate electrophoresis using everyday items. Uses adding machine paper to construct a set of DNA fingerprints that can be used to solve crime cases designed by students in any biology class. (JRH)

  18. DNA sequencing conference, 2

    SciTech Connect

    Cook-Deegan, R.M.; Venter, J.C.; Gilbert, W.; Mulligan, J.; Mansfield, B.K.

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  19. FBI's DNA analysis program

    NASA Astrophysics Data System (ADS)

    Brown, John R.

    1994-03-01

    Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

  20. Aniline mustard analogues of the DNA-intercalating agent amsacrine: DNA interaction and biological activity.

    PubMed

    Fan, J Y; Valu, K K; Woodgate, P D; Baguley, B C; Denny, W A

    1997-04-01

    Two series of analogues of the clinical antileukemic drug and DNA-intercalating ligand amsacrine have been prepared, containing aniline mustard sidechains of varying reactivity, linked either at the 4-position of the intercalating acridine chromophore (type A) or at the 1'-position of the 9-anilino group (type B). DNase I footprinting assays showed that compounds of type B had stronger reversible binding to DNA than did compounds of type A. Compounds of each type showed similar patterns of alkylation-induced cleavage of DNA, and alkylate at the N7 of guanines in runs of guanines (similar to the pattern for untargeted mustards) as well as some adenines. Both classes of compounds crosslinked DNA, although those bearing relatively inactive mustards did so only at high drug/base pair ratios. However, while the patterns of DNA alkylation were broadly similar, the compounds were considerably more cytotoxic than analogous untargeted mustards. Comparison of their cytotoxicities in wild-type and DNA repair-deficient lines indicated this toxicity was due to DNA crosslinks (except for the least reactive SO2-linked mustards). The 4-linked analogues showed slightly higher in vivo antileukemic activity than the corresponding 1'-linked analogues.

  1. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  2. Expansion of the DNA Alphabet beyond Natural DNA Recognition.

    PubMed

    Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2016-07-15

    Simple and inexpensive DNA fibres: New, stable DNA structures are created by the binding of a small molecule to poly(A). Because these DNA fibres are formed from inexpensive materials by using very simple methods, DNA materials suitable for practical use such as information storage should be possible in the near future. PMID:27061868

  3. Blowing DNA bubbles.

    PubMed

    Severin, N; Zhuang, W; Ecker, C; Kalachev, A A; Sokolov, I M; Rabe, J P

    2006-11-01

    We report here experimental observations which indicate that topologically or covalently formed polymer loops embedded in an ultrathin liquid film on a solid substrate can be "blown" into circular "bubbles" during scanning force microscopy (SFM) imaging. In particular, supercoiled vector DNA has been unraveled, moved, stretched, and overstretched to two times its B-form length and then torn apart. We attribute the blowing of the DNA bubbles to the interaction of the tapping SFM tip with the ultrathin liquid film.

  4. Das DNA-Puzzle

    NASA Astrophysics Data System (ADS)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  5. Database for bacterial group II introns.

    PubMed

    Candales, Manuel A; Duong, Adrian; Hood, Keyar S; Li, Tony; Neufeld, Ryan A E; Sun, Runda; McNeil, Bonnie A; Wu, Li; Jarding, Ashley M; Zimmerly, Steven

    2012-01-01

    The Database for Bacterial Group II Introns (http://webapps2.ucalgary.ca/~groupii/index.html#) provides a catalogue of full-length, non-redundant group II introns present in bacterial DNA sequences in GenBank. The website is divided into three sections. The first section provides general information on group II intron properties, structures and classification. The second and main section lists information for individual introns, including insertion sites, DNA sequences, intron-encoded protein sequences and RNA secondary structure models. The final section provides tools for identification and analysis of intron sequences. These include a step-by-step guide to identify introns in genomic sequences, a local BLAST tool to identify closest intron relatives to a query sequence, and a boundary-finding tool that predicts 5' and 3' intron-exon junctions in an input DNA sequence. Finally, selected intron data can be downloaded in FASTA format. It is hoped that this database will be a useful resource not only to group II intron and RNA researchers, but also to microbiologists who encounter these unexpected introns in genomic sequences.

  6. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

    PubMed Central

    Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

    2015-01-01

    The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

  7. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage.

    PubMed

    Fu, Haiqing; Martin, Melvenia M; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I

    2015-01-01

    The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

  8. Glycidol-carbohydrate hybrids: a new family of DNA alkylating agents.

    PubMed

    Toshima, Kazunobu; Okuno, Yukiko; Matsumura, Shuichi

    2003-10-01

    Novel and chiral glycidol-carbohydrate hybrids possessing an epoxy group as a DNA alkylating moiety were designed and synthesized. These artificial hybrids selectively alkylated DNA at the N-7 sites of the guanines and cleaved DNA without any additives. The binding ability of the glycidol was significantly enhanced by the attachment of the carbohydrate.

  9. DNA as a Binary Code: How the Physical Structure of Nucleotide Bases Carries Information

    ERIC Educational Resources Information Center

    McCallister, Gary

    2005-01-01

    The DNA triplet code also functions as a binary code. Because double-ring compounds cannot bind to double-ring compounds in the DNA code, the sequence of bases classified simply as purines or pyrimidines can encode for smaller groups of possible amino acids. This is an intuitive approach to teaching the DNA code. (Contains 6 figures.)

  10. Influence of killing method on Lepidoptera DNA barcode recovery.

    PubMed

    Willows-Munro, Sandi; Schoeman, M Corrie

    2015-05-01

    The global DNA barcoding initiative has revolutionized the field of biodiversity research. Such large-scale sequencing projects require the collection of large numbers of specimens, which need to be killed and preserved in a way that is both DNA-friendly and which will keep voucher specimens in good condition for later study. Factors such as time since collection, correct storage (exposure to free water and heat) and DNA extraction protocol are known to play a role in the success of downstream molecular applications. Limited data are available on the most efficient, DNA-friendly protocol for killing. In this study, we evaluate the quality of DNA barcode (cytochrome oxidase I) sequences amplified from DNA extracted from specimens collected using three different killing methods (ethyl acetate, cyanide and freezing). Previous studies have suggested that chemicals, such as ethyl acetate and formaldehyde, degraded DNA and as such may not be appropriate for the collection of insects for DNA-based research. All Lepidoptera collected produced DNA barcodes of good quality, and our study found no clear difference in nucleotide signal strength, probability of incorrect base calling and phylogenetic utility among the three different treatment groups. Our findings suggest that ethyl acetate, cyanide and freezing can all be used to collect specimens for DNA analysis.

  11. DNA biosensors that reason.

    PubMed

    Sainz de Murieta, Iñaki; Rodríguez-Patón, Alfonso

    2012-08-01

    Despite the many designs of devices operating with the DNA strand displacement, surprisingly none is explicitly devoted to the implementation of logical deductions. The present article introduces a new model of biosensor device that uses nucleic acid strands to encode simple rules such as "IF DNA_strand(1) is present THEN disease(A)" or "IF DNA_strand(1) AND DNA_strand(2) are present THEN disease(B)". Taking advantage of the strand displacement operation, our model makes these simple rules interact with input signals (either DNA or any type of RNA) to generate an output signal (in the form of nucleotide strands). This output signal represents a diagnosis, which either can be measured using FRET techniques, cascaded as the input of another logical deduction with different rules, or even be a drug that is administered in response to a set of symptoms. The encoding introduces an implicit error cancellation mechanism, which increases the system scalability enabling longer inference cascades with a bounded and controllable signal-noise relation. It also allows the same rule to be used in forward inference or backward inference, providing the option of validly outputting negated propositions (e.g. "diagnosis A excluded"). The models presented in this paper can be used to implement smart logical DNA devices that perform genetic diagnosis in vitro.

  12. Variations in brain DNA

    PubMed Central

    Avila, Jesús; Gómez-Ramos, Alberto; Soriano, Eduardo

    2014-01-01

    It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain) of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain. PMID:25505410

  13. DNA biosensors that reason.

    PubMed

    Sainz de Murieta, Iñaki; Rodríguez-Patón, Alfonso

    2012-08-01

    Despite the many designs of devices operating with the DNA strand displacement, surprisingly none is explicitly devoted to the implementation of logical deductions. The present article introduces a new model of biosensor device that uses nucleic acid strands to encode simple rules such as "IF DNA_strand(1) is present THEN disease(A)" or "IF DNA_strand(1) AND DNA_strand(2) are present THEN disease(B)". Taking advantage of the strand displacement operation, our model makes these simple rules interact with input signals (either DNA or any type of RNA) to generate an output signal (in the form of nucleotide strands). This output signal represents a diagnosis, which either can be measured using FRET techniques, cascaded as the input of another logical deduction with different rules, or even be a drug that is administered in response to a set of symptoms. The encoding introduces an implicit error cancellation mechanism, which increases the system scalability enabling longer inference cascades with a bounded and controllable signal-noise relation. It also allows the same rule to be used in forward inference or backward inference, providing the option of validly outputting negated propositions (e.g. "diagnosis A excluded"). The models presented in this paper can be used to implement smart logical DNA devices that perform genetic diagnosis in vitro. PMID:22406690

  14. Complex kinetics of DNA condensation revealed through DNA twist tracing.

    PubMed

    Li, Wei; Wong, Wei Juan; Lim, Ci Ji; Ju, Hai-Peng; Li, Ming; Yan, Jie; Wang, Peng-Ye

    2015-08-01

    Toroid formation is an important mechanism for DNA condensation in cells. The length change during DNA condensation was investigated in previous single-molecule experiments. However, DNA twist is key to understanding the topological kinetics of DNA condensation. In this study, DNA twist as well as DNA length was traced during the DNA condensation by the freely orbiting magnetic tweezers and the tilted magnetic tweezers combined with Brownian dynamics simulations. The experimental results disclose the complex relationship between DNA extension and backbone rotation. Brownian dynamics simulations show that the toroid formation follows a wiggling pathway which leads to the complex DNA backbone rotation as revealed in our experiments. These findings provide the complete description of multivalent cation-dependent DNA toroid formation under tension.

  15. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  16. DNA vaccines and intradermal vaccination by DNA tattooing.

    PubMed

    Oosterhuis, K; van den Berg, J H; Schumacher, T N; Haanen, J B A G

    2012-01-01

    Over the past two decades, DNA vaccination has been developed as a method for the induction of immune responses. However, in spite of high expectations based on their efficacy in preclinical models, immunogenicity of first generation DNA vaccines in clinical trials was shown to be poor, and no DNA vaccines have yet been licensed for human use. In recent years significant progress has been made in the development of second generation DNA vaccines and DNA vaccine delivery methods. Here we review the key characteristics of DNA vaccines as compared to other vaccine platforms, and recent insights into the prerequisites for induction of immune responses by DNA vaccines will be discussed. We illustrate the development of second generation DNA vaccines with the description of DNA tattooing as a novel DNA delivery method. This technique has shown great promise both in a small animal model and in non-human primates and is currently under clinical evaluation.

  17. Voltammetric detection of damage to DNA caused by nitro derivatives of fluorene using an electrochemical DNA biosensor.

    PubMed

    Vyskocil, Vlastimil; Labuda, Ján; Barek, Jirí

    2010-05-01

    An electrochemical DNA biosensor based on the screen printed carbon paste electrode (SPCPE) with an immobilized layer of calf thymus double-stranded DNA has been used for in vitro investigation of the interaction between genotoxic nitro derivatives of fluorene (namely 2-nitrofluorene and 2,7-dinitrofluorene) and DNA. Two types of DNA damage have been detected at the DNA/SPCPE biosensor: first, that caused by direct association of the nitrofluorenes, for which an intercalation association has been found using the known DNA intercalators [Cu(phen)(2)](2+) and [Co(phen)(3)](3+) as competing agents, and, second, that caused by short-lived radicals generated by electrochemical reduction of the nitro group (observable under specific conditions only). PMID:20186538

  18. A strategy for development of electrochemical DNA biosensor based on site-specific DNA cleavage of restriction endonuclease.

    PubMed

    Chen, Jinghua; Zhang, Jing; Yang, Huanghao; Fu, Fengfu; Chen, Guonan

    2010-09-15

    A new strategy for development of electrochemical DNA biosensor based on site-specific DNA cleavage of restriction endonuclease and using quantum dots as reporter was reported in this paper. The biosensor was fabricated by immobilizing a capture hairpin probe, thiolated single strand DNA labeled with biotin group, on a gold electrode. BfuCI nuclease, which is able to specifically cleave only double strand DNA but not single strand DNA, was used to reduce background current and improve the sensitivity. We demonstrated that the capture hairpin probe can be cleaved by BfuCI nuclease in the absence of target DNA, but cannot be cleaved in the presence of target DNA. The difference before and after enzymatic cleavage was then monitored by electrochemical method after the quantum dots were dissolved from the hybrids. Our results suggested that the usage of BfuCI nuclease obviously improved the sensitivity and selectivity of the biosensor. We successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target DNA including a single-base mismatched target DNA, and detected as low as 3.3 × 10(-14) M of complementary target DNA. Furthermore, our above strategy was also verified with fluorescent method by designing a fluorescent molecular beacon (MB), which combined the capture hairpin probe and a pair of fluorophore (TAMRA) and quencher (DABCYL). The fluorescent results are consistent with that of electroanalysis, further indicating that the proposed new strategy indeed works as we expected.

  19. Nonlinear Model of the Specificity of DNA-Protein Interactions and Its Stability

    NASA Astrophysics Data System (ADS)

    Dwiputra, D.; Hidayat, W.; Khairani, R.; Zen, F. P.

    2016-08-01

    Specific DNA-protein interactions are fundamental processes of living cells. We propose a new model of DNA-protein interactions to explain the site specificity of the interactions. The hydrogen bonds between DNA base pairs and between DNA-protein peptide groups play a significant role in determination of the specific binding site. We adopt the Morse potential with coupling terms to construct the Hamiltonian of coupled oscillators representing the hydrogen bonds in which the depth of the potentials vary in the DNA chain. In this paper we investigate the stability of the model to determine the conditions satisfying the biological circumstances of the DNA-protein interactions.

  20. A compendium of human mitochondrial DNA control region: development of an international standard forensic database.

    PubMed

    Miller, K W; Budowle, B

    2001-06-01

    A compendium of human mitochondrial DNA (mtDNA) control region types has been constructed. This updated compilation indexes over 10,000 population-specific mtDNA nucleotide sequences in a standardized format. The sequences represent mtDNA types from the Scientific Working Group on DNA Analysis Methods (SWGDAM) mtDNA database and from the public literature. The SWGDAM data are considered to be of higher quality than the public data, particularly for counting the number of times a particular haplotype has been observed. PMID:11387646

  1. DNA repair genes in the Megavirales pangenome.

    PubMed

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  2. DNA barcodes for ecology, evolution, and conservation.

    PubMed

    Kress, W John; García-Robledo, Carlos; Uriarte, Maria; Erickson, David L

    2015-01-01

    The use of DNA barcodes, which are short gene sequences taken from a standardized portion of the genome and used to identify species, is entering a new phase of application as more and more investigations employ these genetic markers to address questions relating to the ecology and evolution of natural systems. The suite of DNA barcode markers now applied to specific taxonomic groups of organisms are proving invaluable for understanding species boundaries, community ecology, functional trait evolution, trophic interactions, and the conservation of biodiversity. The application of next-generation sequencing (NGS) technology will greatly expand the versatility of DNA barcodes across the Tree of Life, habitats, and geographies as new methodologies are explored and developed.

  3. Elastic correlations in nucleosomal DNA structure.

    PubMed

    Mohammad-Rafiee, Farshid; Golestanian, Ramin

    2005-06-17

    The structure of DNA in the nucleosome core particle is studied using an elastic model that incorporates anisotropy in the bending energetics and twist-bend coupling. Using the experimentally determined structure of nucleosomal DNA [T. J. Richmond and C. A. Davey, Nature (London) 423, 145 (2003)], it is shown that elastic correlations exist between twist, roll, tilt, and stretching of DNA, as well as the distance between phosphate groups. The twist-bend coupling term is shown to be able to capture these correlations to a large extent, and a fit to the experimental data yields a new estimate of G = 25 nm for the value of the twist-bend coupling constant.

  4. Acoustic stimulation promotes DNA fragmentation in the Guinea pig cochlea.

    PubMed

    Kamio, Tomonobu; Watanabe, Ken-Ichi; Okubo, Kimihiro

    2012-01-01

    Apoptosis can be described as programmed cell death. Apoptosis regulates cell turnover and is involved in various pathological conditions. The characteristic features of apoptosis are shrinkage of the cell body, chromatin condensation, and nucleic acid fragmentation. During apoptosis, double-stranded DNA is broken down into single-stranded DNA (ssDNA) by proteases. Acoustic trauma is commonly encountered in otorhinolaryngology clinics. Intense noise can cause inner ear damage, such as hearing disturbance, tinnitus, ear fullness, and decreased speech discrimination. In this study, we used immunohistochemical and electrophysiological methods to examine the fragmentation of DNA in the cochleas of guinea pigs that had been exposed to intense noise. Twenty-four guinea pigs weighing 250 to 350 g were used. The animals were divided into 4 groups: (I) a control group (n=6), (II) a group that was exposed to noise for 2 hours (n=6), (III) a group that was exposed to noise for 5 hours (n=6), and (IV) a group that was exposed to noise for 20 hours. The stimulus was a pure tone delivered at a frequency of 2 kHz. The sound pressure level was 120 dBSPL. No threshold shifts were apparent in group I. Group II showed a significant elevation of the hearing threshold (ANOVA, p<0.05(*)). The ABR threshold level was also significantly elevated immediately after the acoustic stimulation in groups III and IV (ANOVA, p<0.01(**)). In groups I, II, and IV, the lateral wall of the ear did not show immunoreactivity to ssDNA but did in group III. No immunoreactivity was apparent in the organ of Corti in group I or II. However, the supporting cells and outer hair cells in groups III and IV showed reactions for ssDNA. The fine structure of the organ of Corti had been destroyed in group IV. The lateral wall showed immunoreactivity for ssDNA only in group III, whereas the organ of Corti showed reactions for ssDNA in groups III and IV. Our study suggests that apoptotic changes occur in patients that

  5. Acoustic stimulation promotes DNA fragmentation in the Guinea pig cochlea.

    PubMed

    Kamio, Tomonobu; Watanabe, Ken-Ichi; Okubo, Kimihiro

    2012-01-01

    Apoptosis can be described as programmed cell death. Apoptosis regulates cell turnover and is involved in various pathological conditions. The characteristic features of apoptosis are shrinkage of the cell body, chromatin condensation, and nucleic acid fragmentation. During apoptosis, double-stranded DNA is broken down into single-stranded DNA (ssDNA) by proteases. Acoustic trauma is commonly encountered in otorhinolaryngology clinics. Intense noise can cause inner ear damage, such as hearing disturbance, tinnitus, ear fullness, and decreased speech discrimination. In this study, we used immunohistochemical and electrophysiological methods to examine the fragmentation of DNA in the cochleas of guinea pigs that had been exposed to intense noise. Twenty-four guinea pigs weighing 250 to 350 g were used. The animals were divided into 4 groups: (I) a control group (n=6), (II) a group that was exposed to noise for 2 hours (n=6), (III) a group that was exposed to noise for 5 hours (n=6), and (IV) a group that was exposed to noise for 20 hours. The stimulus was a pure tone delivered at a frequency of 2 kHz. The sound pressure level was 120 dBSPL. No threshold shifts were apparent in group I. Group II showed a significant elevation of the hearing threshold (ANOVA, p<0.05(*)). The ABR threshold level was also significantly elevated immediately after the acoustic stimulation in groups III and IV (ANOVA, p<0.01(**)). In groups I, II, and IV, the lateral wall of the ear did not show immunoreactivity to ssDNA but did in group III. No immunoreactivity was apparent in the organ of Corti in group I or II. However, the supporting cells and outer hair cells in groups III and IV showed reactions for ssDNA. The fine structure of the organ of Corti had been destroyed in group IV. The lateral wall showed immunoreactivity for ssDNA only in group III, whereas the organ of Corti showed reactions for ssDNA in groups III and IV. Our study suggests that apoptotic changes occur in patients that

  6. Chromosomal DNA Replication Pattern in Human Tumour Cells in vitro

    PubMed Central

    Kucheria, Kiran

    1970-01-01

    The present paper deals with the chromosomal DNA replication pattern in human solid tumour cells in vitro. This was studied at the terminal stages of the S-period. All the cell lines of female origin showed a late replicating chromosome in group XX6-12. In cell lines of male origin one of the chromosomes of group 21-22Y was later replicating than the rest of the members of the group. The DNA replication pattern of the autosomes and the sex chromosomes was similar to that of the cultured human leucocytes. The results of the present study show that the DNA replication pattern of the chromosome in neoplastic cells is basically unchanged despite the changes in the chromosome number and morphology. Therefore the abnormal behaviour of the neoplastic cells cannot be related to the changes in the pattern of the chromosomal DNA replication. ImagesFig. 5Fig. 1Fig. 6Fig. 2Fig. 3Fig. 4 PMID:5475754

  7. MtDNA ancestry of Rio de Janeiro population, Brazil.

    PubMed

    Bernardo, Suellen; Hermida, Rose; Desidério, Márcia; Silva, Dayse A; de Carvalho, Elizeu F

    2014-01-01

    Polymorphism studies concerning HVI and HVII regions of mitochondrial DNA (mtDNA) have improved the understanding of the admixture genetic process related to the occupation of the continents by human population groups. We have analyzed the mtDNA lineages of 190 healthy and maternally unrelated individuals born in the metropolitan region of the Rio de Janeiro city, the capital of the State of Rio de Janeiro, southeastern Brazil. The data showing that 57.9, 25.3 and 16.8 % of the matrilineages found in Rio de Janeiro come from African, Amerindian and European population groups. They are, respectively, in close agreement with historical records which indicate that the admixture population of Brazil is the resulting of interethnic asymmetry crosses between individuals from those population groups. The high proportion of African mtDNA lineages in the population of Rio de Janeiro is in accordance with studies related to other Brazilian states.

  8. Simultaneous RNA-DNA FISH.

    PubMed

    Lai, Lan-Tian; Meng, Zhenyu; Shao, Fangwei; Zhang, Li-Feng

    2016-01-01

    A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.

  9. Multicolor and Erasable DNA Photolithography

    PubMed Central

    2015-01-01

    The immobilization of DNA molecules onto a solid support is a crucial step in biochip research and related applications. In this work, we report a DNA photolithography method based on photocleavage of 2-nitrobenzyl linker-modified DNA strands. These strands were subjected to ultraviolet light irradiation to generate multiple short DNA strands in a programmable manner. Coupling the toehold-mediated DNA strand-displacement reaction with DNA photolithography enabled the fabrication of a DNA chip surface with multifunctional DNA patterns having complex geometrical structures at the microscale level. The erasable DNA photolithography strategy was developed to allow different paintings on the same chip. Furthermore, the asymmetrical modification of colloidal particles was carried out by using this photolithography strategy. This strategy has broad applications in biosensors, nanodevices, and DNA-nanostructure fabrication. PMID:24988147

  10. Multicolor and erasable DNA photolithography.

    PubMed

    Huang, Fujian; Xu, Huaguo; Tan, Weihong; Liang, Haojun

    2014-07-22

    The immobilization of DNA molecules onto a solid support is a crucial step in biochip research and related applications. In this work, we report a DNA photolithography method based on photocleavage of 2-nitrobenzyl linker-modified DNA strands. These strands were subjected to ultraviolet light irradiation to generate multiple short DNA strands in a programmable manner. Coupling the toehold-mediated DNA strand-displacement reaction with DNA photolithography enabled the fabrication of a DNA chip surface with multifunctional DNA patterns having complex geometrical structures at the microscale level. The erasable DNA photolithography strategy was developed to allow different paintings on the same chip. Furthermore, the asymmetrical modification of colloidal particles was carried out by using this photolithography strategy. This strategy has broad applications in biosensors, nanodevices, and DNA-nanostructure fabrication.

  11. Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity

    PubMed Central

    Qin, Wei Jie; Yung, Lin Yue Lanry

    2007-01-01

    Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Here we report a gold nanoparticle-based method that allows DNA detection and quantification and is capable of single nucleotide polymorphism (SNP) discrimination. The precise quantification of single-stranded DNA is due to the formation of defined nanoparticle-DNA conjugate groupings in the presence of target/linker DNA. Conjugate groupings were characterized and quantified by gel electrophoresis. A linear correlation between the amount of target DNA and conjugate groupings was found. For SNP detection, single base mismatch discrimination was achieved for both the end- and center-base mismatch. The method described here may be useful for the development of a simple and quantitative DNA detection assay. PMID:17720714

  12. The interaction of polyamines with DNA: a 23Na NMR study.

    PubMed Central

    Burton, D R; Forsén, S; Reimarsson, P

    1981-01-01

    The interaction between a variety of polyamines, both naturally occurring and synthetic, and calf thymus DNA has been studied using 23Na NMR. The relaxation behaviour of 23Na reflects the extent of interaction of Na+ with DNA phosphate groups and therefore the extent of charge neutralisation of DNA phosphate groups (P) by polyamine amino and imino groups (N) in solutions of DNa, polyamine and Na+. The studies reveal that whereas spermine and spermidine are capable of expelling nearly all of the Na+ ions from DNA at N/P approximately 1, diamines such as putrescine and homologues of spermine and spermidine are capable of neutralising only roughly 50% of DNA phosphates. The results provide a challenge to current models of DNA-polyamine interactions. PMID:7232215

  13. Statistical analysis of molecular nanotemplate driven DNA adsorption on graphite.

    PubMed

    Dubrovin, E V; Speller, S; Yaminsky, I V

    2014-12-30

    In this work, we have studied the conformation of DNA molecules aligned on the nanotemplates of octadecylamine, stearyl alcohol, and stearic acid on highly oriented pyrolytic graphite (HOPG). For this purpose, fluctuations of contours of adsorbed biopolymers obtained from atomic force microscopy (AFM) images were analyzed using the wormlike chain model. Moreover, the conformations of adsorbed biopolymer molecules were characterized by the analysis of the scaling exponent ν, which relates the mean squared end-to-end distance and contour length of the polymer. During adsorption on octadecylamine and stearyl alcohol nanotemplates, DNA forms straight segments, which order along crystallographic axes of graphite. In this case, the conformation of DNA molecules can be described using two different length scales. On a large length scale (at contour lengths l > 200-400 nm), aligned DNA molecules have either 2D compact globule or partially relaxed 2D conformation, whereas on a short length scale (at l ≤ 200-400 nm) their conformation is close to that of rigid rods. The latter type of conformation can be also assigned to DNA adsorbed on a stearic acid nanotemplate. The different conformation of DNA molecules observed on the studied monolayers is connected with the different DNA-nanotemplate interactions associated with the nature of the functional group of the alkane derivative in the nanotemplate (amine, alcohol, or acid). The persistence length of λ-DNA adsorbed on octadecylamine nanotemplates is 31 ± 2 nm indicating the loss of DNA rigidity in comparison with its native state. Similar values of the persistence length (34 ± 2 nm) obtained for 24-times shorter DNA molecules adsorbed on an octadecylamine nanotemplate demonstrate that this rigidity change does not depend on biopolymer length. Possible reasons for the reduction of DNA persistence length are discussed in view of the internal DNA structure and DNA-surface interaction.

  14. Sanger dideoxy sequencing of DNA.

    PubMed

    Walker, Sarah E; Lorsch, Jon

    2013-01-01

    While the ease and reduced cost of automated DNA sequencing has largely obviated the need for manual dideoxy sequencing for routine purposes, specific applications require manual DNA sequencing. For instance, in studies of enzymes or proteins that bind or modify DNA, a DNA ladder is often used to map the site at which an enzyme is bound or a modification occurs. In these cases, the Sanger method for dideoxy sequencing provides a rapid and facile method for producing a labeled DNA ladder.

  15. Forensic DNA profiling and database.

    PubMed

    Panneerchelvam, S; Norazmi, M N

    2003-07-01

    The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection.

  16. DNA damage phenotype and prostate cancer risk

    PubMed Central

    Kosti, O.; Goldman, L.; Saha, D.T.; Orden, R.A.; Pollock, A.J.; Madej, H.L.; Hsing, A.W.; Chu, L.W.; Lynch, J.H.; Goldman, R.

    2010-01-01

    The capacity of an individual to process DNA damage is considered a crucial factor in carcinogenesis. The comet assay is a phenotypic measure of the combined effects of sensitivity to a mutagen exposure and repair capacity. In this paper, we evaluate the association of the DNA repair kinetics, as measured by the comet assay, with prostate cancer risk. In a pilot study of 55 men with prostate cancer, 53 men without the disease, and 71 men free of cancer at biopsy, we investigated the association of DNA damage with prostate cancer risk at early (0-15 min) and later (15-45 min) stages following gamma-radiation exposure. Although residual damage within 45 min was the same for all groups (65% of DNA in comet tail disappeared), prostate cancer cases had a slower first phase (38% vs 41%) and faster second phase (27% vs 22%) of the repair response compared to controls. When subjects were categorized into quartiles, according to efficiency of repairing DNA damage, high repair-efficiency within the first 15 min after exposure was not associated with prostate cancer risk while higher at the 15-45 min period was associated with increased risk (OR for highest-to-lowest quartiles = 3.24, 95% CI=0.98-10.66, p-trend =0.04). Despite limited sample size, our data suggest that DNA repair kinetics marginally differ between prostate cancer cases and controls. This small difference could be associated with differential responses to DNA damage among susceptible individuals. PMID:21095241

  17. Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

    PubMed Central

    Patel, Yogin; Gowda, Vrushab; Yan, Shan

    2012-01-01

    On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates

  18. Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

    PubMed

    Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E

    2016-08-16

    Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures. PMID:27441419

  19. Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers.

    PubMed Central

    Song, Z; Antzutkin, O N; Lee, Y K; Shekar, S C; Rupprecht, A; Levitt, M H

    1997-01-01

    Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure. Images FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 14 PMID:9284321

  20. Human DNA polymerase α in binary complex with a DNA:DNA template-primer

    PubMed Central

    Coloma, Javier; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2016-01-01

    The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes. PMID:27032819

  1. A Nuclear Family A DNA Polymerase from Entamoeba histolytica Bypasses Thymine Glycol

    PubMed Central

    Pastor-Palacios, Guillermo; Azuara-Liceaga, Elisa; Brieba, Luis G.

    2010-01-01

    Background Eukaryotic family A DNA polymerases are involved in mitochondrial DNA replication or translesion DNA synthesis. Here, we present evidence that the sole family A DNA polymerase from the parasite protozoan E. histolytica (EhDNApolA) localizes to the nucleus and that its biochemical properties indicate that this DNA polymerase may be involved in translesion DNA synthesis. Methodology and Results EhDNApolA is the sole family A DNA polymerase in E. histolytica. An in silico analysis places family A DNA polymerases from the genus Entamoeba in a separate branch of a family A DNA polymerases phylogenetic tree. Biochemical studies of a purified recombinant EhDNApolA demonstrated that this polymerase is active in primer elongation, is poorly processive, displays moderate strand displacement, and does not contain 3′–5′ exonuclease or editing activity. Importantly, EhDNApolA bypasses thymine glycol lesions with high fidelity, and confocal microscopy demonstrates that this polymerase is translocated into the nucleus. These data suggest a putative role of EhDNApolA in translesion DNA synthesis in E. histolytica. Conclusion This is the first report of the biochemical characterization of a DNA polymerase from E. histolytica. EhDNApolA is a family A DNA polymerase that is grouped into a new subfamily of DNA polymerases with translesion DNA synthesis capabilities similar to DNA polymerases from subfamily ν. PMID:20706627

  2. DNA templates silver clusters with magic sizes and colors for multi-cluster fluorescent assemblies

    NASA Astrophysics Data System (ADS)

    Copp, Stacy

    2015-03-01

    The natural inclusion of information in DNA, a vital part of life's rich complexity, can also be exploited to create diverse structures with multiple scales of complexity. Now emerging in novel photonic applications, DNA-stabilized silver clusters (AgN-DNA) are compelling examples of multi-scale DNA-directed assembly: individual fluorescent clusters, each templated by specific DNA base motifs, can then be arranged together in DNA-mediated multi-cluster assemblies with nanoscale precision. We discuss how DNA imbues AgN-DNA with unique features. Our optical data on pure AgN-DNA show that DNA base-cationic silver ligands impose rod-like shapes for neutral silver clusters, whose length primarily determines fluorescence color. This shape anisotropy leads to the aspherical AgN-DNA magic number cluster sizes and ``magic color'' groupings. We exploit DNA's sequence properties to extract multi-base motifs that select certain magic cluster sizes, using machine learning algorithms applied to large data sets. With these base motifs, we design DNA scaffolds to arrange multiple atomically precise AgN together in nanoscale proximity. We demonstrate that clusters are stable when held at separations below 10 nm, both in bicolor, dual cluster DNA clamp assemblies and in one-dimensional assemblies of atomically precise clusters arrayed on DNA nanotubes. Supported by NSF-CHE-1213895 and NSF-DMR-1309410. SMC acknowledges NSF-DGE-1144085, a NSF GRFP.

  3. DNA methyltransferases and epigenetic regulation in bacteria.

    PubMed

    Adhikari, Satish; Curtis, Patrick D

    2016-09-01

    Epigenetics is a change in gene expression that is heritable without a change in DNA sequence itself. This phenomenon is well studied in eukaryotes, particularly in humans for its role in cellular differentiation, X chromosome inactivation and diseases like cancer. However, comparatively little is known about epigenetic regulation in bacteria. Bacterial epigenetics is mainly present in the form of DNA methylation where DNA methyltransferases add methyl groups to nucleotides. This review focuses on two methyltransferases well characterized for their roles in gene regulation: Dam and CcrM. Dam methyltransferase in Escherichia coli is important for expression of certain genes such as the pap operon, as well as other cellular processes like DNA replication initiation and DNA repair. In Caulobacter crescentus and other Alphaproteobacteria, the methyltransferase CcrM is cell cycle regulated and is involved in the cell-cycle-dependent regulation of several genes. The diversity of regulatory targets as well as regulatory mechanisms suggests that gene regulation by methylation could be a widespread and potent method of regulation in bacteria. PMID:27476077

  4. Flow cytometric DNA analysis of corneal epithelium.

    PubMed

    Burns, E R; Roberson, M C; Brown, M F; Shock, J P; Pipkin, J L; Hinson, W G; Anson, J F

    1990-03-01

    We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.

  5. DNA in nanofluidic devices

    NASA Astrophysics Data System (ADS)

    Riehn, Robert

    2006-03-01

    Nanochannels with a channel cross-section of around 100 nm x 100 nm or less are emerging as a powerful new technique for single-molecule DNA analysis. In these nanochannels, DNA is linearized to a constant fraction of its contour length, and thus spatial locations measured by fluorescence microscopy can be directly related to genomic locations. Because the stretching in nanochannels is caused by lateral confinement, molecules are free to undergo longitudinal fluctuations. Hence, time-averaging over a single molecule is meaningful, and a high resolution can be achieved even using few molecules. We will present how DNA imaging in nanochannels can be applied to common tasks in molecular biology that go beyond simple sizing. In particular, we will discuss the genomic identification of human DNA fragments using fluorescent markers, and how to perform enzymatic reactions, such as restriction mapping using endonucleases, in nanochannels. We will also present our recent progress in the development of ``nanoplumbing'', that is devices that contain junctions of nanochannels. We will show how device dimensions influence the transport of DNA at those nanochannel junctions, and how those properties can be utilized in the design of devices and exotic materials.

  6. Beyond DNA repair: DNA-PK function in cancer

    PubMed Central

    Goodwin, Jonathan F.; Knudsen, Karen E.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, further underscoring the importance of understanding its role in disease. Herein, the molecular and cellular consequence of DNA-PK will be considered, with an eye toward discerning the rationale for therapeutic targeting of DNA-PK. PMID:25168287

  7. Dynamic Modulation of DNA Hybridization Using Allosteric DNA Tetrahedral Nanostructures.

    PubMed

    Song, Ping; Li, Min; Shen, Juwen; Pei, Hao; Chao, Jie; Su, Shao; Aldalbahi, Ali; Wang, Lihua; Shi, Jiye; Song, Shiping; Wang, Lianhui; Fan, Chunhai; Zuo, Xiaolei

    2016-08-16

    The fixed dynamic range of traditional biosensors limits their utility in several real applications. For example, viral load monitoring requires the dynamic range spans several orders of magnitude; whereas, monitoring of drugs requires extremely narrow dynamic range. To overcome this limitation, here, we devised tunable biosensing interface using allosteric DNA tetrahedral bioprobes to tune the dynamic range of DNA biosensors. Our strategy takes the advantage of the readily and flexible structure design and predictable geometric reconfiguration of DNA nanotechnology. We reconfigured the DNA tetrahedral bioprobes by inserting the effector sequence into the DNA tetrahedron, through which, the binding affinity of DNA tetrahedral bioprobes can be tuned. As a result, the detection limit of DNA biosensors can be programmably regulated. The dynamic range of DNA biosensors can be tuned (narrowed or extended) for up to 100-fold. Using the regulation of binding affinity, we realized the capture and release of biomolecules by tuning the binding behavior of DNA tetrahedral bioprobes. PMID:27435955

  8. Strandwise translocation of a DNA glycosylase on undamaged DNA

    SciTech Connect

    Qi, Yan; Nam, Kwangho; Spong, Marie C.; Banerjee, Anirban; Sung, Rou-Jia; Zhang, Michael; Karplus, Martin; Verdine, Gregory L.

    2012-05-14

    Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.

  9. Molecular dynamics simulations demonstrate the regulation of DNA-DNA attraction by H4 histone tail acetylations and mutations.

    PubMed

    Korolev, Nikolay; Yu, Hang; Lyubartsev, Alexander P; Nordenskiöld, Lars

    2014-10-01

    The positively charged N-terminal histone tails play a crucial role in chromatin compaction and are important modulators of DNA transcription, recombination, and repair. The detailed mechanism of the interaction of histone tails with DNA remains elusive. To model the unspecific interaction of histone tails with DNA, all-atom molecular dynamics (MD) simulations were carried out for systems of four DNA 22-mers in the presence of 20 or 16 short fragments of the H4 histone tail (variations of the 16-23 a. a. KRHRKVLR sequence, as well as the unmodified fragment a. a.13-20, GGAKRHRK). This setup with high DNA concentration, explicit presence of DNA-DNA contacts, presence of unstructured cationic peptides (histone tails) and K(+) mimics the conditions of eukaryotic chromatin. A detailed account of the DNA interactions with the histone tail fragments, K(+) and water is presented. Furthermore, DNA structure and dynamics and its interplay with the histone tail fragments binding are analysed. The charged side chains of the lysines and arginines play major roles in the tail-mediated DNA-DNA attraction by forming bridges and by coordinating to the phosphate groups and to the electronegative sites in the minor groove. Binding of all species to DNA is dynamic. The structure of the unmodified fully-charged H4 16-23 a.a. fragment KRHRKVLR is dominated by a stretched conformation. The H4 tail a. a. fragment GGAKRHRK as well as the H4 Lys16 acetylated fragment are highly flexible. The present work allows capturing typical features of the histone tail-counterion-DNA structure, interaction and dynamics.

  10. Forensic applications of DNA fingerprinting.

    PubMed

    Sullivan, K M

    1994-02-01

    In many ways, DNA profiling technology is very similar to the conventional techniques used for forensic identification. As with, for example, blood grouping techniques, the molecular characteristics of the scene of crime sample may be determined and compared with those of the scene of reference samples from suspects and victim. If the molecular characteristics of the crime sample and the suspect are different, then they cannot be from the same person, whereas if they match, then the possibility remains that they may be from a single source. Similar material, such as blood or semen stains, may be used for both biochemical and genetic tests, and the main applications of identification and relationship testing are shared by both techniques. At this point, the similarity ends; DNA profiling has the following characteristics: 1. It is more sensitive, being able to generate sound data from only a tiny amount of even partially degraded biological material. 2. It is capable of resolving mixtures of semen or tissue from up to several individuals. 3. It has a far greater power of discrimination between individuals--sometimes up to 1 millionfold higher than conventional techniques. 4. It provides considerably more information on the nature of relationships, particularly in cases of incest. As such, the technique represents a quantum leap in forensic identification and relationship testing.

  11. Active DNA demethylation by DNA repair: Facts and uncertainties.

    PubMed

    Schuermann, David; Weber, Alain R; Schär, Primo

    2016-08-01

    Pathways that control and modulate DNA methylation patterning in mammalian cells were poorly understood for a long time, although their importance in establishing and maintaining cell type-specific gene expression was well recognized. The discovery of proteins capable of converting 5-methylcytosine (5mC) to putative substrates for DNA repair introduced a novel and exciting conceptual framework for the investigation and ultimate discovery of molecular mechanisms of DNA demethylation. Against the prevailing notion that DNA methylation is a static epigenetic mark, it turned out to be dynamic and distinct mechanisms appear to have evolved to effect global and locus-specific DNA demethylation. There is compelling evidence that DNA repair, in particular base excision repair, contributes significantly to the turnover of 5mC in cells. By actively demethylating DNA, DNA repair supports the developmental establishment as well as the maintenance of DNA methylation landscapes and gene expression patterns. Yet, while the biochemical pathways are relatively well-established and reviewed, the biological context, function and regulation of DNA repair-mediated active DNA demethylation remains uncertain. In this review, we will thus summarize and critically discuss the evidence that associates active DNA demethylation by DNA repair with specific functional contexts including the DNA methylation erasure in the early embryo, the control of pluripotency and cellular differentiation, the maintenance of cell identity, and the nuclear reprogramming. PMID:27247237

  12. Energy and Technology Review: Unlocking the mysteries of DNA repair

    SciTech Connect

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  13. Molecular dynamics simulations of DNA-polycation complexes

    NASA Astrophysics Data System (ADS)

    Ziebarth, Jesse; Wang, Yongmei

    2008-03-01

    A necessary step in the preparation of DNA for use in gene therapy is the packaging of DNA with a vector that can condense DNA and provide protection from degrading enzymes. Because of the immunoresponses caused by viral vectors, there has been interest in developing synthetic gene therapy vectors, with polycations emerging as promising candidates. Molecular dynamics simulations of the DNA duplex CGCGAATTCGCG in the presence of 20 monomer long sequences of the polycations, poly-L-lysine (PLL) and polyethyleneimine (PEI), with explicit counterions and TIP3P water, are performed to provide insight into the structure and formation of DNA polyplexes. After an initial separation of approximately 50 å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes upon complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, and rarely occupy electronegative sites in either the major or minor grooves. Differences between complexation with PEI and PLL will be discussed.

  14. Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η.

    PubMed

    Su, Yan; Egli, Martin; Guengerich, F Peter

    2016-02-19

    Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.

  15. Circadian Modulation of 8-Oxoguanine DNA Damage Repair

    PubMed Central

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  16. Optimality in DNA repair.

    PubMed

    Richard, Morgiane; Fryett, Matthew; Miller, Samantha; Booth, Ian; Grebogi, Celso; Moura, Alessandro

    2012-01-01

    DNA within cells is subject to damage from various sources. Organisms have evolved a number of mechanisms to repair DNA damage. The activity of repair enzymes carries its own risk, however, because the repair of two nearby lesions may lead to the breakup of DNA and result in cell death. We propose a mathematical theory of the damage and repair process in the important scenario where lesions are caused in bursts. We use this model to show that there is an optimum level of repair enzymes within cells which optimises the cell's response to damage. This optimal level is explained as the best trade-off between fast repair and a low probability of causing double-stranded breaks. We derive our results analytically and test them using stochastic simulations, and compare our predictions with current biological knowledge. PMID:21945337

  17. Duplication in DNA Sequences

    NASA Astrophysics Data System (ADS)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  18. Transposon facilitated DNA sequencing

    SciTech Connect

    Berg, D.E.; Berg, C.M.; Huang, H.V.

    1990-01-01

    The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses, and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.

  19. The origin of DNA genomes and DNA replication proteins.

    PubMed

    Forterre, Patrick

    2002-10-01

    In recent years, it has became clear that most proteins involved in cellular DNA precursor synthesis or DNA replication have been 'invented' more than once, indicating that the transition from RNA to DNA genomes was more complex than previously thought. Several authors have suggested that DNA viruses, which often encode their own version of these proteins, played an important role in this process. The nature of the genome of the last universal cellular ancestor (LUCA) -- that is, RNA or DNA, prokaryotic-like or eukaryotic-like -- remains in dispute. A hyperthermophilic LUCA would have suggested a circular, double-stranded DNA genome; however, recent data favor a mesophilic or moderately thermophilic LUCA.

  20. DNA templated magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Kinsella, Joseph M.

    Recent discoveries in nanoscience are predicted to potentially revolutionize future technologies in an extensive number of fields. These developments are contingent upon discovering new and often unconventional methods to synthesize and control nanoscale components. Nature provides several examples of working nanotechnology such as the use of programmed self assembly to build and deconstruct complex molecular systems. We have adopted a method to control the one dimensional assembly of magnetic nanoparticles using DNA as a scaffold molecule. With this method we have demonstrated the ability to organize 5 nm particles into chains that stretch up to ˜20 mum in length. One advantage of using DNA compared is the ability of the molecule to interact with other biomolecules. After assembling particles onto DNA we have been able to cleave the molecule into smaller fragments using restriction enzymes. Using ligase enzymes we have re-connected these fragments, coated with either gold or iron oxide, to form long one-dimensional arrangements of the two different types of nanoparticles on a single molecular guide. We have also created a sensitive magnetic field sensor by incorporating magnetic nanoparticle coated DNA strands with microfabricated electrodes. The IV characteristics of the aligned nanoparticles are dependant on the magnitude of an externally applied magnetic field. This transport phenomenon known as tunneling magnetoresistance (TMR) shows room temperature resistance of our devices over 80% for cobalt ferrite coated DNA when a field of 20 kOe is applied. In comparison, studies using two dimensional nanoparticle films of irox oxides xii only exhibit a 35% MR effect. Confinement into one dimension using the DNA guide produces a TMR mechanism which produces significant increases in magnetoresistance. This property can be utilized for applications in magnetic field sensing, data storage, and logic elements.

  1. An Electrochemical DNA Microbiosensor Based on Succinimide-Modified Acrylic Microspheres

    PubMed Central

    Ulianas, Alizar; Heng, Lee Yook; Hanifah, Sharina Abu; Ling, Tan Ling

    2012-01-01

    An electrochemical microbiosensor for DNA has been fabricated based on new acrylic microspheres modified with reactive N-acryloxysuccinimide (NAS) functional groups. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized in an emulsion form with a simple one-step photopolymerization technique. Aminated DNA probe was attached to the succinimde functional group of the acrylic microspheres via covalent bonding. The hybridization of the immobilized DNA probe with the complementary DNA was studied by differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, pH, type of ions, buffer concentrations, ionic strength, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a linear response range to target DNA over a wide concentration range of 1.0 × 10−16 and 1.0 × 10−8 M with a lower limit of detection (LOD) of 9.46 × 10−17 M (R2 = 0.97). This DNA microbiosensor showed good reproducibility with 2.84% RSD (relative standard deviation) (n = 3). Application of the NAS-modified acrylic microspheres in the construction of DNA microbiosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices. PMID:22778594

  2. Effect of salt concentration on the stability of heterogeneous DNA

    NASA Astrophysics Data System (ADS)

    Singh, Amar; Singh, Navin

    2015-02-01

    We study the role of cations on the stability of double stranded DNA (dsDNA) molecules. It is known that the two strands of double stranded DNA (dsDNA) have negative charge due to phosphate group. Cations in the form of salt in the solution, act as shielding agents thereby reducing the repulsion between these strands. We study several heterogeneous DNA molecules. We calculate the phase diagrams for DNA molecules in thermal as well as in force ensembles using Peyrard-Bishop-Dauxois (PBD) model. The dissociation and the stacking energies are the two most important factors that play an important role in the DNA stability. With suitable modifications in the model parameters we investigate the role of cation concentration on the stability of different heterogeneous DNA molecules. The objective of this work is to understand how these cations modify the strength of different pairs or bases along the strand. The phase diagram for the force ensemble case (a dsDNA is pulled from an end) is compared with the experimental results.

  3. Kinetoplast DNA minicircles: regions of extensive sequence divergence.

    PubMed Central

    Rogers, W O; Wirth, D F

    1987-01-01

    Previous work has shown that the kinetoplast minicircle DNA of Leishmania species exhibits species-specific sequence divergence and this observation has led to the development of a DNA probe-based diagnostic test for leishmaniasis. In the work reported here, we demonstrate that the minicircle is composed of three types of DNA sequences with differing specificities reflecting different rates of DNA sequence change. A library of cloned fragments of kinetoplast DNA (kDNA) from Leishmania mexicana amazonensis was prepared and the cloned subfragments were found to contain DNA sequences with different taxonomic specificities based on hybridization analysis with various species of Leishmania. Four groups of subfragments were found, those that hybridized with a large number of Leishmania sp. as well as sequences unique to the species, subspecies, or isolate. Analysis of nested deletions of a single, full-length minicircle demonstrates that these different taxonomic specificities are contained within a single minicircle. This implies that different regions of a single minicircle have DNA sequences that diverge at different rates. These sequences represent potentially valuable tools in diagnostic, epidemiologic, and ecological studies of leishmaniasis and provide the basis for a model of kDNA sequence evolution. Images PMID:3025880

  4. Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity.

    PubMed

    Shen, Qingming; Fan, Mengxing; Yang, Yin; Zhang, Hui

    2016-08-31

    DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL(-1) with a limit of detection as low as 0.17 ng mL(-1). With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening. PMID:27506345

  5. Rigidity of melting DNA.

    PubMed

    Pal, Tanmoy; Bhattacharjee, Somendra M

    2016-05-01

    The temperature dependence of DNA flexibility is studied in the presence of stretching and unzipping forces. Two classes of models are considered. In one case the origin of elasticity is entropic due to the polymeric correlations, and in the other the double-stranded DNA is taken to have an intrinsic rigidity for bending. In both cases single strands are completely flexible. The change in the elastic constant for the flexible case due to thermally generated bubbles is obtained exactly. For the case of intrinsic rigidity, the elastic constant is found to be proportional to the square root of the bubble number fluctuation. PMID:27300825

  6. Rigidity of melting DNA

    NASA Astrophysics Data System (ADS)

    Pal, Tanmoy; Bhattacharjee, Somendra M.

    2016-05-01

    The temperature dependence of DNA flexibility is studied in the presence of stretching and unzipping forces. Two classes of models are considered. In one case the origin of elasticity is entropic due to the polymeric correlations, and in the other the double-stranded DNA is taken to have an intrinsic rigidity for bending. In both cases single strands are completely flexible. The change in the elastic constant for the flexible case due to thermally generated bubbles is obtained exactly. For the case of intrinsic rigidity, the elastic constant is found to be proportional to the square root of the bubble number fluctuation.

  7. The GROOP Effect: Groups Mimic Group Actions

    ERIC Educational Resources Information Center

    Tsai, Jessica Chia-Chin; Sebanz, Natalie; Knoblich, Gunther

    2011-01-01

    Research on perception-action links has focused on an interpersonal level, demonstrating effects of observing individual actions on performance. The present study investigated perception-action matching at an inter-group level. Pairs of participants responded to hand movements that were performed by two individuals who used one hand each or they…

  8. An Arabidopsis cDNA encoding a DNA-binding protein that is highly similar to the DEAH family of RNA/DNA helicase genes.

    PubMed

    Isono, K; Yamamoto, H; Satoh, K; Kobayashi, H

    1999-09-15

    A cDNA encoding a putative RNA and/or DNA helicase has been isolated from Arabidopsis thaliana cDNA libraries. The cloned cDNA is 5166 bases long, and its largest open reading frame encodes 1538 amino acids. The central region of the predicted protein is homologous to a group of nucleic acid helicases from the DEAD/H family. However, the N- and C-terminal regions of the Arabidopsis cDNA product are distinct from these animal DEIH proteins. We have found that the C-terminal region contains three characteristic sequences: (i) two DNA-binding segments that form a probe helix (PH) involved in DNA recognition; (ii) an SV40-type nuclear localization signal; and (iii) 11 novel tandem-repeat sequences each consisting of about 28 amino acids. We have designated this cDNA as NIH (nuclear DEIH-boxhelicase). Functional character-ization of a recombinant fusion product containing the repeated region indicates that NIH may form homodimers, and that this is the active form in solution. Based on this information and the observation that the sequence homology is limited to the DEAH regions, we conclude that the biological roles of the plant helicase NIH differ from those of the animal DEIH family. PMID:10471743

  9. The effect of local melting of DNA on DNA loop formation

    NASA Astrophysics Data System (ADS)

    Jeong, Jiyoun; Kim, Harold

    Statistical mechanics of double-stranded DNA (dsDNA) is well described by the wormlike chain model (WLC) which assumes a harmonic bending potential. Such smooth bending potential may no longer be valid for large bending angles to form small loops (<100 bp). Instead, DNA may rely on rare structural transitions such as local melting (opening) of base pairs to lower the energetic cost. In theory, open base pairs called bubbles can increase the looping probability of short DNA molecules by a few orders of magnitude, but a robust experimental validation of this theoretical prediction is lacking. Here, we investigated the correlation between local melting probability and looping dynamics of dsDNA using single-molecule fluorescence resonance energy transfer (FRET). We designed two groups of short DNA molecules with low and high melting probabilities around their center and measured their looping and unlooping rates in equilibrium. Our data allow rigorous tests of meltable wormlike chain (MWLC) models at short length scales for setting ranges of acceptable free energy cost of bubble formation and flexibility values of a bubble.

  10. DNA binding induces conformational transition within human DNA topoisomerase I in solution.

    PubMed

    Oleinikov, Vladimir; Sukhanova, Alyona; Mochalov, Konstantin; Ustinova, Olga; Kudelina, Irina; Bronstein, Igor; Nabiev, Igor

    2002-01-01

    We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex. PMID:12209444

  11. Defects in mitochondrial DNA replication and oxidative damage in muscle of mtDNA mutator mice.

    PubMed

    Kolesar, Jill E; Safdar, Adeel; Abadi, Arkan; MacNeil, Lauren G; Crane, Justin D; Tarnopolsky, Mark A; Kaufman, Brett A

    2014-10-01

    A causal role for mitochondrial dysfunction in mammalian aging is supported by recent studies of the mtDNA mutator mouse ("PolG" mouse), which harbors a defect in the proofreading-exonuclease activity of mitochondrial DNA polymerase gamma. These mice exhibit accelerated aging phenotypes characteristic of human aging, including systemic mitochondrial dysfunction, exercise intolerance, alopecia and graying of hair, curvature of the spine, and premature mortality. While mitochondrial dysfunction has been shown to cause increased oxidative stress in many systems, several groups have suggested that PolG mutator mice show no markers of oxidative damage. These mice have been presented as proof that mitochondrial dysfunction is sufficient to accelerate aging without oxidative stress. In this study, by normalizing to mitochondrial content in enriched fractions we detected increased oxidative modification of protein and DNA in PolG skeletal muscle mitochondria. We separately developed novel methods that allow simultaneous direct measurement of mtDNA replication defects and oxidative damage. Using this approach, we find evidence that suggests PolG muscle mtDNA is indeed oxidatively damaged. We also observed a significant decrease in antioxidants and expression of mitochondrial biogenesis pathway components and DNA repair enzymes in these mice, indicating an association of maladaptive gene expression with the phenotypes observed in PolG mice. Together, these findings demonstrate the presence of oxidative damage associated with the premature aging-like phenotypes induced by mitochondrial dysfunction.

  12. Entropic Enhancement of Protein-DNA Affinity by Oxygen-to-Sulfur Substitution in DNA Phosphate.

    PubMed

    Zandarashvili, Levani; Nguyen, Dan; Anderson, Kurtis M; White, Mark A; Gorenstein, David G; Iwahara, Junji

    2015-09-01

    Dithioation of DNA phosphate is known to enhance binding affinities, at least for some proteins. We mechanistically characterized this phenomenon for the Antennapedia homeodomain-DNA complex by integrated use of fluorescence, isothermal titration calorimetry, NMR spectroscopy, and x-ray crystallography. By fluorescence and isothermal titration calorimetry, we found that this affinity enhancement is entropy driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH3(+) groups involved in ion pairs with DNA. By x-ray crystallography, we compared the structures of the complexes with and without dithioation of the phosphate. Our NMR and x-ray data show that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively suggest that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface. PMID:26331260

  13. Entropic Enhancement of Protein-DNA Affinity by Oxygen-to-Sulfur Substitution in DNA Phosphate

    PubMed Central

    Zandarashvili, Levani; Nguyen, Dan; Anderson, Kurtis M.; White, Mark A.; Gorenstein, David G.; Iwahara, Junji

    2015-01-01

    Dithioation of DNA phosphate is known to enhance binding affinities, at least for some proteins. We mechanistically characterized this phenomenon for the Antennapedia homeodomain-DNA complex by integrated use of fluorescence, isothermal titration calorimetry, NMR spectroscopy, and x-ray crystallography. By fluorescence and isothermal titration calorimetry, we found that this affinity enhancement is entropy driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH3+ groups involved in ion pairs with DNA. By x-ray crystallography, we compared the structures of the complexes with and without dithioation of the phosphate. Our NMR and x-ray data show that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively suggest that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface. PMID:26331260

  14. Rapid and effective DNA extraction method with bead grinding for a large amount of fungal DNA.

    PubMed

    Watanabe, M; Lee, K; Goto, K; Kumagai, S; Sugita-Konishi, Y; Hara-Kudo, Y

    2010-06-01

    To identify a rapid method for extracting a large amount of DNA from fungi associated with food hygiene, extraction methods were compared using fungal pellets formed rapidly in liquid media. Combinations of physical and chemical methods or commercial kits were evaluated with 3 species of yeast, 10 species of ascomycetous molds, and 4 species of zygomycetous molds. Bead grinding was the physical method, followed by chemical methods involving sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), and benzyl chloride and two commercial kits. Quantity was calculated by UV absorbance at 260 nm, quality was determined by the ratio of UV absorbance at 260 and 280 nm, and gene amplifications and electrophoresis profiles of whole genomes were analyzed. Bead grinding with the SDS method was the most effective for DNA extraction for yeasts and ascomycetous molds, and bead grinding with the CTAB method was most effective with zygomycetous molds. For both groups of molds, bead grinding with the CTAB method was the best approach for DNA extraction. Because this combination also is relatively effective for yeasts, it can be used to extract a large amount of DNA from a wide range of fungi. The DNA extraction methods are useful for developing gene indexes to identify fungi with molecular techniques, such as DNA fingerprinting.

  15. Gestalt Interactional Groups

    ERIC Educational Resources Information Center

    Harman, Robert L.; Franklin, Richard W.

    1975-01-01

    Gestalt therapy in groups is not limited to individual work in the presence of an audience. Describes several ways to involve gestalt groups interactionally. Interactions described focus on learning by doing and discovering, and are noninterpretive. (Author/EJT)

  16. What Makes Groups Tick.

    ERIC Educational Resources Information Center

    Allcorn, Seth

    1985-01-01

    By reviewing this analysis of the behavior of both groups and individuals in groups, human resources managers can learn to tell whether committees, task forces, and departments may be encouraging or inhibiting the work they set out to do. (Author)

  17. Nilpotent -local finite groups

    NASA Astrophysics Data System (ADS)

    Cantarero, José; Scherer, Jérôme; Viruel, Antonio

    2014-10-01

    We provide characterizations of -nilpotency for fusion systems and -local finite groups that are inspired by known result for finite groups. In particular, we generalize criteria by Atiyah, Brunetti, Frobenius, Quillen, Stammbach and Tate.

  18. DNA banking and DNA databanking by academic and commercial laboratories

    SciTech Connect

    McEwen, J.E. |; Reilly, P.R.

    1994-09-01

    The advent of DNA-based testing is giving rise to DNA banking (the long-term storage of cells, transformed cell lines, or extracted DNA for subsequent retrieval and analysis) and DNA data banking (the indefinite storage of information derived from DNA analysis). Large scale acquisition and storage of DNA and DNA data has important implications for the privacy rights of individuals. A survey of 148 academically based and commercial DNA diagnostic laboratories was conducted to determine: (1) the extent of their DNA banking activities; (2) their policies and experiences regarding access to DNA samples and data; (3) the quality assurance measures they employ; and (4) whether they have written policies and/or depositor`s agreements addressing specific issues. These issues include: (1) who may have access to DNA samples and data; (2) whether scientists may have access to anonymous samples or data for research use; (3) whether they have plans to contact depositors or retest samples if improved tests for a disorder become available; (4) disposition of samples at the end of the contract period if the laboratory ceases operations, if storage fees are unpaid, or after a death or divorce; (5) the consequence of unauthorized release, loss, or accidental destruction of samples; and (6) whether depositors may share in profits from the commercialization of tests or treatments developed in part from studies of stored DNA. The results suggest that many laboratories are banking DNA, that many have already amassed a large number of samples, and that a significant number plan to further develop DNA banking as a laboratory service over the next two years. Few laboratories have developed written policies governing DNA banking, and fewer still have drafted documents that define the rights and obligations of the parties. There may be a need for increased regulation of DNA banking and DNA data banking and for better defined policies with respect to protecting individual privacy.

  19. Liposome-mediated DNA immunisation via the subcutaneous route.

    PubMed

    Perrie, Y; McNeil, S; Vangala, A

    2003-01-01

    liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 microg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. PMID:15203925

  20. Symmetry of electrostatic interaction between pyrophosphate DNA molecules.

    PubMed

    Golo, V L; Kats, E I; Kuznetsova, S A; Volkov, Yu S

    2010-01-01

    We study chiral electrostatic interaction between artificial ideal homopolymer DNA-like molecules in which a number of phosphate groups of the sugar-phosphate backbone are exchanged for the pyrophosphate ones. We employ a model in which the DNA is considered as a one-dimensional lattice of dipoles and charges corresponding to base pairs and (pyro)phosphate groups, respectively. The interaction between molecules of the DNA is described by a pair potential U of electrostatic forces between the two sets of dipoles and charges belonging to respective lattices describing the molecules. Minima of the potential U indicate orientational ordering of the molecules and thus liquid crystalline phases of the DNA. We use numerical methods for finding the set of minima in conjunction with symmetries verified by the potential U . The symmetries form a non-commutative group of 8th order, S . Using the group S we suggest a classification of liquid crystalline phases of the DNA, which allows several cholesteric phases, that is polymorphism. Pyrophosphate forms of the DNA could clarify the role played by charges in their liquid crystalline phases, and open experimental research, important for nano-technological and bio-medical applications.