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Sample records for cytosine nucleotides

  1. Solution structures of oligonucleotides containing either a guanine or a cytosine in front of a gap of one nucleotide

    NASA Astrophysics Data System (ADS)

    Boulard, Y.; Faibis, V.; Fazakerley, G. V.

    1999-10-01

    We report NMR and molecular modelling studies on two DNA duplexes containing a gap of one nucleotides. The difference between the two oligonucleotides lies in the central base face to the gap, a guanine or a cytosine. For the gapG, we observed in solution a B-form conformation where the guanine stacks in the helix. For the gapC, we reveal the existence of two species, one majority where the cytosine is inside the helix and a second for which the cytosine is extrahelical. Nous présentons une étude par RMN et modélisation moléculaire sur deux duplexes d'ADN contenant une lacune de un nucléotide. La différence entre les deux oligonucléotides réside dans la base centrale en face de la lacune, une guanine ou une cytosine. Pour le duplex appelé gapG, nous observons en solution une hélice de type B dans laquelle la guanine est empilée à l'intérieur de l'hélice. Dans le cas du duplex gapC, nous montrons l'existence de deux formes, l'une où la cytosine est à l'intérieur de l'hélice; la seconde où la cytosine est extra hélicale.

  2. Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5‐Methylcytosine

    PubMed Central

    von Watzdorf, Janina; Leitner, Kim

    2016-01-01

    Abstract 5‐Methyl‐2′‐deoxycytosine, the most common epigenetic marker of DNA in eukaryotic cells, plays a key role in gene regulation and affects various cellular processes such as development and carcinogenesis. Therefore, the detection of 5mC can serve as an important biomarker for diagnostics. Here we describe that modified dGTP analogues as well as modified primers are able to sense the presence or absence of a single methylation of C, even though this modification does not interfere directly with Watson–Crick nucleobase pairing. By screening several modified nucleotide scaffolds, O6‐modified 2′‐deoxyguanosine analogues were identified as discriminating between C and 5mC. These modified nucleotides might find application in site‐specific 5mC detection, for example, through real‐time PCR approaches. PMID:26835661

  3. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases.

    PubMed

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-03-15

    New types of double-headed 2'-deoxycytidine 5'-O-triphosphates (dC(XC)TPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC(XC)TPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins. PMID:26899597

  4. Inheritance of Cytosine Methylation.

    PubMed

    Tillo, Desiree; Mukherjee, Sanjit; Vinson, Charles

    2016-11-01

    There are numerous examples of parental transgenerational inheritance that is epigenetic. The informational molecules include RNA, chromatin modifications, and cytosine methylation. With advances in DNA sequencing technologies, the molecular and epigenetic mechanisms mediating these effects are now starting to be uncovered. This mini-review will highlight some of the examples of epigenetic inheritance, the establishment of cytosine methylation in sperm, and recent genomic studies linking sperm cytosine methylation to epigenetic effects on offspring. A recent paper examining changes in diet and sperm cytosine methylation from pools of eight animals each, found differences between a normal diet, a high fat diet, and a low protein diet. However, epivariation between individuals within a group was greater than the differences between groups obscuring any potential methylation changes linked to diet. Learning more about epivariation may help unravel the mechanisms that regulate cytosine methylation. In addition, other experimental and genetic systems may also produce more dramatic changes in the sperm methylome, making it easier to unravel potential transgenerational phenomena. J. Cell. Physiol. 231: 2346-2352, 2016. © 2016 Wiley Periodicals, Inc. PMID:26910768

  5. Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

    PubMed Central

    Smith, Rick W. A.; Monroe, Cara; Bolnick, Deborah A.

    2015-01-01

    While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches. PMID:26016479

  6. Detection of Cytosine methylation in ancient DNA from five native american populations using bisulfite sequencing.

    PubMed

    Smith, Rick W A; Monroe, Cara; Bolnick, Deborah A

    2015-01-01

    While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches. PMID:26016479

  7. The control of natural variation in cytosine methylation in Arabidopsis.

    PubMed Central

    Riddle, Nicole C; Richards, Eric J

    2002-01-01

    We explore the extent and sources of epigenetic variation in cytosine methylation in natural accessions of the flowering plant, Arabidopsis thaliana, by focusing on the methylation of the major rRNA gene repeats at the two nucleolus organizer regions (NOR). Our findings indicate that natural variation in NOR methylation results from a combination of genetic and epigenetic mechanisms. Genetic variation in rRNA gene copy number and trans-acting modifier loci account for some of the natural variation in NOR methylation. Our results also suggest that divergence and inheritance of epigenetic information, independent of changes in underlying nucleotide sequence, may play an important role in maintaining natural variation in cytosine methylation. PMID:12242246

  8. Electron attachment to the cytosine-centered DNA single strands: does base stacking matter?

    PubMed

    Gu, Jiande; Wang, Jing; Leszczynski, Jerzy

    2012-02-01

    Electron attachment to the trimer of nucleotide, dGpdCpdG, has been investigated by a quantum mechanical approach at a reliable level of theory. The study of the electron attached dGpdCpdG species demonstrates that cytosine contained DNA single strands have a strong tendency to capture low-energy electrons and to form electronically stable cytosine-centered radical anions. The comparative study of the model molecules pdCpdG and dGpdCp reveals that base stacking has little contribution to the adiabatic electron affinity (AEA) of cytosine in DNA single strands. Additionally, the base-base stacking does not affect the vertical detachment energy (VDE) of the cytosine-centered radicals. Intrastrand H-bonding is found to be critical in increasing the values of the AEA and VDE. However, base-base stacking is revealed to be important in enlarging the vertical electron affinity (VEA) of cytosine. The electron attachment to the cytosine moiety intensifies the intrastrand H-bonding between the neighboring G and C bases. This process disrupts the base-base stacking interaction in the radical anion of dGpdCpdG. PMID:22225006

  9. The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation.

    PubMed

    Zakrzewski, Falk; Schubert, Veit; Viehoever, Prisca; Minoche, André E; Dohm, Juliane C; Himmelbauer, Heinz; Weisshaar, Bernd; Schmidt, Thomas

    2014-06-01

    Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next-generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome-wide fluorescent in situ hybridization complemented with immunostaining and super-resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44-52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes. PMID:24661787

  10. Comparative study of spontaneous deamination of adenine and cytosine in unbuffered aqueous solution at room temperature

    NASA Astrophysics Data System (ADS)

    Wang, Shiliang; Hu, Anguang

    2016-06-01

    Adenine in unbuffered nanopure water at a concentration of 2 mM is completely deaminated (>99%) to hypoxanthine at room temperature in ca. 10 weeks, with an estimated half-life (t1/2) less than 10 days, about six orders of magnitude faster than previously reported. Cytosine is not deaminated under the same condition, even after 3 years. This is in contrast to previous observations that cytosine deaminates 20-40 times faster than adenine free base, in nucleoside, in nucleotide and in single-stranded DNA in buffered neutral aqueous solutions.

  11. Linking short tandem repeat polymorphisms with cytosine modifications in human lymphoblastoid cell lines.

    PubMed

    Zhang, Zhou; Zheng, Yinan; Zhang, Xu; Liu, Cong; Joyce, Brian Thomas; Kibbe, Warren A; Hou, Lifang; Zhang, Wei

    2016-02-01

    Inter-individual variation in cytosine modifications has been linked to complex traits in humans. Cytosine modification variation is partially controlled by single nucleotide polymorphisms (SNPs), known as modified cytosine quantitative trait loci (mQTL). However, little is known about the role of short tandem repeat polymorphisms (STRPs), a class of structural genetic variants, in regulating cytosine modifications. Utilizing the published data on the International HapMap Project lymphoblastoid cell lines (LCLs), we assessed the relationships between 721 STRPs and the modification levels of 283,540 autosomal CpG sites. Our findings suggest that, in contrast to the predominant cis-acting mode for SNP-based mQTL, STRPs are associated with cytosine modification levels in both cis-acting (local) and trans-acting (distant) modes. In local scans within the ±1 Mb windows of target CpGs, 21, 9, and 21 cis-acting STRP-based mQTL were detected in CEU (Caucasian residents from Utah, USA), YRI (Yoruba people from Ibadan, Nigeria), and the combined samples, respectively. In contrast, 139,420, 76,817, and 121,866 trans-acting STRP-based mQTL were identified in CEU, YRI, and the combined samples, respectively. A substantial proportion of CpG sites detected with local STRP-based mQTL were not associated with SNP-based mQTL, suggesting that STRPs represent an independent class of mQTL. Functionally, genetic variants neighboring CpG-associated STRPs are enriched with genome-wide association study (GWAS) loci for a variety of complex traits and diseases, including cancers, based on the National Human Genome Research Institute (NHGRI) GWAS Catalog. Therefore, elucidating these STRP-based mQTL in addition to SNP-based mQTL can provide novel insights into the genetic architectures of complex traits. PMID:26714498

  12. Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

    PubMed Central

    Wang, Yong; Luan, Bin-Quan; Yang, Zhiyu; Zhang, Xinyue; Ritzo, Brandon; Gates, Kent; Gu, Li-Qun

    2014-01-01

    Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair, and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications. PMID:25103463

  13. Cytosine modifications in neurodevelopment and diseases

    PubMed Central

    Yao, Bing; Jin, Peng

    2013-01-01

    DNA methylation has been studied comprehensively and linked to both normal neurodevelopment and neurological diseases. The recent identification of several new DNA modifications, including 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), has given us a new perspective on the previously observed plasticity in 5mC-dependent regulatory processes. Here we review the latest research into these cytosine modifications, focusing mainly on their roles in neurodevelopment and diseases. PMID:23912899

  14. Linking the genetic architecture of cytosine modifications with human complex traits

    PubMed Central

    Zhang, Xu; Moen, Erika L.; Liu, Cong; Mu, Wenbo; Gamazon, Eric R.; Delaney, Shannon M.; Wing, Claudia; Godley, Lucy A.; Dolan, M. Eileen; Zhang, Wei

    2014-01-01

    Interindividual variation in cytosine modifications could contribute to heterogeneity in disease risks and other complex traits. We assessed the genetic architecture of cytosine modifications at 283 540 CpG sites in lymphoblastoid cell lines (LCLs) derived from independent samples of European and African descent. Our study suggests that cytosine modification variation was primarily controlled in local by single major modification quantitative trait locus (mQTL) and additional minor loci. Local genetic epistasis was detectable for a small proportion of CpG sites, which were enriched by more than 9-fold for CpG sites mapped to population-specific mQTL. Genetically dependent CpG sites whose modification levels negatively (repressive sites) or positively (facilitative sites) correlated with gene expression levels significantly co-localized with transcription factor binding, with the repressive sites predominantly associated with active promoters whereas the facilitative sites rarely at active promoters. Genetically independent repressive or facilitative sites preferentially modulated gene expression variation by influencing local chromatin accessibility, with the facilitative sites primarily antagonizing H3K27me3 and H3K9me3 deposition. In comparison with expression quantitative trait loci (eQTL), mQTL detected from LCLs were enriched in associations for a broader range of disease categories including chronic inflammatory, autoimmune and psychiatric disorders, suggesting that cytosine modification variation, while possesses a degree of cell linage specificity, is more stably inherited over development than gene expression variation. About 11% of unique single-nucleotide polymorphisms reported in the Genome-Wide Association Study Catalog were annotated, 78% as mQTL and 31% as eQTL in LCLs, which covered 37% of the investigated diseases/traits and provided insights to the biological mechanisms. PMID:24943591

  15. NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

    PubMed Central

    Hussain, Shobbir; Sajini, Abdulrahim A.; Blanco, Sandra; Dietmann, Sabine; Lombard, Patrick; Sugimoto, Yoichiro; Paramor, Maike; Gleeson, Joseph G.; Odom, Duncan T.; Ule, Jernej; Frye, Michaela

    2013-01-01

    Summary Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs), yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs). Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders. PMID:23871666

  16. Information Thermodynamics of Cytosine DNA Methylation.

    PubMed

    Sanchez, Robersy; Mackenzie, Sally A

    2016-01-01

    Cytosine DNA methylation (CDM) is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background ("noise") induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1) the adherence to Landauer's principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2) whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic rules as do current

  17. Information Thermodynamics of Cytosine DNA Methylation

    PubMed Central

    Sanchez, Robersy; Mackenzie, Sally A.

    2016-01-01

    Cytosine DNA methylation (CDM) is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background (“noise”) induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1) the adherence to Landauer’s principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2) whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic rules as do

  18. Cytosines, but not purines, determine recombination activating gene (RAG)-induced breaks on heteroduplex DNA structures: implications for genomic instability.

    PubMed

    Naik, Abani Kanta; Lieber, Michael R; Raghavan, Sathees C

    2010-03-01

    The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C((d))C((S))C((S))" (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability. PMID:20051517

  19. Spatial and Functional Relationships Among Pol V-Associated loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the Arabidopsis Epigenome

    SciTech Connect

    Wierzbicki, A. T.; Cocklin, Ross; Mayampurath, Anoop; Lister, Ryan; Rowley, M. J.; Gregory, Brian D.; Ecker, Joseph R.; Tang, Haixu; Pikaard, Craig S.

    2012-08-15

    Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

  20. Genetic code correlations - Amino acids and their anticodon nucleotides

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Lacey, J. C., Jr.

    1978-01-01

    The data here show direct correlations between both the hydrophobicity and the hydrophilicity of the homocodonic amino acids and their anticodon nucleotides. While the differences between properties of uracil and cytosine derivatives are small, further data show that uracil has an affinity for charged species. Although these data suggest that molecular relationships between amino acids and anticodons were responsible for the origin of the code, it is not clear what the mechanism of the origin might have been.

  1. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  2. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  3. Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD).

    PubMed

    Zhang, Xin; Zhao, Yuan; Yan, Honggao; Cao, Zexing; Mo, Yirong

    2016-05-15

    Extensive combined quantum mechanical (B3LYP/6-31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton-transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2-pyrimidinone. The rate-determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc. PMID:26813441

  4. Photophysical properties of pyrrolocytosine, a cytosine fluorescent base analogue.

    PubMed

    Nguyen, Quynh L; Spata, Vincent A; Matsika, Spiridoula

    2016-07-27

    The photophysical behavior of pyrrolocytosine (PC), a fluorescent base analogue of cytosine, has been investigated using theoretical approaches. The similarities between the PC and cytosine structures allow PC to maintain the pseudo-Watson-Crick base-pairing arrangement with guanine. Cytosine, similar to the other natural nucleobases, is practically non-fluorescent, because of ultrafast radiationless decay occurring through conical intersections. PC displays a much higher fluorescence quantum yield than cytosine, making it an effective fluorescent marker to study the structure, function, and dynamics of DNA/RNA complexes. Similar to 2-aminopurine, a constitutional isomer of adenine that base-pairs with thymine, PC's fluorescence is quenched when it is incorporated into a dinucleotide or a trinucleotide. In this work we examine the photophysical properties of isolated PC, microhydrated PC, as well as, complexes where PC is either base-stacked or hydrogen-bonded with guanine. Our results indicate that hydration affects the radiationless decay pathways in PC by destabilizing conical intersections. The calculations of dimers and trimers show that the radiative decay is affected by π stacking, while the presence of charge transfer states between PC and guanine may contribute to radiationless decay. PMID:27251599

  5. Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli

    PubMed Central

    Bhagwat, Ashok S.; Hao, Weilong; Townes, Jesse P.; Lee, Heewook; Tang, Haixu; Foster, Patricia L.

    2016-01-01

    The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in an Escherichia coli strain defective in uracil repair (ung mutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome. PMID:26839411

  6. Nucleotide discrimination with DNA immobilized in the MspA nanopore.

    PubMed

    Manrao, Elizabeth A; Derrington, Ian M; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H

    2011-01-01

    Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices. PMID:21991340

  7. Pathways of Nucleotide Biosynthesis in Mycoplasma mycoides subsp. mycoides

    PubMed Central

    Mitchell, Alana; Finch, Lloyd R.

    1977-01-01

    By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of 14C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5′-monophosphate to adenosine 5′-monophosphate via the intermediate inosine 5′-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter. PMID:324972

  8. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

    NASA Astrophysics Data System (ADS)

    Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

    2015-01-01

    Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

  9. Communication: UV photoionization of cytosine catalyzed by Ag+

    NASA Astrophysics Data System (ADS)

    Taccone, Martín I.; Féraud, Geraldine; Berdakin, Matías; Dedonder-Lardeux, Claude; Jouvet, Christophe; Pino, Gustavo A.

    2015-07-01

    The photo-induced damages of DNA in interaction with metal cations, which are found in various environments, still remain to be characterized. In this paper, we show how the complexation of a DNA base (cytosine (Cyt)) with a metal cation (Ag+) changes its electronic properties. By means of UV photofragment spectroscopy of cold ions, it was found that the photoexcitation of the CytAg+ complex at low energy (315-282) nm efficiently leads to ionized cytosine (Cyt+) as the single product. This occurs through a charge transfer state in which an electron from the p orbital of Cyt is promoted to Ag+, as confirmed by ab initio calculations at the TD-DFT/B3LYP and RI-ADC(2) theory level using the SV(P) basis set. The low ionization energy of Cyt in the presence of Ag+ could have important implications as point mutation of DNA upon sunlight exposition.

  10. Non-symmetrical cytosine methylation in tobacco pollen DNA.

    PubMed

    Oakeley, E J; Jost, J P

    1996-07-01

    We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region. 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1 x m5CpTpC, 1 x m5CpGpT, 1 x m5CpCpT, 2 x m5CpTpT, 2 x m5CpGpG, and 3 x m5CpApT of which only m5CpGpG and m5CpGpT fitted the consensus sequence for symmetrical methylation in plants. PMID:8806424

  11. High-throughput sequencing of cytosine methylation in plant DNA

    PubMed Central

    2013-01-01

    Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field. PMID:23758782

  12. Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

    SciTech Connect

    R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

  13. An efficient prebiotic synthesis of cytosine and uracil

    NASA Astrophysics Data System (ADS)

    Robertson, Michael P.; Miller, Stanley L.

    1995-06-01

    IN contrast to the purines1 3, the routes that have been proposed for the prebiotic synthesis of pyrimidines from simple precursors give only low yields. Cytosine can be synthesized from cyano-acetylene and cyanate4,5; the former precursor is produced from a spark discharge in a CH4/N2 mixture4,5 and is an abundant interstellar molecule6. But this reaction requires relatively high concentrations of cyanate (>0.1 M), which are unlikely to occur in aqueous media as cyanate is hydrolysed rapidly to CO2 and NH3. An alternative route that has been explored7 is the reaction of cyanoacetaldehyde (formed by hydrolysis of cyanoacetylene8) with urea. But at low concentrations of urea, this reaction produces no detectable quantities of cytosine7. Here we show that in concentrated urea solution-such as might have been found in an evaporating lagoon or in pools on drying beaches on the early Earth-cyanoacetaldehyde reacts to form cytosine in yields of 30-50%, from which uracil can be formed by hydrolysis. These reactions provide a plausible route to the pyrimidine bases required in the RNA world9.

  14. Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry.

    PubMed

    Abakir, Abdulkadir; Wheldon, Lee; Johnson, Andrew D; Laurent, Patrick; Ruzov, Alexey

    2016-01-01

    Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts. PMID:27585398

  15. An efficient prebiotic synthesis of cytosine and uracil

    NASA Technical Reports Server (NTRS)

    Robertson, M. P.; Miller, S. L.

    1995-01-01

    In contrast to the purines, the routes that have been proposed for the prebiotic synthesis of pyrimidines from simple precursors give only low yields. Cytosine can be synthesized from cyanoacetylene and cyanate; the former precursor is produced from a spark discharge in a CH4/N2 mixture and is an abundant interstellar molecule. But this reaction requires relatively high concentrations of cyanate (> 0.1 M), which are unlikely to occur in aqueous media as cyanate is hydrolysed rapidly to CO2 and NH3. An alternative route that has been explored is the reaction of cyanoacetaldehyde (formed by hydrolysis of cyanoacetylene) with urea. But at low concentrations of urea, this reaction produces no detectable quantities of cytosine. Here we show that in concentrated urea solution--such as might have been found in an evaporating lagoon or in pools on drying beaches on the early Earth--cyanoacetaldehyde reacts to form cytosine in yields of 30-50%, from which uracil can be formed by hydrolysis. These reactions provide a plausible route to the pyrimidine bases required in the RNA world.

  16. Updating Our View of Organelle Genome Nucleotide Landscape

    PubMed Central

    Smith, David Roy

    2012-01-01

    Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT) content. Recent studies, however, have uncovered guanine and cytosine (GC)-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits. PMID:22973299

  17. Low energy electron induced cytosine base release in 2′-deoxycytidine-3′-monophosphate via glycosidic bond cleavage: A time-dependent wavepacket study

    SciTech Connect

    Bhaskaran, Renjith; Sarma, Manabendra

    2014-09-14

    Low energy electron (LEE) induced cytosine base release in a selected pyrimidine nucleotide, viz., 2′-deoxycytidine-3′-monophosphate is investigated using ab initio electronic structure methods and time dependent quantum mechanical calculations. It has been noted that the cytosine base scission is comparatively difficult process than the 3′ C–O bond cleavage from the lowest π{sup *} shape resonance in energy region <1 eV. This is mainly due to the high activation energy barrier associated with the electron transfer from the π{sup *} orbital of the base to the σ{sup *} orbital of the glycosidic N–C bond. In addition, the metastable state formed after impinging LEE (0–1 eV) has very short lifetime (10 fs) which may decay in either of the two competing auto-detachment or dissociation process simultaneously. On the other hand, the selected N–C mode may cleave to form the cytosine base anion at higher energy regions (>2 eV) via tunneling of the glycosidic bond. Resonance states generated within this energy regime will exist for a duration of ∼35–55 fs. Comparison of salient features of the two dissociation events, i.e., 3′ C–O single strand break and glycosidic N–C bond cleavage in 3′-dCMPH molecule are also provided.

  18. Low energy electron induced cytosine base release in 2'-deoxycytidine-3'-monophosphate via glycosidic bond cleavage: A time-dependent wavepacket study

    NASA Astrophysics Data System (ADS)

    Bhaskaran, Renjith; Sarma, Manabendra

    2014-09-01

    Low energy electron (LEE) induced cytosine base release in a selected pyrimidine nucleotide, viz., 2'-deoxycytidine-3'-monophosphate is investigated using ab initio electronic structure methods and time dependent quantum mechanical calculations. It has been noted that the cytosine base scission is comparatively difficult process than the 3' C-O bond cleavage from the lowest π* shape resonance in energy region <1 eV. This is mainly due to the high activation energy barrier associated with the electron transfer from the π* orbital of the base to the σ* orbital of the glycosidic N-C bond. In addition, the metastable state formed after impinging LEE (0-1 eV) has very short lifetime (10 fs) which may decay in either of the two competing auto-detachment or dissociation process simultaneously. On the other hand, the selected N-C mode may cleave to form the cytosine base anion at higher energy regions (>2 eV) via tunneling of the glycosidic bond. Resonance states generated within this energy regime will exist for a duration of ˜35-55 fs. Comparison of salient features of the two dissociation events, i.e., 3' C-O single strand break and glycosidic N-C bond cleavage in 3'-dCMPH molecule are also provided.

  19. Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

    PubMed Central

    Pedersen, Jakob Skou; Valen, Eivind; Velazquez, Amhed M. Vargas; Parker, Brian J.; Rasmussen, Morten; Lindgreen, Stinus; Lilje, Berit; Tobin, Desmond J.; Kelly, Theresa K.; Vang, Søren; Andersson, Robin; Jones, Peter A.; Hoover, Cindi A.; Tikhonov, Alexei; Prokhortchouk, Egor; Rubin, Edward M.; Sandelin, Albin; Gilbert, M. Thomas P.; Krogh, Anders; Willerslev, Eske; Orlando, Ludovic

    2014-01-01

    Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics. PMID:24299735

  20. Electronic excited states of guanine-cytosine hairpins and duplexes studied by fluorescence spectroscopy.

    PubMed

    Brazard, Johanna; Thazhathveetil, Arun K; Vayá, Ignacio; Lewis, Frederick D; Gustavsson, Thomas; Markovitsi, Dimitra

    2013-08-01

    Guanine-cytosine hairpins, containing a hexaethylene glycol bridge, are studied by steady-state fluorescence spectroscopy and time-correlated single photon counting; their properties are compared to those of duplexes with the same sequence. It is shown that, both in hairpins and in duplexes, base pairing induces quenching of the ππ* fluorescence, the quantum yield decreasing by at least two orders of magnitude. When the size of the systems increases from two to ten base pairs, a fluorescent component decaying on the nanosecond time-scale appears at energy higher than that stemming from the bright states of non-interacting mono-nucleotides (ca. 330 nm). For ten base pairs, this new fluorescence forms a well-defined band peaking at 305 nm. Its intensity is about 20% higher for the hairpin compared to the duplex. Its position (red-shifted by 1600 cm(-1)) and width (broader by 1800 cm(-1) FWHM) differ from those observed for large duplexes containing 1000 base pairs, suggesting the involvement of electronic coupling. Fluorescence anisotropy reveals that the excited states responsible for high energy emission are not populated directly upon photon absorption but are reached during a relaxation process. They are assigned to charge transfer states. According to the emerging picture, the amplitude of conformational motions determines whether instantaneous deactivation to the ground state or emission from charge transfer states will take place, while ππ* fluorescence is associated to imperfect base-pairing. PMID:23736116

  1. Effects of cytosine methylation on DNA charge transport

    NASA Astrophysics Data System (ADS)

    Hihath, Joshua; Guo, Shaoyin; Zhang, Peiming; Tao, Nongjian

    2012-04-01

    The methylation of cytosine bases in DNA commonly takes place in the human genome and its abnormality can be used as a biomarker in the diagnosis of genetic diseases. In this paper we explore the effects of cytosine methylation on the conductance of DNA. Although the methyl group is a small chemical modification, and has a van der Waals radius of only 2 Å, its presence significantly changes the duplex stability, and as such may also affect the conductance properties of DNA. To determine if charge transport through the DNA stack is sensitive to this important biological modification we perform multiple conductance measurements on a methylated DNA molecule with an alternating G:C sequence and its non-methylated counterpart. From these studies we find a measurable difference in the conductance between the two types of molecules, and demonstrate that this difference is statistically significant. The conductance values of these molecules are also compared with a similar sequence that has been previously studied to help elucidate the charge transport mechanisms involved in direct DNA conductance measurements.

  2. Arabidopsis PAI gene arrangements, cytosine methylation and expression.

    PubMed Central

    Melquist, S; Luff, B; Bender, J

    1999-01-01

    Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression. PMID:10471722

  3. Arabidopsis PAI gene arrangements, cytosine methylation and expression.

    PubMed

    Melquist, S; Luff, B; Bender, J

    1999-09-01

    Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression. PMID:10471722

  4. Communication: UV photoionization of cytosine catalyzed by Ag{sup +}

    SciTech Connect

    Taccone, Martín I.; Berdakin, Matías; Pino, Gustavo A.; Féraud, Geraldine; Dedonder-Lardeux, Claude; Jouvet, Christophe

    2015-07-28

    The photo-induced damages of DNA in interaction with metal cations, which are found in various environments, still remain to be characterized. In this paper, we show how the complexation of a DNA base (cytosine (Cyt)) with a metal cation (Ag{sup +}) changes its electronic properties. By means of UV photofragment spectroscopy of cold ions, it was found that the photoexcitation of the CytAg{sup +} complex at low energy (315-282) nm efficiently leads to ionized cytosine (Cyt{sup +}) as the single product. This occurs through a charge transfer state in which an electron from the p orbital of Cyt is promoted to Ag{sup +}, as confirmed by ab initio calculations at the TD-DFT/B3LYP and RI-ADC(2) theory level using the SV(P) basis set. The low ionization energy of Cyt in the presence of Ag{sup +} could have important implications as point mutation of DNA upon sunlight exposition.

  5. OmpF, a nucleotide-sensing nanoprobe, computational evaluation of single channel activities

    NASA Astrophysics Data System (ADS)

    Abdolvahab, R. H.; Mobasheri, H.; Nikouee, A.; Ejtehadi, M. R.

    2016-09-01

    The results of highthroughput practical single channel experiments should be formulated and validated by signal analysis approaches to increase the recognition precision of translocating molecules. For this purpose, the activities of the single nano-pore forming protein, OmpF, in the presence of nucleotides were recorded in real time by the voltage clamp technique and used as a means for nucleotide recognition. The results were analyzed based on the permutation entropy of current Time Series (TS), fractality, autocorrelation, structure function, spectral density, and peak fraction to recognize each nucleotide, based on its signature effect on the conductance, gating frequency and voltage sensitivity of channel at different concentrations and membrane potentials. The amplitude and frequency of ion current fluctuation increased in the presence of Adenine more than Cytosine and Thymine in milli-molar (0.5 mM) concentrations. The variance of the current TS at various applied voltages showed a non-monotonic trend whose initial increasing slope in the presence of Thymine changed to a decreasing one in the second phase and was different from that of Adenine and Cytosine; e.g., by increasing the voltage from 40 to 140 mV in the 0.5 mM concentration of Adenine or Cytosine, the variance decreased by one third while for the case of Thymine it was doubled. Moreover, according to the structure function of TS, the fractality of current TS differed as a function of varying membrane potentials (pd) and nucleotide concentrations. Accordingly, the calculated permutation entropy of the TS, validated the biophysical approach defined for the recognition of different nucleotides at various concentrations, pd's and polarities. Thus, the promising outcomes of the combined experimental and theoretical methodologies presented here can be implemented as a complementary means in pore-based nucleotide recognition approaches.

  6. Cytosine methylation of sperm DNA in horse semen after cryopreservation.

    PubMed

    Aurich, Christine; Schreiner, Bettina; Ille, Natascha; Alvarenga, Marco; Scarlet, Dragos

    2016-09-15

    Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure. PMID:27242182

  7. Watson-Crick and sugar-edge base pairing of cytosine in the gas phase: UV and infrared spectra of cytosine·2-pyridone.

    PubMed

    Frey, Jann A; Ottiger, Philipp; Leutwyler, Samuel

    2014-01-23

    While keto-amino cytosine is the dominant species in aqueous solution, spectroscopic studies in molecular beams and in noble gas matrices show that other cytosine tautomers prevail in apolar environments. Each of these offers two or three H-bonding sites (Watson-Crick, wobble, sugar-edge). The mass- and isomer-specific S1 ← S0 vibronic spectra of cytosine·2-pyridone (Cyt·2PY) and 1-methylcytosine·2PY are measured using UV laser resonant two-photon ionization (R2PI), UV/UV depletion, and IR depletion spectroscopy. The UV spectra of the Watson-Crick and sugar-edge isomers of Cyt·2PY are separated using UV/UV spectral hole-burning. Five different isomers of Cyt·2PY are observed in a supersonic beam. We show that the Watson-Crick and sugar-edge dimers of keto-amino cytosine with 2PY are the most abundant in the beam, although keto-amino-cytosine is only the third most abundant tautomer in the gas phase. We identify the different isomers by combining three different diagnostic tools: (1) methylation of the cytosine N1-H group prevents formation of both the sugar-edge and wobble isomers and gives the Watson-Crick isomer exclusively. (2) The calculated ground state binding and dissociation energies, relative gas-phase abundances, excitation and the ionization energies are in agreement with the assignment of the dominant Cyt·2PY isomers to the Watson-Crick and sugar-edge complexes of keto-amino cytosine. (3) The comparison of calculated ground state vibrational frequencies to the experimental IR spectra in the carbonyl stretch and NH/OH/CH stretch ranges strengthen this identification. PMID:24383817

  8. Prebiotic cytosine synthesis: A critical analysis and implications for the origin of life

    PubMed Central

    Shapiro, Robert

    1999-01-01

    A number of theories propose that RNA, or an RNA-like substance, played a role in the origin of life. Usually, such hypotheses presume that the Watson–Crick bases were readily available on prebiotic Earth, for spontaneous incorporation into a replicator. Cytosine, however, has not been reported in analyses of meteorites nor is it among the products of electric spark discharge experiments. The reported prebiotic syntheses of cytosine involve the reaction of cyanoacetylene (or its hydrolysis product, cyanoacetaldehyde), with cyanate, cyanogen, or urea. These substances undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation. To favor cytosine formation, reactant concentrations are required that are implausible in a natural setting. Furthermore, cytosine is consumed by deamination (the half-life for deamination at 25°C is ≈340 yr) and other reactions. No reactions have been described thus far that would produce cytosine, even in a specialized local setting, at a rate sufficient to compensate for its decomposition. On the basis of this evidence, it appears quite unlikely that cytosine played a role in the origin of life. Theories that involve replicators that function without the Watson–Crick pairs, or no replicator at all, remain as viable alternatives. PMID:10200273

  9. Cytosine deamination plays a primary role in the evolution of mammalian isochores.

    PubMed

    Fryxell, K J; Zuckerkandl, E

    2000-09-01

    DNA melting is rate-limiting for cytosine deamination, from which we infer that the rate of cytosine deamination should decline twofold for each 10% increase in GC content. Analysis of human DNA sequence data confirms that this is the case for 5-methylcytosine. Several lines of evidence further confirm that it is also the case for unmethylated cytosine and that cytosine deamination causes the majority of all C-->T and G-->A transitions in mammals. Thus, cytosine deamination and DNA base composition each affect the other, forming a positive feedback loop that facilitates divergent genetic drift to high or low GC content. Because a 10 degrees C increase in temperature in vitro increases the rate of cytosine deamination 5. 7-fold, cytosine deamination must be highly dependent on body temperature, which is consistent with the dramatic differences between the isochores of warm-blooded versus cold-blooded vertebrates. Because this process involves both DNA melting and positive feedback, it would be expected to spread progressively (in evolutionary time) down the length of the chromosome, which is consistent with the large size of isochores in modern mammals. PMID:10958853

  10. Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation.

    PubMed

    Buchmann, R Cody; Asad, Shaheen; Wolf, Jamie N; Mohannath, Gireesha; Bisaro, David M

    2009-05-01

    Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS. PMID:19279102

  11. Safety of intrathecal administration of cytosine arabinoside and methotrexate in dogs and cats.

    PubMed

    Genoni, S; Palus, V; Eminaga, S; Cherubini, G B

    2016-09-01

    The objective of the study was to retrospectively evaluate the short-term safety of intrathecal administration of cytosine arabinoside alone or in combination with methotrexate in dogs and cats. One hundred and twelve dogs and eight cats admitted between September 2008 and December 2013, diagnosed with suspected inflammatory (meningoencephalomyelitis of unknown aetiology) or neoplastic disease affecting brain or spinal cord and treated with an intrathecal administration of cytosine arabinoside alone or in combination with methotrexate were included in the study. Recorded information regarding possible adverse events during administration while recovering from anaesthesia and during hospitalization period were evaluated. The results showed that one patient developed generalized tonic-clonic seizure activity after administration of cytosine arabinoside and methotrexate during recovery from anaesthesia, however responded to intravenous administration of diazepam. On the base of our results we can conclude that intrathecal administration of cytosine arabinoside alone or in combination with methotrexate is a safe procedure in dogs and cats. PMID:25041580

  12. Genome-Wide Discriminatory Information Patterns of Cytosine DNA Methylation.

    PubMed

    Sanchez, Robersy; Mackenzie, Sally A

    2016-01-01

    Cytosine DNA methylation (CDM) is a highly abundant, heritable but reversible chemical modification to the genome. Herein, a machine learning approach was applied to analyze the accumulation of epigenetic marks in methylomes of 152 ecotypes and 85 silencing mutants of Arabidopsis thaliana. In an information-thermodynamics framework, two measurements were used: (1) the amount of information gained/lost with the CDM changes I R and (2) the uncertainty of not observing a SNP L C R . We hypothesize that epigenetic marks are chromosomal footprints accounting for different ontogenetic and phylogenetic histories of individual populations. A machine learning approach is proposed to verify this hypothesis. Results support the hypothesis by the existence of discriminatory information (DI) patterns of CDM able to discriminate between individuals and between individual subpopulations. The statistical analyses revealed a strong association between the topologies of the structured population of Arabidopsis ecotypes based on I R and on LCR, respectively. A statistical-physical relationship between I R and L C R was also found. Results to date imply that the genome-wide distribution of CDM changes is not only part of the biological signal created by the methylation regulatory machinery, but ensures the stability of the DNA molecule, preserving the integrity of the genetic message under continuous stress from thermal fluctuations in the cell environment. PMID:27322251

  13. Genome-Wide Discriminatory Information Patterns of Cytosine DNA Methylation

    PubMed Central

    Sanchez, Robersy; Mackenzie, Sally A.

    2016-01-01

    Cytosine DNA methylation (CDM) is a highly abundant, heritable but reversible chemical modification to the genome. Herein, a machine learning approach was applied to analyze the accumulation of epigenetic marks in methylomes of 152 ecotypes and 85 silencing mutants of Arabidopsis thaliana. In an information-thermodynamics framework, two measurements were used: (1) the amount of information gained/lost with the CDM changes IR and (2) the uncertainty of not observing a SNP LCR. We hypothesize that epigenetic marks are chromosomal footprints accounting for different ontogenetic and phylogenetic histories of individual populations. A machine learning approach is proposed to verify this hypothesis. Results support the hypothesis by the existence of discriminatory information (DI) patterns of CDM able to discriminate between individuals and between individual subpopulations. The statistical analyses revealed a strong association between the topologies of the structured population of Arabidopsis ecotypes based on IR and on LCR, respectively. A statistical-physical relationship between IR and LCR was also found. Results to date imply that the genome-wide distribution of CDM changes is not only part of the biological signal created by the methylation regulatory machinery, but ensures the stability of the DNA molecule, preserving the integrity of the genetic message under continuous stress from thermal fluctuations in the cell environment. PMID:27322251

  14. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study.

    PubMed

    Emel'yanenko, Vladimir N; Zaitsau, Dzmitry H; Shoifet, Evgeni; Meurer, Florian; Verevkin, Sergey P; Schick, Christoph; Held, Christoph

    2015-09-17

    The thermochemical properties available in the literature for adenine and cytosine are in disarray. A new condensed phase standard (p° = 0.1 MPa) molar enthalpy of formation at T = 298.15 K was measured by using combustion calorimetry. New molar enthalpies of sublimation were derived from the temperature dependence of vapor pressure measured by transpiration and by the quarz-crystal microbalance technique. The heat capacities of crystalline adenine and cytosine were measured by temperature-modulated DSC. Thermodynamic data on adenine and cytosine available in the literature were collected, evaluated, and combined with our experimental results. Thus, the evaluated collection of data together with the new experimental results reported here has helped to resolve contradictions in the available enthalpies of formation. A set of reliable thermochemical data is recommended for adenine and cytosine for further thermochemical calculations. Quantum-chemical calculations of the gas phase molar enthalpies of formation of adenine and cytosine have been performed by using the G4 method and results were in excellent agreement with the recommended experimental data. The standard molar entropies of formation and the standard molar Gibbs functions of formation in crystal and gas state have been calculated. Experimental vapor-pressure data measured in this work were used to estimate pure-component PC-SAFT parameters. This allowed modeling solubility of adenine and cytosine in water over the temperature interval 278-310 K. PMID:26317826

  15. Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme.

    PubMed Central

    Som, S; Bhagwat, A S; Friedman, S

    1987-01-01

    The gene coding for the EcoRII modification enzyme has been cloned and the nucleotide sequence of 1933 base pairs containing the gene has been determined. The gene codes for a protein of 477 amino acids. Two transcriptional start sites have been mapped by S1 mapping. One deletion that removes 34 N-terminal amino acids was found to have partial enzyme activity. Comparison of the EcoRII methylase sequence with other cytosine methylases revealed several domains of partial homology among all cytosine methylases. Cloning the gene in multicopy pUC vectors increased the expression by 6-18 fold. A 40 fold overproduction of the EcoRII methylase was obtained by cloning the gene in the expression vector carrying the lambda PL promoter. Images PMID:3029675

  16. Plant Cyclic Nucleotide Signalling

    PubMed Central

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc

    2007-01-01

    The presence of the cyclic nucleotides 3′,5′-cyclic adenyl monophosphate (cAMP) and 3′,5′-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed. PMID:19704553

  17. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

    An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

  18. Structure of the 2-Aminopurine-Cytosine Base Pair Formed in the Polymerase Active Site of the RB69 Y567A-DNA Polymerase

    SciTech Connect

    Reha-Krantz, Linda J.; Hariharan, Chithra; Subuddhi, Usharani; Xia, Shuangluo; Zhao, Chao; Beckman, Jeff; Christian, Thomas; Konigsberg, William

    2011-11-21

    The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased 'breathing' at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

  19. A computational study of adenine, uracil, and cytosine adsorption upon AlN and BN nano-cages

    NASA Astrophysics Data System (ADS)

    Baei, Mohammad T.; Taghartapeh, Mohammad Ramezani; Lemeski, E. Tazikeh; Soltani, Alireza

    Density-functional theory calculations are used to investigate the interaction of Al12N12 and B12N12 clusters with the adenine (A), uracil (U), and cytosine (C) molecules. The current calculations demonstrate that these hybrid adsorbent materials are able to adsorb the adenine, uracil, and cytosine molecules through exothermic processes. Our theoretical results reveal improvement in the adsorption of adenine, uracil, and cytosine on Al12N12 and B12N12. It is observed that B12N12 is highly sensitive to adenine, uracil, and cytosine compared with Al12N12 to serve as a biochemical sensor.

  20. Cytochrome b nucleotide sequence variation among the Atlantic Alcidae.

    PubMed

    Friesen, V L; Montevecchi, W A; Davidson, W S

    1993-01-01

    Analysis of cytochrome b nucleotide sequences of the six extant species of Atlantic alcids and a gull revealed an excess of adenines and cytosines and a deficit of guanines at silent sites on the coding strand. Phylogenetic analyses grouped the sequences of the common (Uria aalge) and Brünnich's (U. lomvia) guillemots, followed by the razorbill (Alca torda) and little auk (Alle alle). The black guillemot (Cepphus grylle) sequence formed a sister taxon, and the puffin (Fratercula arctica) fell outside the other alcids. Phylogenetic comparisons of substitutions indicated that mutabilities of bases did not differ, but that C was much more likely to be incorporated than was G. Imbalances in base composition appear to result from a strand bias in replication errors, which may result from selection on secondary RNA structure and/or the energetics of codon-anticodon interactions. PMID:7916741

  1. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes.

    PubMed

    Shi, Xianzong; Jarvis, Donald L

    2006-09-15

    Rapid amplification of cDNA ends (RACE) is widely used to determine the 5'- and 3'-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none addresses the difficult problems that arise when trying to isolate the ends of extremely guanine plus cytosine (GC)-rich genes. In this study, we found that we were unable to isolate the correct 5' or 3' end of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first-strand cDNA synthesis at 70 degrees C with Thermo-X reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98 degrees C denaturation steps and Phusion DNA polymerase in the presence of 1M betaine and 5% dimethyl sulfoxide (DMSO). The use of these conditions yielded 5'- and 3'-RACE products that were approximately 80% GC over 213 and 162bp, respectively, and included shorter internal regions of 82 to 89% GC. PMID:16875657

  2. Modulation of metabolism and cytotoxicity of cytosine arabinoside with N-(phosphon)-acetyl-L-aspartate in human leukemic blast cells and cell lines.

    PubMed

    Noordhuis, P; Kazemier, K M; Kasperrs, G J; Peters, G J

    1996-02-01

    Cytosine arabinoside (Ara-C) activation to cytosine arabinoside triphosphate (Ara-CTP) and subsequent incorporation into DNA is regulated by the pyrimidine nucleotides UTP, CTP and dCTP. Inhibition of the de novo synthesis of these pyrimidine nucleotides by N-(phosphon)-acetyl-L-aspartate (PALA) may enhance the cytotoxicity of Ara-C. We therefore studied the effect of PALA on Ara-C cytotoxicity and on Ara-CTP accumulation and incorporation into DNA on cell lines and patient samples. Fifty micromolar PALA increased the growth inhibitory effect of Ara-C in U937 cells several fold both with pre- and coincubation. Ara-C cytotoxicity was not potentiated by PALA in Hl60 cells. However, coincubation with PALA did not enhance Ara-CTP accumulation both in HL60 and U937 cells, nor affect Ara-C incorporation into DNA. Ara-C cytotoxicity to leukemic blast cells from 11 untreated patients with different types of leukemia was only modulated to a small extent by high PALA concentrations in only two cases. Ara-CTP accumulation in leukemic blast cells varied from non-detectable levels to 200 pmol/10(6) cells. Fifty micromolar PALA enhanced the accumulation of Ara-CTP significantly in only one patient with no apparent effect on UTP and CTP levels. Raising PALA to 500 microM decreased UTP and CTP levels to 50% but had no effect on Ara-CTP levels. In conclusion, modulation by PALA of Ara-C cytotoxicity and metabolism is limited in leukemic cells, both in culture and from patients. This suggests the possibility for selective modulation of other agents by PALA on non-hematological cells. PMID:8628011

  3. Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging.

    PubMed

    Wang, Xiaolei; Cui, Yi; Irudayaraj, Joseph

    2015-12-22

    Epigenetic modifications on DNA, especially on cytosine, play a critical role in regulating gene expression and genome stability. It is known that the levels of different cytosine derivatives are highly dynamic and are regulated by a variety of factors that act on the chromatin. Here we report an optical methodology based on hyperspectral dark-field imaging (HSDFI) using plasmonic nanoprobes to quantify the recently identified cytosine modifications on DNA in single cells. Gold (Au) and silver (Ag) nanoparticles (NPs) functionalized with specific antibodies were used as contrast-generating agents due to their strong local surface plasmon resonance (LSPR) properties. With this powerful platform we have revealed the spatial distribution and quantity of 5-carboxylcytosine (5caC) at the different stages in cell cycle and demonstrated that 5caC was a stably inherited epigenetic mark. We have also shown that the regional density of 5caC on a single chromosome can be mapped due to the spectral sensitivity of the nanoprobes in relation to the interparticle distance. Notably, HSDFI enables an efficient removal of the scattering noises from nonspecifically aggregated nanoprobes, to improve accuracy in the quantification of different cytosine modifications in single cells. Further, by separating the LSPR fingerprints of AuNPs and AgNPs, multiplex detection of two cytosine modifications was also performed. Our results demonstrate HSDFI as a versatile platform for spatial and spectroscopic characterization of plasmonic nanoprobe-labeled nuclear targets at the single-cell level for quantitative epigenetic screening. PMID:26505210

  4. Hydroxymethylated Cytosines Are Associated with Elevated C to G Transversion Rates

    PubMed Central

    Warnecke, Tobias

    2014-01-01

    It has long been known that methylated cytosines deaminate at higher rates than unmodified cytosines and constitute mutational hotspots in mammalian genomes. The repertoire of naturally occurring cytosine modifications, however, extends beyond 5-methylcytosine to include its oxidation derivatives, notably 5-hydroxymethylcytosine. The effects of these modifications on sequence evolution are unknown. Here, we combine base-resolution maps of methyl- and hydroxymethylcytosine in human and mouse with population genomic, divergence and somatic mutation data to show that hydroxymethylated and methylated cytosines show distinct patterns of variation and evolution. Surprisingly, hydroxymethylated sites are consistently associated with elevated C to G transversion rates at the level of segregating polymorphisms, fixed substitutions, and somatic mutations in tumors. Controlling for multiple potential confounders, we find derived C to G SNPs to be 1.43-fold (1.22-fold) more common at hydroxymethylated sites compared to methylated sites in human (mouse). Increased C to G rates are evident across diverse functional and sequence contexts and, in cancer genomes, correlate with the expression of Tet enzymes and specific components of the mismatch repair pathway (MSH2, MSH6, and MBD4). Based on these and other observations we suggest that hydroxymethylation is associated with a distinct mutational burden and that the mismatch repair pathway is implicated in causing elevated transversion rates at hydroxymethylated cytosines. PMID:25211471

  5. Global cytosine methylation in Daphnia magna depends on genotype, environment, and their interaction.

    PubMed

    Asselman, Jana; De Coninck, Dieter I M; Vandegehuchte, Michiel B; Jansen, Mieke; Decaestecker, Ellen; De Meester, Luc; Vanden Bussche, Julie; Vanhaecke, Lynn; Janssen, Colin R; De Schamphelaere, Karel A C

    2015-05-01

    The authors characterized global cytosine methylation levels in 2 different genotypes of the ecotoxicological model organism Daphnia magna after exposure to a wide array of biotic and abiotic environmental stressors. The present study aimed to improve the authors' understanding of the role of cytosine methylation in the organism's response to environmental conditions. The authors observed a significant genotype effect, an environment effect, and a genotype × environment effect. In particular, global cytosine methylation levels were significantly altered after exposure to Triops predation cues, Microcystis, and sodium chloride compared with control conditions. Significant differences between the 2 genotypes were observed when animals were exposed to Triops predation cues, Microcystis, Cryptomonas, and sodium chloride. Despite the low global methylation rate under control conditions (0.49-0.52%), global cytosine methylation levels upon exposure to Triops demonstrated a 5-fold difference between the genotypes (0.21% vs 1.02%). No effects were found in response to arsenic, cadmium, fish, lead, pH of 5.5, pH of 8, temperature, hypoxia, and white fat cell disease. The authors' results point to the potential role of epigenetic effects under changing environmental conditions such as predation (i.e., Triops), diet (i.e., Cryptomonas and Microcystis), and salinity. The results of the present study indicate that, despite global cytosine methylation levels being low, epigenetic effects may be important in environmental studies on Daphnia. PMID:25639773

  6. The effect of sequence context on the activity of cytosine DNA glycosylases.

    PubMed

    Kimber, Scott T; Brown, Tom; Fox, Keith R

    2015-12-01

    We have prepared single (N204D) and double (N204D:L272A) mutants of human uracil DNA glycosylase (hUDG), generating two cytosine DNA glycosylases (hCDG and hCYDG). Both these enzymes are able to excise cytosine (but not 5-methylcytosine), when this base is part of a mismatched base pair. hCDG is more active than the equivalent E. coli enzyme (eCYDG) and also has some activity when the cytosine is paired with guanine, unlike eCYDG. hCDG also has some activity against single stranded DNA, while having poor activity towards an unnatural base pair that forces the cytosine into an extrahelical conformation (in contrast to eCYDG for which a bulky base enhances the enzyme's activity). We also examined how sequence context affects the activity of these enzymes, determining the effect of flanking base pairs on cleavage efficiency. An abasic site or a hexaethylene glycol linker placed opposite the target cytosine, also causes an increase in activity compared with an AC mismatch. Flanking an AC mismatch with GC base pairs resulted in a 100-fold decrease in excision activity relative to flanking AT base pairs and the 5'-flanking base pair had a greater effect on the rate of cleavage. However, this effect is not simply due to the stability of the flanking base pairs as adjacent GT mismatches also produce low cleavage efficiency. PMID:26463365

  7. Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

    2015-11-01

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct

  8. Prokaryotic nucleotide excision repair.

    PubMed

    Kisker, Caroline; Kuper, Jochen; Van Houten, Bennett

    2013-03-01

    Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation. PMID:23457260

  9. Expression of microRNAs in Horse Plasma and Their Characteristic Nucleotide Composition

    PubMed Central

    Lee, Seungwoo; Hwang, Seungwoo; Yu, Hee Jeong; Oh, Dayoung; Choi, Yu Jung; Kim, Myung-Chul; Kim, Yongbaek; Ryu, Doug-Young

    2016-01-01

    MicroRNAs (miRNAs) in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition. PMID:26731407

  10. Expression of microRNAs in Horse Plasma and Their Characteristic Nucleotide Composition.

    PubMed

    Lee, Seungwoo; Hwang, Seungwoo; Yu, Hee Jeong; Oh, Dayoung; Choi, Yu Jung; Kim, Myung-Chul; Kim, Yongbaek; Ryu, Doug-Young

    2016-01-01

    MicroRNAs (miRNAs) in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition. PMID:26731407

  11. Quantum chemical investigations on the nonradiative deactivation pathways of cytosine derivatives.

    PubMed

    Nakayama, Akira; Yamazaki, Shohei; Taketsugu, Tetsuya

    2014-10-01

    The nonradiative deactivation pathways of cytosine derivatives (cytosine, 5-fluorocytosine, 5-methylcytosine, and 1-methycytosine) and their tautomers are investigated by quantum chemical calculations, and the substituent effects on the deactivation process are examined. The MS-CASPT2 method is employed in the excited-state geometry optimization and also in the search for conical intersection points, and the potential energy profiles connecting the Franck-Condon point, excited-state minimum energy structures, and the conical intersection points are investigated. Our calculated vertical and adiabatic excitation energies are in quite good agreement with the experimental results, and the relative barrier heights leading to the conical intersections are correlated with the experimentally observed excite-state lifetimes, where the calculated barrier heights are in the order of cytosine < 5-methylcytosine < 5-fluorocytosine. PMID:25178384

  12. Ionization of cytosine monomer and dimer studied by VUV photoionization and electronic structure calculations

    SciTech Connect

    Kostko, Oleg; Bravaya, Ksenia; Krylov, Anna; Ahmed, Musahid

    2009-12-14

    We report a combined theoretical and experimental study of ionization of cytosine monomers and dimers. Gas-phase molecules are generated by thermal vaporization of cytosine followed by expansion of the vapor in a continuous supersonic jet seeded in Ar. The resulting species are investigated by single photon ionization with tunable vacuum-ultraviolet (VUV) synchrotron radiation and mass analyzed using reflectron mass spectrometry. Energy onsets for the measured photoionization efficiency (PIE) spectra are 8.60+-0.05 eV and 7.6+-0.1 eV for the monomer and the dimer, respectively, and provide an estimate for the adiabatic ionization energies (AIE). The first AIE and the ten lowest vertical ionization energies (VIEs) for selected isomers of cytosine dimer computed using equation-of-motion coupled-cluster (EOM-IP-CCSD) method are reported. The comparison of the computed VIEs with the derivative of the PIE spectra, suggests that multiple isomers of the cytosine dimer are present in the molecular beam. The calculations reveal that the large red shift (0.7 eV) of the first IE of the lowest-energy cytosine dimer is due to strong inter-fragment electrostatic interactions, i.e., the hole localized on one of the fragments is stabilized by the dipole moment of the other. A sharp rise in the CH+ signal at 9.20+-0.05 eV is ascribed to the formation of protonated cytosine by dissociation of the ionized dimers. The dominant role of this channel is supported by the computed energy thresholds for the CH+ appearance and the barrierless or nearly barrierless ionization-induced proton transfer observed for five isomers of the dimer.

  13. Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history.

    PubMed

    Lewis, Charles A; Crayle, Jesse; Zhou, Shuntai; Swanstrom, Ronald; Wolfenden, Richard

    2016-07-19

    The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90 °C to 200 °C and indicated a heat of activation (ΔH(‡)) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 °C to 25 °C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years. PMID:27382162

  14. Influence of C5-methylation of cytosine on the formation of cyclobutane pyrimidine dimers

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyi; Eriksson, Leif A.

    2005-01-01

    The reaction pathways for thermal and photochemical formation of 5-methylcytosine (m 5C) pyrimidine dimers (CPD) are explored using density functional theory techniques. It is shown that the methylation of cytosine does not contribute to an increased yield of CPDs after UV irradiation due to an even lower excitation energy at the reactant complex of m 5C as compared to cytosine, a larger barrier to reach the decay channel corresponding to the transition state structure along the ground state reaction path, and a higher-lying decay channel.

  15. Nucleotide diversity in gorillas.

    PubMed Central

    Yu, Ning; Jensen-Seaman, Michael I; Chemnick, Leona; Ryder, Oliver; Li, Wen-Hsiung

    2004-01-01

    Comparison of the levels of nucleotide diversity in humans and apes may provide valuable information for inferring the demographic history of these species, the effect of social structure on genetic diversity, patterns of past migration, and signatures of past selection events. Previous DNA sequence data from both the mitochondrial and the nuclear genomes suggested a much higher level of nucleotide diversity in the African apes than in humans. Noting that the nuclear DNA data from the apes were very limited, we previously conducted a DNA polymorphism study in humans and another in chimpanzees and bonobos, using 50 DNA segments randomly chosen from the noncoding, nonrepetitive parts of the human genome. The data revealed that the nucleotide diversity (pi) in bonobos (0.077%) is actually lower than that in humans (0.087%) and that pi in chimpanzees (0.134%) is only 50% higher than that in humans. In the present study we sequenced the same 50 segments in 15 western lowland gorillas and estimated pi to be 0.158%. This is the highest value among the African apes but is only about two times higher than that in humans. Interestingly, available mtDNA sequence data also suggest a twofold higher nucleotide diversity in gorillas than in humans, but suggest a threefold higher nucleotide diversity in chimpanzees than in humans. The higher mtDNA diversity in chimpanzees might be due to the unique pattern in the evolution of chimpanzee mtDNA. From the nuclear DNA pi values, we estimated that the long-term effective population sizes of humans, bonobos, chimpanzees, and gorillas are, respectively, 10,400, 12,300, 21,300, and 25,200. PMID:15082556

  16. Global DNA cytosine methylation as an evolving trait: phylogenetic signal and correlated evolution with genome size in angiosperms.

    PubMed

    Alonso, Conchita; Pérez, Ricardo; Bazaga, Pilar; Herrera, Carlos M

    2015-01-01

    DNA cytosine methylation is a widespread epigenetic mechanism in eukaryotes, and plant genomes commonly are densely methylated. Genomic methylation can be associated with functional consequences such as mutational events, genomic instability or altered gene expression, but little is known on interspecific variation in global cytosine methylation in plants. In this paper, we compare global cytosine methylation estimates obtained by HPLC and use a phylogenetically-informed analytical approach to test for significance of evolutionary signatures of this trait across 54 angiosperm species in 25 families. We evaluate whether interspecific variation in global cytosine methylation is statistically related to phylogenetic distance and also whether it is evolutionarily correlated with genome size (C-value). Global cytosine methylation varied widely between species, ranging between 5.3% (Arabidopsis) and 39.2% (Narcissus). Differences between species were related to their evolutionary trajectories, as denoted by the strong phylogenetic signal underlying interspecific variation. Global cytosine methylation and genome size were evolutionarily correlated, as revealed by the significant relationship between the corresponding phylogenetically independent contrasts. On average, a ten-fold increase in genome size entailed an increase of about 10% in global cytosine methylation. Results show that global cytosine methylation is an evolving trait in angiosperms whose evolutionary trajectory is significantly linked to changes in genome size, and suggest that the evolutionary implications of epigenetic mechanisms are likely to vary between plant lineages. PMID:25688257

  17. Global DNA cytosine methylation as an evolving trait: phylogenetic signal and correlated evolution with genome size in angiosperms

    PubMed Central

    Alonso, Conchita; Pérez, Ricardo; Bazaga, Pilar; Herrera, Carlos M.

    2015-01-01

    DNA cytosine methylation is a widespread epigenetic mechanism in eukaryotes, and plant genomes commonly are densely methylated. Genomic methylation can be associated with functional consequences such as mutational events, genomic instability or altered gene expression, but little is known on interspecific variation in global cytosine methylation in plants. In this paper, we compare global cytosine methylation estimates obtained by HPLC and use a phylogenetically-informed analytical approach to test for significance of evolutionary signatures of this trait across 54 angiosperm species in 25 families. We evaluate whether interspecific variation in global cytosine methylation is statistically related to phylogenetic distance and also whether it is evolutionarily correlated with genome size (C-value). Global cytosine methylation varied widely between species, ranging between 5.3% (Arabidopsis) and 39.2% (Narcissus). Differences between species were related to their evolutionary trajectories, as denoted by the strong phylogenetic signal underlying interspecific variation. Global cytosine methylation and genome size were evolutionarily correlated, as revealed by the significant relationship between the corresponding phylogenetically independent contrasts. On average, a ten-fold increase in genome size entailed an increase of about 10% in global cytosine methylation. Results show that global cytosine methylation is an evolving trait in angiosperms whose evolutionary trajectory is significantly linked to changes in genome size, and suggest that the evolutionary implications of epigenetic mechanisms are likely to vary between plant lineages. PMID:25688257

  18. Interaction of cyclic cytosine-, guanine-, thymine-, uracil- and mixed guanine-cytosine base tetrads with K+, Na+ and Li+ ions -- a density functional study.

    PubMed

    Meyer, Michael; Sühnel, Jürgen

    2003-02-01

    We have carried out B3LYP hybrid density functional studies of complexes formed by cyclic cytosine-, guanine-, thymine-, uracil- and mixed guanine cytosine-tetrads with Li+, Na+ and K+ ions to determine their structures and interaction energies. The conformations studied have been restricted to a hydrogen bond pattern closely related to the tetrads observed in experimental nucleic acid structures. A comparison of the alkali metal ion/tetrad complexes with the tetrads without cations indicates that alkali metal ions modulate the tetrad structures significantly and that even the hydrogen bond pattern may change. Guanine-tetrad cation complexes show the strongest interaction energy compared to other tetrads that occur less frequently in experimental structures. The most stable G-tetrad/metal ion structure adopts a nearly planar geometry that is especially suitable for tetraplex formation, which requires approximately parallel tetrad planes. In the cytosine-tetrad there is a very large central cavity suitable for cation recognition, but the complexes adopt a non-planar structure unsuitable for stacking, except possibly for ions with very large radii. Uracil and thymine tetrads show a significant different characteristics which may contribute to the differences between DNA and RNA PMID:12529150

  19. The analytical and biomedical potential of cytosine-rich oligonucleotides: A review.

    PubMed

    Dembska, Anna

    2016-08-01

    Polycytosine DNA strands are often found among natural sequences, including the ends of telomeres, centromeres, and introns or in the regulatory regions of genes. A characteristic feature of oligonucleotides that are rich in cytosine (C-rich) is their ability to associate under acidic conditions to form a tetraplex i-motif consisting of two parallel stranded cytosine-hemiprotonated cytosine (C·C+) base-paired duplexes that are mutually intercalated in an antiparallel orientation. Nanotechnology has been exploiting the advantages of i-motif pH-dependent formation to fabricate nanomachines, nanoswitches, electrodes and intelligent nanosurfaces or nanomaterials. Although a few reviews regarding the structure, properties and applications of i-motifs have been published, this review focuses on recently developed biosensors (e.g., to detect pH, glucose or silver ions) and drug-delivery biomaterials. Furthermore, we have included examples of sensors based on parallel C-rich triplexes and silver nanoclusters (AgNCs) fabricated on cytosine-rich DNA strands. The potential diagnostic and therapeutic applications of this type of material are discussed. PMID:27265899

  20. Comments on `Concentration by Evaporation and the Prebiotic Synthesis of Cytosine'

    NASA Astrophysics Data System (ADS)

    Shapiro, Robert

    2002-06-01

    The claim by Nelson et al. (2001) that the reaction of cyanoacetaldehyde and urea provides `an efficient prebiotic synthesis' of cytosine is disputed. The authors have not dealt with the important points presented in a criticism of this reaction (Shapiro, 1999): (1) The reactants undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation, and (2) No reactions have been described thus far that would produce cytosine at a rate sufficient to compensate for its decomposition by deamination, and permit accumulation over extended periods of time. Instead, Nelson et al. have conducted `drying-down' experiments, in an effort to simulate evaporations on the early Earth, but the design of these experiments is flawed. The initial reactant concentrations are much higher than might be expected in a natural setting, and potentially interfering substances such as glycine, cyanide and thiols have been excluded. `Drying beaches and drying lagoons' have been invoked as sites for such a reaction but no effort has been made to describe the characteristics of such sites or to estimate their frequency with reference to the present Earth. In the absence of contradictory data, the conclusion put forward in Shapiro (1999) remains valid: `It was quite unlikely that cytosine played a role in the origin of life'.

  1. Cytosine methylation of an ancient satellite family in the wild beet Beta procumbens.

    PubMed

    Schmidt, Martin; Hense, Sarah; Minoche, André E; Dohm, Juliane C; Himmelbauer, Heinz; Schmidt, Thomas; Zakrzewski, Falk

    2014-01-01

    DNA methylation is an essential epigenetic feature for the regulation and maintenance of heterochromatin. Satellite DNA is a repetitive sequence component that often occurs in large arrays in heterochromatin of subtelomeric, intercalary and centromeric regions. Knowledge about the methylation status of satellite DNA is important for understanding the role of repetitive DNA in heterochromatization. In this study, we investigated the cytosine methylation of the ancient satellite family pEV in the wild beet Beta procumbens. The pEV satellite is widespread in species-specific pEV subfamilies in the genus Beta and most likely originated before the radiation of the Betoideae and Chenopodioideae. In B. procumbens, the pEV subfamily occurs abundantly and spans intercalary and centromeric regions. To uncover its cytosine methylation, we performed chromosome-wide immunostaining and bisulfite sequencing of pEV satellite repeats. We found that CG and CHG sites are highly methylated while CHH sites show only low levels of methylation. As a consequence of the low frequency of CG and CHG sites and the preferential occurrence of most cytosines in the CHH motif in pEV monomers, this satellite family displays only low levels of total cytosine methylation. PMID:24994030

  2. Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells

    PubMed Central

    Mesa-Pereira, Beatriz; Medina, Carlos; Camacho, Eva María; Flores, Amando; Santero, Eduardo

    2015-01-01

    In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate-inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5-fluorocytosine, a 5-fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures. PMID:25227763

  3. The role of gene body cytosine modifications in MGMT expression and sensitivity to temozolomide

    PubMed Central

    Moen, Erika L.; Stark, Amy L.; Zhang, Wei; Dolan, M. Eileen; Godley, Lucy A.

    2014-01-01

    The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosine modification in regulating levels of MGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, we observed significant variation in MGMT expression levels in patients with an unmethylated promoter, with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across the MGMT gene body with decitabine corresponded with decreased levels of MGMT gene expression in lymphoblastoid and glioblastoma cell lines, indicating an important functional role for gene body cytosine modifications in maintaining gene expression. We reasoned that the decrease in MGMT expression induced by decitabine may render resistant glioblastoma cell lines more sensitive to temozolomide. Consistent with this reasoning, we found that the MGMT-expressing glioblastoma cell lines exhibiting an unmethylated MGMT promoter that were pre-treated with decitabine became significantly more sensitive to temozolomide. Overall, our results suggest a functional role for gene body cytosine modification in regulating gene expression of MGMT and indicate that pre-treating patients whose tumors have an unmethylated MGMT promoter with decitabine prior to temozolomide treatment may increase their response to therapy. PMID:24568970

  4. Nucleotide cleaving agents and method

    DOEpatents

    Que, Jr., Lawrence; Hanson, Richard S.; Schnaith, Leah M. T.

    2000-01-01

    The present invention provides a unique series of nucleotide cleaving agents and a method for cleaving a nucleotide sequence, whether single-stranded or double-stranded DNA or RNA, using and a cationic metal complex having at least one polydentate ligand to cleave the nucleotide sequence phosphate backbone to yield a hydroxyl end and a phosphate end.

  5. Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-Fluorocytosine

    PubMed Central

    Stolworthy, Tiffany S.; Korkegian, Aaron M.; Willmon, Candice L.; Ardiani, Andressa; Cundiff, Jennifer; Stoddard, Barry L.; Black, Margaret E.

    2008-01-01

    SUMMARY Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), an inhibitor of DNA synthesis and RNA function. Over 150 studies of cytosine deaminase-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of cytosine deaminase are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study we stabilized and extended the half-life of yeast cytosine deaminase (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated Tm values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in the temperature that induces protein unfolding and aggregation in thermal denaturation experiments measured by circular dichroism spectroscopy, and an increase in the half-life of enzyme activity at physiological temperature, as well as more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models. PMID:18291415

  6. Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging

    PubMed Central

    Wang, Xiaolei; Cui, Yi; Irudayaraj, Joseph

    2016-01-01

    Epigenetic modifications on DNA, especially on cytosine, play a critical role in regulating gene expression and genome stability. It is known that the levels of different cytosine derivatives are highly dynamic and are regulated by a variety of factors that act on the chromatin. Here we report an optical methodology based on hyperspectral dark-field imaging (HSDFI) using plasmonic nanoprobes to quantify the recently identified cytosine modifications on DNA in single cells. Gold (Au) and silver (Ag) nanoparticles (NPs) functionalized with specific antibodies were used as contrast-generating agents due to their strong Local Surface Plasmon Resonance (LSPR) properties. With this powerful platform we have revealed the spatial distribution and quantity of 5-carboxylcytosine (5caC) at the different stages in cell cycle, and demonstrated that 5caC was a stably inherited epigenetic mark. We have also shown that the regional density of 5caC on a single chromosome can be mapped due to the spectral sensitivity of the nanoprobes in relation to the inter-particle distance. Notably, HSDFI enables an efficient removal of the scattering noises from non-specifically aggregated nanoprobes, to improve accuracy in the quantification of different cytosine modifications in single cells. Further, by separating the LSPR fingerprints of AuNPs and AgNPs, multiplex detection of two cytosine modifications was also performed. Our results demonstrate HSDFI as a versatile platform for spatial and spectroscopic characterization of plasmonic nanoprobe-labeled nuclear targets at the single-cell level for quantitative epigenetic screening. PMID:26505210

  7. Cytosine Methylation Alteration in Natural Populations of Leymus chinensis Induced by Multiple Abiotic Stresses

    PubMed Central

    Yu, Yingjie; Yang, Xuejiao; Wang, Huaying; Shi, Fengxue; Liu, Ying; Liu, Jushan; Li, Linfeng; Wang, Deli; Liu, Bao

    2013-01-01

    Background Human activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N) addition, and warming+nitrogen (N) addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP) and retrotransposon based sequence-specific amplification polymorphism (SSAP) techniques. Methodology/Principal Findings Our results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML) indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses. Conclusions/Significance Our results demonstrated that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid adaptation by

  8. Conformational Variants of Duplex DNA Correlated with Cytosine-rich Chromosomal Fragile Sites*S⃞

    PubMed Central

    Tsai, Albert G.; Engelhart, Aaron E.; Hatmal, Ma'mon M.; Houston, Sabrina I.; Hud, Nicholas V.; Haworth, Ian S.; Lieber, Michael R.

    2009-01-01

    We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2′-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B- or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility. PMID:19106104

  9. Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax

    PubMed Central

    2012-01-01

    Background Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells. Results Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications occur only during nuclear development and programmed genome rearrangement. We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing. We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce demethylation of both somatic and germline sequence elements during genome rearrangements, with consequent elevated levels of germline-limited repetitive elements in exconjugant cells. Conclusions These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we propose that this motif recruits DNA modification machinery to specific chromosomes in the parental macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination during genome rearrangements. PMID:23075511

  10. New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction.

    PubMed

    Mizuta, Masahiro; Banba, Jun-ichi; Kanamori, Takashi; Tawarada, Ryuya; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2008-07-30

    New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC5Ars) and cyclic deoxycytidine derivatives, dCPPP and dCPPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, phi: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies. PMID:18611007

  11. Clinical implications of cytosine deletion of exon 5 of P53 gene in non small cell lung cancer patients

    PubMed Central

    Mir, Rashid; Masroor, Mirza; Javid, Jamsheed; Ahamad, Imtiyaz; Farooq, Shazia; Yadav, Prasant; Zuberi, Mariyam; Lone, Maqbool; Ray, P. C; Saxena, Alpana

    2016-01-01

    Aim: Lung cancer is considered to be the most common cancer in the world. In humans, about 50% or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate the cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Our study was aimed to evaluate the frequency of cytosine deletion in nonsmall cell lung cancer (NSCLC) patients. Methods: One hundred NSCLC patients were genotyped for P53 (exon5, codon168) cytosine deletion leading to loss of its function and activate the target genes by allele-specific polymerase chain reaction. The P53 cytosine deletion was correlated with all the clinicopathological parameters of the patients. Results and Analysis: 59% cases were carrying P53 cytosine deletion. Similarly, the significantly higher incidence of cytosine deletion was reported in current smokers (75%) in comparison to exsmoker and nonsmoker. Significantly higher frequency of cytosine deletion was reported in adenocarcinoma (68.08%) than squamous cell carcinoma (52.83%). Also, a significant difference was reported between p53 cytosine deletion and metastasis (64.28%). Further, the majority of the cases assessed for response carrying P53 cytosine deletion were found to show faster disease progression. Conclusion: The data suggests that there is a significant association of the P53 exon 5 deletion of cytosine in codon 168 with metastasis and staging of the disease. PMID:27169122

  12. Prokaryotic Nucleotide Composition Is Shaped by Both Phylogeny and the Environment

    PubMed Central

    Reichenberger, Erin R.; Rosen, Gail; Hershberg, Uri; Hershberg, Ruth

    2015-01-01

    The causes of the great variation in nucleotide composition of prokaryotic genomes have long been disputed. Here, we use extensive metagenomic and whole-genome data to demonstrate that both phylogeny and the environment shape prokaryotic nucleotide content. We show that across environments, various phyla are characterized by different mean guanine and cytosine (GC) values as well as by the extent of variation on that mean value. At the same time, we show that GC-content varies greatly as a function of environment, in a manner that cannot be entirely explained by disparities in phylogenetic composition. We find environmentally driven differences in nucleotide content not only between highly diverged environments (e.g., soil, vs. aquatic vs. human gut) but also within a single type of environment. More specifically, we demonstrate that some human guts are associated with a microbiome that is consistently more GC-rich across phyla, whereas others are associated with a more AT-rich microbiome. These differences appear to be driven both by variations in phylogenetic composition and by environmental differences—which are independent of these phylogenetic composition differences. Combined, our results demonstrate that both phylogeny and the environment significantly affect nucleotide composition and that the environmental differences affecting nucleotide composition are far subtler than previously appreciated. PMID:25861819

  13. Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere

    NASA Technical Reports Server (NTRS)

    Kang, C.; Berger, I.; Lockshin, C.; Ratliff, R.; Moyzis, R.; Rich, A.

    1995-01-01

    In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

  14. Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase) from Streptomyces griseochromogenes, the first prokaryotic UDP-glucuronosyltransferase.

    PubMed Central

    Gould, S J; Guo, J

    1994-01-01

    Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase), the first prokaryotic UDP-GT and a key enzyme in the biosynthesis of the antibiotic blasticidin S, was purified 870-fold. It has optimum activity at a pH of 8.4 to 8.6, Kms of 6.0 (UDP-glucuronic acid) and 243 (cytosine) microM, and a maximum rate of metabolism of 14.6 mumol/min/mg. The apparent M(r) is 43,000. Activity was slightly enhanced by Mg2+ or Ca2+ but was not inhibited by EDTA. Activity was strongly inhibited by UDP. Cytosylglucuronic acid differs from eukaryotic UDP-glucuronosyltransferases in being a soluble protein with no apparent phospholipid requirement. Images PMID:8113166

  15. The role of cytosine methylation on charge transport through a DNA strand.

    PubMed

    Qi, Jianqing; Govind, Niranjan; Anantram, M P

    2015-09-01

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. PMID:26342369

  16. The role of cytosine methylation on charge transport through a DNA strand

    NASA Astrophysics Data System (ADS)

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    2015-09-01

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  17. O2 Protonation Controls Threshold Behavior for N-Glycosidic Bond Cleavage of Protonated Cytosine Nucleosides.

    PubMed

    Wu, R R; Rodgers, M T

    2016-06-01

    IRMPD action spectroscopy studies of protonated 2'-deoxycytidine and cytidine, [dCyd+H](+) and [Cyd+H](+), have established that both N3 and O2 protonated conformers coexist in the gas phase. Threshold collision-induced dissociation (CID) of [dCyd+H](+) and [Cyd+H](+) is investigated here using guided ion beam tandem mass spectrometry techniques to elucidate the mechanisms and energetics for N-glycosidic bond cleavage. N-Glycosidic bond cleavage is observed as the major dissociation pathways resulting in competitive elimination of either protonated or neutral cytosine for both protonated cytosine nucleosides. Electronic structure calculations are performed to map the potential energy surfaces (PESs) for both N-glycosidic bond cleavage pathways observed. The molecular parameters derived from theoretical calculations are employed for thermochemical analysis of the energy-dependent CID data to determine the minimum energies required to cleave the N-glycosidic bond along each pathway. B3LYP and MP2(full) computed activation energies for N-glycosidic bond cleavage associated with elimination of protonated and neutral cytosine, respectively, are compared to measured values to evaluate the efficacy of these theoretical methods in describing the dissociation mechanisms and PESs for N-glycosidic bond cleavage. The 2'-hydroxyl of [Cyd+H](+) is found to enhance the stability of the N-glycosidic bond vs that of [dCyd+H](+). O2 protonation is found to control the threshold energies for N-glycosidic bond cleavage as loss of neutral cytosine from the O2 protonated conformers is found to require ∼25 kJ/mol less energy than the N3 protonated analogues, and the activation energies and reaction enthalpies computed using B3LYP exhibit excellent agreement with the measured thresholds for the O2 protonated conformers. PMID:27159774

  18. The Role of Cytosine Methylation on Charge Transport through a DNA Strand

    SciTech Connect

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    2015-09-04

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modifi-cation remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Buttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. Specifically, we compare the results generated with the widely used B3LYP exchange-correlation (XC) functional and CAM-B3LYP based tuned range-separated hybrid density functional. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that with both functionals, the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and interstrand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital (HOMO) level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with both functionals. We also study the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. Our results suggest that the effect of the two different functionals is to alter the on-site energies of the DNA bases at the HOMO level, while the transport properties don't depend much on the two functionals.

  19. The role of cytosine methylation on charge transport through a DNA strand

    SciTech Connect

    Qi, Jianqing Anantram, M. P.; Govind, Niranjan

    2015-09-07

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  20. Molecular energetics of cytosine revisited: a joint computational and experimental study.

    PubMed

    Gomes, José R B; Ribeiro da Silva, Maria D M C; Freitas, Vera L S; Ribeiro da Silva, Manuel A V

    2007-08-01

    A static bomb calorimeter has been used to measure the standard molar energy of combustion, in oxygen, at T = 298.15 K, of a commercial sample of cytosine. From this energy, the standard (p degrees = 0.1 MPa) molar enthalpy of formation in the crystalline state was derived as -(221.9 +/- 1.7) kJ.mol(-1). This value confirms one experimental value already published in the literature but differs from another literature value by 13.5 kJ.mol(-1). Using the present standard molar enthalpy of formation in the condensed phase and the enthalpy of sublimation due to Burkinshaw and Mortimer [J. Chem. Soc., Dalton Trans. 1984, 75], (155.0 +/- 3.0) kJ.mol(-1), results in a value for the gas-phase standard molar enthalpy of formation for cytosine of -66.9 kJ.mol(-1). A similar value, -65.1 kJ.mol(-1), has been estimated after G3MP2B3 calculations combined with the reaction of atomization on three different tautomers of cytosine. In agreement with experimental evidence, the hydroxy-amino tautomer is the most stable form of cytosine in the gas phase. The enthalpies of formation of the other two tautomers were also estimated as -60.7 kJ.mol(-1) and -57.2 kJ.mol(-1) for the oxo-amino and oxo-imino tautomers, respectively. The same composite approach was also used to compute other thermochemical data, which is difficult to be measured experimentally, such as C-H, N-H, and O-H bond dissociation enthalpies, gas-phase acidities, and ionization enthalpies. PMID:17616179

  1. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability

    PubMed Central

    Ngo, Thuy T. M.; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility. PMID:26905257

  2. Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy

    PubMed Central

    Nemani, Krishnamurthy V.; Ennis, Riley C.; Griswold, Karl E.; Gimi, Barjor

    2015-01-01

    Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42 °C as compared to 30 °C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions. PMID:25820125

  3. Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands.

    PubMed

    Schreiber, Jacob; Wescoe, Zachary L; Abu-Shumays, Robin; Vivian, John T; Baatar, Baldandorj; Karplus, Kevin; Akeson, Mark

    2013-11-19

    Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ~3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine-, and 5-hydroxymethylcytosine-bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5' and 3' of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5-19 times depending upon sequence context. PMID:24167260

  4. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability

    NASA Astrophysics Data System (ADS)

    Ngo, Thuy T. M.; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-02-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

  5. Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

    PubMed

    Nemani, Krishnamurthy V; Ennis, Riley C; Griswold, Karl E; Gimi, Barjor

    2015-06-10

    Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions. PMID:25820125

  6. Nucleotide sequence analysis of the hypervariable region III of mitochondrial DNA in Thais.

    PubMed

    Thongngam, Punlop; Leewattanapasuk, Worraanong; Bhoopat, Tanin; Sangthong, Padchanee

    2016-07-01

    This study analyzed the nucleotide sequences of the hypervariable region III (HVRIII) of mitochondrial DNA in Thai individuals. Buccal swab samples were randomly obtained from 100 healthy, unrelated, adult (18-60 years old), volunteer donors living in Thailand. Eighteen different haplotypes were found, of which 11 haplotypes were unique. The most frequent haplotypes observed were 522D-523D. Nucleotide transition from Thymine (T) to Cytosine (C) at position 489 (43%) was the most frequent substitution. Nucleotide transversions were also observed at position 433 (Adenine (A) to C, 1%) and position 499 (Guanine (G) to C, 1%). Fifty-three samples presented nucleotide insertion and deletion of C and A (CA) at position 514-523. Insertion of 1AC (3%) and 2AC (2%) were observed. Deletion of 1CA (53%) and 2CA (2%) at position 514-523 were revealed. The deletion of T at position 459 was observed. The haplotype diversity, random match probability, and discrimination power were calculated to be 0.7770, 0.2308, and 0.7692, respectively. PMID:27107562

  7. Cytosine hypomethylation at CHG and CHH sites in the pleiotropic mutants of Mendelian inheritance in Catharanthus roseus.

    PubMed

    Kumari, Renu; Yadav, Gitanjali; Sharma, Vishakha; Sharma, Vinay; Kumar, Sushil

    2013-12-01

    The 5S and 18S rDNA sequences of Catharanthus roseus cv 'Nirmal' (wild type) and its leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill) single mutants and lli egd, lli ill and egd ill double mutants were characterized. The lli, egd and ill mutants of Mendelian inheritance bore the names after their most conspicuous morphological feature(s). They had been chemically induced and isolated for their salt tolerance. The double mutants were isolated as morphological segregants from crosses between single mutants. The morphological features of the two parents accompanied salt tolerance in the double mutants. All the six mutants were hypomethylated at repeat sequences, upregulated and downregulated for many genes and carried pleiotropic alterations for several traits. Here the 5S and 18S rDNAs of C. roseus were found to be relatively low in cytosine content. Cytosines were preponderantly in CG context (53%) and almost all of them were methylated (97%). The cytosines in CHH and CHG (where H = A, T or C) contexts were largely demethylated (92%) in mutants. The demethylation was attributable to reduced expression of RDR2 and DRM2 led RNA dependant DNA methylation and CMT3 led maintenance methylation pathways. Mutants had gained some cytosines by substitution of C at T sites. These perhaps arose on account of errors in DNA replication, mediated by widespread cytosine demethylation at CHG and CHH sites. It was concluded that the regulation of cytosine ethylation mechanisms was disturbed in the mutants. ILL, EGD and LLI genes were identified as the positive regulators of other genes mediating the RdDM and CMT3 pathways, for establishment and maintenance of cytosine methylation in C. roseus. PMID:24371171

  8. Labeled nucleotide phosphate (NP) probes

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  9. [Interaction of divalent cadmium ions with nucleotides and native DNA].

    PubMed

    Sorokin, V A; Valeev, V A; Gladchenko, G O; Sysa, I V

    1997-01-01

    Complex formation of Cd2+ ions with 2'-deoxy-5'-phosphates of canonical bases and their riboanalogues in water solution is studied by the method of differential UV-spectroscopy. It is stated that the atoms coordinating Cd2+ in complexes are N7 of dGMP and GMP, dAMP and AMP, N1 of adenine derivatives, N3 of dCMP. No interaction with base heteroatoms of UMP and dTMP is found. O2' present in the structure of the sugar ring has a weak influence on the Cd2+ ion binding to purine nucleotides. It manifests itself strongly in the complexes with cytosine derivatives: cadmium does not interact with N3 of CMP and poly-C practically. In the last case O2 is a centre coordinating Cd2+ ions. The interaction with this atom induces the melting of polynucleotide helical parts. At the high cadmium concentration poly-C forms compact particles. The main centre binding Cd2+ ions in DNA is N7 of guanines. Noncooperative interaction with these centres results in the internal protonation of N3C of GC-pairs. This is not followed with the disordering of the DNA helical structure. PMID:9181783

  10. Methylated Cytosines Mutate to Transcription Factor Binding Sites that Drive Tetrapod Evolution

    PubMed Central

    He, Ximiao; Tillo, Desiree; Vierstra, Jeff; Syed, Khund-Sayeed; Deng, Callie; Ray, G. Jordan; Stamatoyannopoulos, John; FitzGerald, Peter C.; Vinson, Charles

    2015-01-01

    In mammals, the cytosine in CG dinucleotides is typically methylated producing 5-methylcytosine (5mC), a chemically less stable form of cytosine that can spontaneously deaminate to thymidine resulting in a T•G mismatched base pair. Unlike other eukaryotes that efficiently repair this mismatched base pair back to C•G, in mammals, 5mCG deamination is mutagenic, sometimes producing TG dinucleotides, explaining the depletion of CG dinucleotides in mammalian genomes. It was suggested that new TG dinucleotides generate genetic diversity that may be critical for evolutionary change. We tested this conjecture by examining the DNA sequence properties of regulatory sequences identified by DNase I hypersensitive sites (DHSs) in human and mouse genomes. We hypothesized that the new TG dinucleotides generate transcription factor binding sites (TFBS) that become tissue-specific DHSs (TS-DHSs). We find that 8-mers containing the CG dinucleotide are enriched in DHSs in both species. However, 8-mers containing a TG and no CG dinucleotide are preferentially enriched in TS-DHSs when compared with 8-mers with neither a TG nor a CG dinucleotide. The most enriched 8-mer with a TG and no CG dinucleotide in tissue-specific regulatory regions in both genomes is the AP-1 motif (TGAC/GTCAN), and we find evidence that TG dinucleotides in the AP-1 motif arose from CG dinucleotides. Additional TS-DHS-enriched TFBS containing the TG/CA dinucleotide are the E-Box motif (GCAGCTGC), the NF-1 motif (GGCA—TGCC), and the GR (glucocorticoid receptor) motif (G-ACA—TGT-C). Our results support the suggestion that cytosine methylation is mutagenic in tetrapods producing TG dinucleotides that create TFBS that drive evolution. PMID:26507798

  11. Methylated Cytosines Mutate to Transcription Factor Binding Sites that Drive Tetrapod Evolution.

    PubMed

    He, Ximiao; Tillo, Desiree; Vierstra, Jeff; Syed, Khund-Sayeed; Deng, Callie; Ray, G Jordan; Stamatoyannopoulos, John; FitzGerald, Peter C; Vinson, Charles

    2015-11-01

    In mammals, the cytosine in CG dinucleotides is typically methylated producing 5-methylcytosine (5mC), a chemically less stable form of cytosine that can spontaneously deaminate to thymidine resulting in a T•G mismatched base pair. Unlike other eukaryotes that efficiently repair this mismatched base pair back to C•G, in mammals, 5mCG deamination is mutagenic, sometimes producing TG dinucleotides, explaining the depletion of CG dinucleotides in mammalian genomes. It was suggested that new TG dinucleotides generate genetic diversity that may be critical for evolutionary change. We tested this conjecture by examining the DNA sequence properties of regulatory sequences identified by DNase I hypersensitive sites (DHSs) in human and mouse genomes. We hypothesized that the new TG dinucleotides generate transcription factor binding sites (TFBS) that become tissue-specific DHSs (TS-DHSs). We find that 8-mers containing the CG dinucleotide are enriched in DHSs in both species. However, 8-mers containing a TG and no CG dinucleotide are preferentially enriched in TS-DHSs when compared with 8-mers with neither a TG nor a CG dinucleotide. The most enriched 8-mer with a TG and no CG dinucleotide in tissue-specific regulatory regions in both genomes is the AP-1 motif ( TG: A(C)/GT CA: N), and we find evidence that TG dinucleotides in the AP-1 motif arose from CG dinucleotides. Additional TS-DHS-enriched TFBS containing the TG/CA dinucleotide are the E-Box motif (G CA: GC TG: C), the NF-1 motif (GG CATG: CC), and the GR (glucocorticoid receptor) motif (G-A CATG: T-C). Our results support the suggestion that cytosine methylation is mutagenic in tetrapods producing TG dinucleotides that create TFBS that drive evolution. PMID:26507798

  12. Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes

    PubMed Central

    2013-01-01

    Background The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. Results Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and ‘Turbellaria’) contain methylated cytosines within their genome compartments

  13. Some new reaction pathways for the formation of cytosine in interstellar space - A quantum chemical study

    NASA Astrophysics Data System (ADS)

    Gupta, V. P.; Tandon, Poonam; Mishra, Priti

    2013-03-01

    The detection of nucleic acid bases in carbonaceous meteorites suggests that their formation and survival is possible outside of the Earth. Small N-heterocycles, including pyrimidine, purines and nucleobases, have been extensively sought in the interstellar medium. It has been suggested theoretically that reactions between some interstellar molecules may lead to the formation of cytosine, uracil and thymine though these processes involve significantly high potential barriers. We attempted therefore to use quantum chemical techniques to explore if cytosine can possibly form in the interstellar space by radical-radical and radical-molecule interaction schemes, both in the gas phase and in the grains, through barrier-less or low barrier pathways. Results of DFT calculations for the formation of cytosine starting from some of the simple molecules and radicals detected in the interstellar space are being reported. Global and local descriptors such as molecular hardness, softness and electrophilicity, and condensed Fukui functions and local philicity indices were used to understand the mechanistic aspects of chemical reaction. The presence and nature of weak bonds in the molecules and transition states formed during the reaction process have been ascertained using Bader's quantum theory of atoms in molecules (QTAIMs). Two exothermic reaction pathways starting from propynylidyne (CCCH) and cyanoacetylene (HCCCN), respectively, have been identified. While the first reaction path is found to be totally exothermic, it involves a barrier of 12.5 kcal/mol in the gas phase against the lowest value of about 32 kcal/mol reported in the literature. The second path is both exothermic and barrier-less. The later has, therefore, a greater probability of occurrence in the cold interstellar clouds (10-50 K).

  14. Ambivalent incorporation of the fluorescent cytosine analogues tC and tCo by human DNA polymerase alpha and Klenow fragment.

    PubMed

    Stengel, Gudrun; Purse, Byron W; Wilhelmsson, L Marcus; Urban, Milan; Kuchta, Robert D

    2009-08-11

    We studied the incorporation of the fluorescent cytidine analogues 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) by human DNA polymerase alpha and Klenow fragment of DNA polymerase I (Escherichia coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly expanded in size toward the major groove. Despite the size alteration, both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only approximately 4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs and can incorporate at least four consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. Klenow fragment inserts dGTP with a 4-9-fold higher probability than dATP, while polymerase alpha favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as a templating base and as an incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o). PMID:19580325

  15. The mechanism of M.HhaI DNA C5 cytosine methyltransferase enzyme: A quantum mechanics/molecular mechanics approach

    PubMed Central

    Zhang, Xiaodong; Bruice, Thomas C.

    2006-01-01

    The mechanism of DNA cytosine-5-methylation catalyzed by the bacterial M.HhaI enzyme has been considered as a stepwise nucleophilic addition of Cys-81-S− to cytosine C6 followed by C5 nucleophilic replacement of the methyl of S-adenosyl-l-methionine to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. In this study, we show that the reaction is concerted from a series of energy calculations by using the quantum mechanical/molecular mechanical hybrid method. Deprotonation of 5-methyl-6-Cys-81-S-5,6-dihydrocytosine and expulsion of Cys-81-S− provides the product DNA 5-methylcytosine. A required base catalyst for this deprotonation is not available as a member of the active site structure. A water channel between the active site and bulk water allows entrance of solvent to the active site. Hydroxide at 10−7 mole fraction (pH = 7) is shown to be sufficient for the required catalysis. We also show that Glu-119-CO2H can divert the reaction by protonating cytosine N3 when Cys-81-S− attacks cytosine, to form the 6-Cys-81-S-3-hydrocytosine. The reactants and 6-Cys-81-S-3-hydrocytosine product are in rapid equilibrium, and this explains the observed hydrogen exchange of cytosine with solvent. PMID:16606828

  16. Recent Trends in Nucleotide Synthesis.

    PubMed

    Roy, Béatrice; Depaix, Anaïs; Périgaud, Christian; Peyrottes, Suzanne

    2016-07-27

    Focusing on the recent literature (since 2000), this review outlines the main synthetic approaches for the preparation of 5'-mono-, 5'-di-, and 5'-triphosphorylated nucleosides, also known as nucleotides, as well as several derivatives, namely, cyclic nucleotides and dinucleotides, dinucleoside 5',5'-polyphosphates, sugar nucleotides, and nucleolipids. Endogenous nucleotides and their analogues can be obtained enzymatically, which is often restricted to natural substrates, or chemically. In chemical synthesis, protected or unprotected nucleosides can be used as the starting material, depending on the nature of the reagents selected from P(III) or P(V) species. Both solution-phase and solid-support syntheses have been developed and are reported here. Although a considerable amount of research has been conducted in this field, further work is required because chemists are still faced with the challenge of developing a universal methodology that is compatible with a large variety of nucleoside analogues. PMID:27319940

  17. Effects of high dose intraperitoneal cytosine arabinoside on the radiation tolerance of the rat spinal cord

    SciTech Connect

    Menten, J.; Landuyt, W.; van der Kogel, A.J.; Ang, K.K.; van der Schueren, E.

    1989-07-01

    The effect of intraperitoneal high dose (9 g/kg) cytosine arabinoside (Ara-C) on the early delayed radiation response of the rat cervical spinal cord has been studied. When given 2 hrs before irradiation, systemically administered Ara-C significantly reduces the isoeffect doses for the induction of paralysis due to white matter necrosis by a factor of approximately 1.2 for both a single irradiation treatment and for a two fraction irradiation with 24 hr interval. No effect on the latency time to develop paralysis was recorded.

  18. A nucleotide deletion and frame-shift cause analbuminemia in a Turkish family.

    PubMed

    Caridi, Gianluca; Gulec, Elif Yilmaz; Campagnoli, Monica; Lugani, Francesca; Onal, Hasan; Kilic, Duzgun; Galliano, Monica; Minchiotti, Lorenzo

    2016-01-01

    Congenital analbuminemia is an autosomal recessive disorder, in which albumin, the major blood protein, is present only in a minute amount. The condition is a rare allelic heterogeneous defect, only about seventy cases have been reported worldwide. To date, more than twenty different mutations within the albumin gene have been found to cause the trait. In our continuing study of the molecular genetics of congenital analbuminemia, we report here the clinical and biochemical findings and the mutation analysis of the gene in two Turkish infants. For the molecular analysis, we used our strategy, based on the screening of the gene by single-strand conformation polymorphism, heteroduplex analysis and direct DNA sequencing. The results showed that both patients are homozygous for the deletion of a cytosine residue in exon 5, in a stretch of four cytosines starting from nucleotide position 524 and ending at position 527 (NM_000477.5(ALB):c.527delC). The subsequent frame-shift inserts a stop codon in position 215, markedly reducing the size of the predicted protein product. The parents are both heterozygous for the same mutation, for which we propose the name Erzurum from the city of origin of the family. In conclusion, our results show that in this family congenital analbuminemia is caused by a novel frame-shift/deletion defect, confirm the inheritance of the trait, and contribute to advance our understanding of the molecular basis underlying this condition. PMID:27346974

  19. A nucleotide deletion and frame-shift cause analbuminemia in a Turkish family

    PubMed Central

    Caridi, Gianluca; Gulec, Elif Yilmaz; Campagnoli, Monica; Lugani, Francesca; Onal, Hasan; Kilic, Duzgun; Galliano, Monica; Minchiotti, Lorenzo

    2016-01-01

    Congenital analbuminemia is an autosomal recessive disorder, in which albumin, the major blood protein, is present only in a minute amount. The condition is a rare allelic heterogeneous defect, only about seventy cases have been reported worldwide. To date, more than twenty different mutations within the albumin gene have been found to cause the trait. In our continuing study of the molecular genetics of congenital analbuminemia, we report here the clinical and biochemical findings and the mutation analysis of the gene in two Turkish infants. For the molecular analysis, we used our strategy, based on the screening of the gene by single-strand conformation polymorphism, heteroduplex analysis and direct DNA sequencing. The results showed that both patients are homozygous for the deletion of a cytosine residue in exon 5, in a stretch of four cytosines starting from nucleotide position 524 and ending at position 527 (NM_000477.5(ALB):c.527delC). The subsequent frame-shift inserts a stop codon in position 215, markedly reducing the size of the predicted protein product. The parents are both heterozygous for the same mutation, for which we propose the name Erzurum from the city of origin of the family. In conclusion, our results show that in this family congenital analbuminemia is caused by a novel frame-shift/deletion defect, confirm the inheritance of the trait, and contribute to advance our understanding of the molecular basis underlying this condition. PMID:27346974

  20. Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.

    PubMed Central

    Penázová, H; Vorlicková, M

    1997-01-01

    Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure. PMID:9336200

  1. Cytosine methylation of plastid genome in higher plants. Fact or artefact?

    PubMed

    Fojtová, M; Kovarík, A; Matyásek, R

    2001-03-01

    DNA methylation of chloroplast genome has been studied in a large variety of angiosperm species using restriction enzyme analysis of three genomic loci (totally encompassing about 10% of chloroplast genome) and bisulfite genomic sequencing of tobacco ribulose bisphosphate carboxylase/oxygenase (large subunit) gene (rbcL). Except for CCWGG (W=A or T) sites that were partially refractory to the cleavage with methylation sensitive EcoRII in all loci, no cytosine methylation was found at the CCGG (MspI/HpaII) and several other restriction sites tested. However, EcoRII was unable to completely digest an unmethylated CCWGG site in the cloned rbcL gene on plasmid. Further a bisulfite genomic sequencing performed on EcoRII-restricted DNA failed to show any 5-methylcytosine either within or outside inspected EcoRII sites along the 3' end of rbcL coding region. In conclusion our results do not support evidence for methylated cytosine residues in plant chloroplast genomes and we suggest that results obtained with EcoRII should be interpreted with great care especially when small differences in methylation levels are analysed. PMID:11448733

  2. Functional and Structural Characterization of DR_0079 from Deinococcus radiodurans, a Novel Nudix Hydrolase with a Preference for Cytosine (Deoxy)ribonucleoside 5 -Di- and Triphophates

    SciTech Connect

    Buchko,G.; Litvinova, O.; Robinson, H.; Yakunin, A.; Kennedy, M.

    2008-01-01

    The genome of the extremely radiation resistant bacterium Deinococcus radiodurans encodes 21 Nudix hydrolases, of which only two have been characterized in detail. Here we report the activity and crystal structure for DR{_}0079, the first Nudix hydrolase observed to have a marked preference for cytosine ribonucleoside 5'-diphosphate (CDP) and cytosine ribonucleoside 5'-triphosphate (CTP). After CDP and CTP, the next most preferred substrates for DR{_}0079, with a relative activity of <50%, were the corresponding deoxyribose nucleotides, dCDP and dCTP. Hydrolase activity at the site of the phosphodiester bond was corroborated using 31P NMR spectroscopy to follow the phosphorus resonances for three substrates, CDP, IDP, and CTP, and their hydrolysis products, CMP + Pi, IMP + Pi, and CMP + PPi, respectively. Nucleophilic substitution at the {beta}-phosphorus of CDP and CTP was established, using 31P NMR spectroscopy, by the appearance of an upfield-shifted Pi resonance and line-broadened PPi resonance, respectively, when the hydrolysis was performed in 40% H218O-enriched water. The optimal activity for CDP was at pH 9.0-9.5 with the reaction requiring divalent metal cation (Mg2+ > Mn2+ > Co2+). The biochemical data are discussed with reference to the crystal structure for DR{_}0079 that was determined in the metal-free form at 1.9 Angstroms resolution. The protein contains nine {beta}-strands, three a-helices, and two 310-helices organized into three subdomains: an N-terminal {beta}-sheet, a central Nudix core, and a C-terminal helix-turn-helix motif. As observed for all known structures of Nudix hydrolases, the a-helix of the 'Nudix box' is one of two helices that sandwich a 'four-strand' mixed {beta}-sheet. To identify residues potentially involved in metal and substrate binding, NMR chemical shift mapping experiments were performed on 15N-labeled DR{_}0079 with the paramagnetic divalent cation Co2+ and the nonhydrolyzable substrate thymidine 5'-O-({alpha}, {beta

  3. Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

    PubMed

    Wang, Jifeng; Sklenak, Stepan; Liu, Aizhuo; Felczak, Krzysztof; Wu, Yan; Li, Yue; Yan, Honggao

    2012-01-10

    Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and

  4. Role of Glutamate 64 in the Activation of the Prodrug 5-fluorocytosine by Yeast Cytosine Deaminase†

    PubMed Central

    Wang, Jifeng; Sklenak, Stepan; Liu, Aizhuo; Felczak, Krzysztof; Wu, Yan; Li, Yue; Yan, Honggao

    2012-01-01

    Yeast cytosine deaminase catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, NMR, and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in kcat and a dramatic increase in Km, indicating Glu64 is important for both binding and catalysis in the activation of 5FC. 19F-NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. 1H and 15N NMR analysis revealed trans-hydrogen-bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has higher activation energy as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by 1H and 15N NMR analysis. To explore the functional role of Glu64 in catalysis, deamination of cytosine catalyzed by the E64A mutant was investigated by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction

  5. The EMBL Nucleotide Sequence Database.

    PubMed

    Stoesser, G; Tuli, M A; Lopez, R; Sterk, P

    1999-01-01

    The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl.html) constitutes Europe's primary nucleotide sequence resource. Main sources for DNA and RNA sequences are direct submissions from individual researchers, genome sequencing projects and patent applications. While automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO), the preferred submission tool for individual submitters is Webin (WWW). Through all stages, dataflow is monitored by EBI biologists communicating with the sequencing groups. In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute (EBI). Database releases are produced quarterly and are distributed on CD-ROM. Network services allow access to the most up-to-date data collection via Internet and World Wide Web interface. EBI's Sequence Retrieval System (SRS) is a Network Browser for Databanks in Molecular Biology, integrating and linking the main nucleotide and protein databases, plus many specialised databases. For sequence similarity searching a variety of tools (e.g. Blitz, Fasta, Blast etc) are available for external users to compare their own sequences against the most currently available data in the EMBL Nucleotide Sequence Database and SWISS-PROT. PMID:9847133

  6. Metal Ion Induced Pairing of Cytosine Bases: Formation of I-Motif Structures Identified by IR Ion Spectroscopy

    NASA Astrophysics Data System (ADS)

    Gao, Juehan; Berden, Giel; Oomens, J.

    2015-06-01

    While the Watson-Crick structure of DNA is among the most well-known molecular structures of our time, alternative base-pairing motifs are also known to occur, often depending on base sequence, pH, or presence of cations. Pairing of two cytosine (C) bases induced by the sharing of a single proton (C-H^+-C) gives rise to the so-called i-motif, occurring particularly in the telomeric region of DNA, and particularly at low pH. At physiological pH, silver cations were recently suggested to form cytosine dimers in a C-Ag^+-C structure analogous to the hemiprotonated cytosine dimer, which was later confirmed by IR spectroscopy.^1 Here we investigate whether Ag^+ is unique in this behavior. Using infrared action spectroscopy employing the free-electron laser FELIX and a tandem mass spectrometer in combination with quantum-chemical computations, we investigate a series of C-M^+-C complexes, where M is Cu, Li and Na. The complexes are formed by electrospray ionization (ESI) from a solution of cytosine and the metal chloride salt in acetonitrile/water. The complexes of interest are mass-isolated in the cell of a FT ion cyclotron resonance mass spectrometer, where they are irradiated with the tunable IR radiation from FELIX in the 600 - 1800 wn range. Spectra in the H-stretching range are obtained with a LaserVision OPO. Both experimental spectra as well as theoretical calculations indicate that while Cu behaves as Ag, the alkali metal ions induce a clearly different dimer structure, in which the two cytosine units are parallelly displaced. In addition to coordination to the ring nitrogen atom, the alkali metal ions coordinate to the carbonyl oxygen atoms of both cytosine bases, indicating that the alkali metal ion coordination favorably competes with hydrogen bonding between the two cytosine sub-units of the i-motif like structure. 1. Berdakin, Steinmetz, Maitre, Pino, J. Phys. Chem. A 2014, 118, 3804

  7. Redetermination of cytosinium hydrogen maleate–cytosine (1/1) from the original data

    PubMed Central

    Fábry, Jan

    2016-01-01

    The title salt, C4H6N3O+·C4H3O4 −·C4H5N3O, has been redetermined from the data published by Benali-Cherif, Falek & Direm [Acta Cryst. (2009), E65, o3058–o3059]. The improvement of the present redetermination consists in the discovery of the splitting of one of the H atoms into two disordered positions, the occupancies of which are equal to 0.55 (2) and 0.45 (2). These H atoms are involved in an N⋯N hydrogen bond and are shifted towards its centre. The disorder of these H atoms is in agreement with a similar environment of the two independent, but chemically equivalent, cytosinium/cytosine mol­ecules. PMID:27375877

  8. Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs.

    PubMed

    Vande Voorde, Johan; Vervaeke, Peter; Liekens, Sandra; Balzarini, Jan

    2015-01-01

    Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency. PMID:26322268

  9. Redetermination of cytosinium hydrogen maleate-cytosine (1/1) from the original data.

    PubMed

    Fábry, Jan

    2016-04-01

    The title salt, C4H6N3O(+)·C4H3O4 (-)·C4H5N3O, has been redetermined from the data published by Benali-Cherif, Falek & Direm [Acta Cryst. (2009), E65, o3058-o3059]. The improvement of the present redetermination consists in the discovery of the splitting of one of the H atoms into two disordered positions, the occupancies of which are equal to 0.55 (2) and 0.45 (2). These H atoms are involved in an N⋯N hydrogen bond and are shifted towards its centre. The disorder of these H atoms is in agreement with a similar environment of the two independent, but chemically equivalent, cytosinium/cytosine mol-ecules. PMID:27375877

  10. Cytosine chemoreceptor McpC in Pseudomonas putida F1 also detects nicotinic acid

    PubMed Central

    Nesteryuk, Vasyl; Hughes, Jonathan G.; Luu, Rita A.; Ditty, Jayna L.

    2014-01-01

    Soil bacteria are generally capable of growth on a wide range of organic chemicals, and pseudomonads are particularly adept at utilizing aromatic compounds. Pseudomonads are motile bacteria that are capable of sensing a wide range of chemicals, using both energy taxis and chemotaxis. Whilst the identification of specific chemicals detected by the ≥26 chemoreceptors encoded in Pseudomonas genomes is ongoing, the functions of only a limited number of Pseudomonas chemoreceptors have been revealed to date. We report here that McpC, a methyl-accepting chemotaxis protein in Pseudomonas putida F1 that was previously shown to function as a receptor for cytosine, was also responsible for the chemotactic response to the carboxylated pyridine nicotinic acid. PMID:25294107

  11. Absolute cross sections for vibrational excitations of cytosine by low energy electron impact

    PubMed Central

    Michaud, M.; Bazin, M.; Sanche, L.

    2013-01-01

    The absolute cross sections (CSs) for vibrational excitations of cytosine by electron impact between 0.5 and 18 eV were measured by electron-energy loss (EEL) spectroscopy of the molecule deposited at monolayer coverage on an inert Ar substrate. The vibrational energies compare to those that have been reported from IR spectroscopy of cytosine isolated in Ar matrix, IR and Raman spectra of poly-crystalline cytosine, and ab initio calculation. The CSs for the various H bending modes at 142 and 160 meV are both rising from their energy threshold up to 1.7 and 2.1 × 10−17 cm2 at about 4 eV, respectively, and then decrease moderately while maintaining some intensity at 18 eV. The latter trend is displayed as well for the CS assigned to the NH2 scissor along with bending of all H at 179 meV. This overall behavior in electron-molecule collision is attributed to direct processes such as the dipole, quadrupole, and polarization contributions, etc. of the interaction of the incident electron with a molecule. The CSs for the ring deformation at 61 meV, the ring deformation with N-H symmetric wag at 77 meV, and the ring deformations with symmetric bending of all H at 119 meV exhibit common enhancement maxima at 1.5, 3.5, and 5.5 eV followed by a broad hump at about 12 eV, which are superimposed on the contribution due to the direct processes. At 3.5 eV, the CS values for the 61-, 77-, and 119-meV modes reach 4.0, 3.0, and 4.5 × 10−17 cm2, respectively. The CS for the C-C and C-O stretches at 202 meV, which dominates in the intermediate EEL region, rises sharply until 1.5 eV, reaches its maximum of 5.7 × 10−17 cm2 at 3.5 eV and then decreases toward 18 eV. The present vibrational enhancements, correspond to the features found around 1.5 and 4.5 eV in electron transmission spectroscopy (ETS) and those lying within 1.5–2.1 eV, 5.2–6.8 eV, and 9.5–10.9 eV range in dissociative electron attachment (DEA) experiments with cytosine in gas phase. While the ETS features

  12. Guanosine potentiates the antiproliferative effect of cytosine-beta-D-arabinofuranoside in melanoma cell lines.

    PubMed

    Sidi, Y; Panet, C; Cyjon, A; Fenig, E; Beery, E; Nordenberg, J

    1993-01-01

    Guanosine is shown to potentiate markedly the antiproliferative effect of cytosine-beta-D-arabinoside (ara-C) on B16 F10 mouse and SKMEL-28 human melanoma cell lines. Several metabolic consequences of the synergistic interaction between ara-C and guanosine on cell growth were determined in B16 F10 mouse melanoma cells. Treatment of the cells with guanosine for 24 hr resulted in an increase in the percentage of cells in the S phase of the cell cycle, a threefold increase in intracellular GTP concentration, and an increase in the incorporation of ara-C into acid-insoluble material and phosphorylated metabolites. These findings suggest that guanosine potentiates the growth-inhibitory effect of ara-C in B16 F10 melanoma cells by increasing the intracellular concentration of its active metabolites. PMID:8402221

  13. Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population

    PubMed Central

    Schmitz, Robert J.; He, Yupeng; Valdés-López, Oswaldo; Khan, Saad M.; Joshi, Trupti; Urich, Mark A.; Nery, Joseph R.; Diers, Brian; Xu, Dong; Stacey, Gary; Ecker, Joseph R.

    2013-01-01

    Cytosine DNA methylation is one avenue for passing information through cell divisions. Here, we present epigenomic analyses of soybean recombinant inbred lines (RILs) and their parents. Identification of differentially methylated regions (DMRs) revealed that DMRs mostly cosegregated with the genotype from which they were derived, but examples of the uncoupling of genotype and epigenotype were identified. Linkage mapping of methylation states assessed from whole-genome bisulfite sequencing of 83 RILs uncovered widespread evidence for local methylQTL. This epigenomics approach provides a comprehensive study of the patterns and heritability of methylation variants in a complex genetic population over multiple generations, paving the way for understanding how methylation variants contribute to phenotypic variation. PMID:23739894

  14. Mapping global changes in nuclear cytosine base modifications in the early mouse embryo

    PubMed Central

    Li, Y; Seah, Michelle K Y; O'Neill, C

    2016-01-01

    Reprogramming epigenetic modifications to cytosine is required for normal embryo development. We used improved immunolocalization techniques to simultaneously map global changes in the levels of 5′-methylcytosine (5meC) and 5′-hydroxymethylcytosine (5hmC) in each cell of the embryo from fertilization through the first rounds of cellular differentiation. The male and female pronuclei of the zygote showed similar staining levels, and these remained elevated over the next three cell cycles. The inner cells of the morula showed a progressive reduction in global levels of both 5meC and 5hmC and further losses occurred in the pluripotent inner cell mass (ICM) of the blastocyst. This was accompanied by undetectable levels of DNA methyltransferase of each class in the nuclei of the ICM, while DNA methyltransferase 3B was elevated in the hypermethylated nuclei of the trophectoderm (TE). Segregation of the ICM into hypoblast and epiblast was accompanied by increased levels in the hypoblast compared with the epiblast. Blastocyst outgrowth in vitro is a model for implantation and showed that a demethylated state persisted in the epiblast while the hypoblast had higher levels of both 5meC and 5hmC staining. The high levels of 5meC and 5hmC evident in the TE persisted in trophoblast and trophoblast giant cells after attachment of the blastocyst to the substratum in vitro. This study shows that global cytosine hypomethylation and hypohydroxymethylation accompanied the formation of the pluripotent ICM and this persisted into the epiblast after blastocyst outgrowth, and each differentiated lineage formed in the early embryo showed higher global levels of 5meC and 5hmC. PMID:26660107

  15. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B

    PubMed Central

    Quist, Jelmar S.; Temiz, Nuri A.; Tutt, Andrew N. J.; Grigoriadis, Anita; Harris, Reuben S.

    2016-01-01

    Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80–90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells. PMID:27163364

  16. Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma.

    PubMed

    Kaliberov, S A; Market, J M; Gillespie, G Y; Krendelchtchikova, V; Della Manna, D; Sellers, J C; Kaliberova, L N; Black, M E; Buchsbaum, D J

    2007-07-01

    Combined treatment using adenoviral (Ad)-directed enzyme/prodrug therapy and radiation therapy has the potential to become a powerful method of cancer therapy. We have developed an Ad vector encoding a mutant bacterial cytosine deaminase (bCD) gene (AdbCD-D314A), which has a higher affinity for cytosine than wild-type bCD (bCDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdbCD-D314A with the prodrug 5-fluorocytosine (5-FC) and ionizing radiation against human glioma. The present study demonstrates that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Furthermore, a significant increase in cytotoxicity following AdbCD-D314A and radiation treatment of glioma cells in vitro was demonstrated as compared to AdbCDwt. Animal studies showed significant inhibition of subcutaneous or intracranial tumor growth of D54MG glioma xenografts by the combination of AdbCD-D314A/5-FC with ionizing radiation as compared with either agent alone, and with AdbCDwt/5-FC plus radiation. The results suggest that the combination of AdbCD-D314A/5-FC with radiation produces markedly increased cytotoxic effects in cancer cells in vitro and in vivo. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for cancer therapy. PMID:17495948

  17. Tissue-Specific Differences in Cytosine Methylation and Their Association with Differential Gene Expression in Sorghum1[W

    PubMed Central

    Zhang, Meishan; Xu, Chunming; von Wettstein, Diter; Liu, Bao

    2011-01-01

    It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5′-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5′-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum. PMID:21632971

  18. Identification of a nucleotide in 5' untranslated region contributing to virus replication and virulence of Coxsackievirus A16.

    PubMed

    Li, Zhaolong; Liu, Xin; Wang, Shaohua; Li, Jingliang; Hou, Min; Liu, Guanchen; Zhang, Wenyan; Yu, Xiao-Fang

    2016-01-01

    Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are two main causative pathogens of hand, foot and mouth disease (HFMD). Unlike EV71, virulence determinants of CA16, particularly within 5' untranslated region (5'UTR), have not been investigated until now. Here, a series of nucleotides present in 5'UTR of lethal but not in non-lethal CA16 strains were screened by aligning nucleotide sequences of lethal circulating Changchun CA16 and the prototype G10 as well as non-lethal SHZH05 strains. A representative infectious clone based on a lethal Changchun024 sequence and infectious mutants with various nucleotide alterations in 5'UTR were constructed and further investigated by assessing virus replication in vitro and virulence in neonatal mice. Compared to the lethal infectious clone, the M2 mutant with a change from cytosine to uracil at nucleotide 104 showed weaker virulence and lower replication capacity. The predicted secondary structure of the 5'UTR of CA16 RNA showed that M2 mutant located between the cloverleaf and stem-loop II, affected interactions between the 5'UTR and the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and A1 (hnRNP A1) that are important for translational activity. Thus, our research determined a virulence-associated site in the 5'UTR of CA16, providing a crucial molecular target for antiviral drug development. PMID:26861413

  19. Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    PubMed Central

    Li, Zhaolong; Liu, Xin; Wang, Shaohua; Li, Jingliang; Hou, Min; Liu, Guanchen; Zhang, Wenyan; Yu, Xiao-Fang

    2016-01-01

    Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are two main causative pathogens of hand, foot and mouth disease (HFMD). Unlike EV71, virulence determinants of CA16, particularly within 5′ untranslated region (5′UTR), have not been investigated until now. Here, a series of nucleotides present in 5′UTR of lethal but not in non-lethal CA16 strains were screened by aligning nucleotide sequences of lethal circulating Changchun CA16 and the prototype G10 as well as non-lethal SHZH05 strains. A representative infectious clone based on a lethal Changchun024 sequence and infectious mutants with various nucleotide alterations in 5′UTR were constructed and further investigated by assessing virus replication in vitro and virulence in neonatal mice. Compared to the lethal infectious clone, the M2 mutant with a change from cytosine to uracil at nucleotide 104 showed weaker virulence and lower replication capacity. The predicted secondary structure of the 5′UTR of CA16 RNA showed that M2 mutant located between the cloverleaf and stem-loop II, affected interactions between the 5′UTR and the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and A1 (hnRNP A1) that are important for translational activity. Thus, our research determined a virulence-associated site in the 5′UTR of CA16, providing a crucial molecular target for antiviral drug development. PMID:26861413

  20. Effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation.

    PubMed

    Ji, Debin; Lin, Krystal; Song, Jikui; Wang, Yinsheng

    2014-07-01

    We investigated systematically the effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation in synthetic duplex DNA. We found that the replacement of 5-methylcytosine at a CpG site with a 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine or 5-hydroxymethyluracil resulted in altered methylation of cytosine at both the opposite and the neighboring CpG sites. Our results provided important new knowledge about the implications of the 5-methylcytosine oxidation products in maintenance cytosine methylation. PMID:24789765

  1. Applications of adenine nucleotide measurements in oceanography

    NASA Technical Reports Server (NTRS)

    Holm-Hansen, O.; Hodson, R.; Azam, F.

    1975-01-01

    The methodology involved in nucleotide measurements is outlined, along with data to support the premise that ATP concentrations in microbial cells can be extrapolated to biomass parameters. ATP concentrations in microorganisms and nucleotide analyses are studied.

  2. Nucleotide sequences 1986/1987

    SciTech Connect

    Not Available

    1987-01-01

    These eight volumes are the third annual published compendium of nucleic acid sequences included in the European Molecular Biology Laboratory Nucleotide Sequence Data Library and the GenBank Genetic Sequences Data Bank. Each volume surveys one or more subdivisions of the database. The volume subtitles are: Primates; Rodents; Other Vertebrates and Invertebrates, Plants and Organelles, Bacteria and Bacteriophage, Viruses, Structural RNA, Synthetic and Unannotated Sequences, and Database Directory and Master Indices.

  3. The Single Nucleotide Polymorphism Consortium

    NASA Technical Reports Server (NTRS)

    Morgan, Michael

    2003-01-01

    I want to discuss both the Single Nucleotide Polymorphism (SNP) Consortium and the Human Genome Project. I am afraid most of my presentation will be thin on law and possibly too high on rhetoric. Having been engaged in a personal and direct way with these issues as a trained scientist, I find it quite difficult to be always as objective as I ought to be.

  4. Nucleotides in neuroregeneration and neuroprotection.

    PubMed

    Miras-Portugal, M Teresa; Gomez-Villafuertes, Rosa; Gualix, Javier; Diaz-Hernandez, Juan Ignacio; Artalejo, Antonio R; Ortega, Felipe; Delicado, Esmerilda G; Perez-Sen, Raquel

    2016-05-01

    Brain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-associated genes expression. Finally, general signaling pathways triggered by nucleotide receptors in neuronal populations converge on several intracellular kinases, such as PI3K/Akt, GSK3 and ERK1,2, as well as the Nrf-2/heme oxigenase-1 axis, which specifically link them to neuroprotection. In this regard, regulation of dual specificity protein phosphatases can become novel mechanism of actions for nucleotide receptors that associate them to cell homeostasis regulation. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'. PMID:26359530

  5. Necessary relations for nucleotide frequencies.

    PubMed

    Sinclair, Robert

    2015-06-01

    Genome composition analysis of di-, tri- and tetra-nucleotide frequencies is known to be evolutionarily informative, and useful in metagenomic studies, where binning of raw sequence data is often an important first step. Patterns appearing in genome composition analysis may be due to evolutionary processes or purely mathematical relations. For example, the total number of dinucleotides in a sequence is equal to the sum of the individual totals of the sixteen types of dinucleotide, and this is entirely independent of any assumptions made regarding mutation or selection, or indeed any physical or chemical process. Before any statistical analysis can be attempted, a knowledge of all necessary mathematical relations is required. I show that 25% of di-, tri- and tetra-nucleotide frequencies can be written as simple sums and differences of the remainder. The vast majority of organisms have circular genomes, for which these relations are exact and necessary. In the case of linear molecules, the absolute error is very nearly zero, and does not grow with contiguous sequence length. As a result of the new, necessary relations presented here, the foundations of the statistical analysis of di-, tri- and tetra-nucleotide frequencies, and k-mer analysis in general, need to be revisited. PMID:25843217

  6. Nucleotides critical for the interaction of the Streptococcus pyogenes Mga virulence regulator with Mga-regulated promoter sequences.

    PubMed

    Hause, Lara L; McIver, Kevin S

    2012-09-01

    The Mga regulator of Streptococcus pyogenes directly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1 binding site was altered and screened for nucleotides important for DNA binding in vitro and for transcriptional activation using a plasmid-based luciferase reporter in vivo. Following this analysis, 34 nucleotides within the Pemm1 binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5' and 3' ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1 minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemm with directed mutagenesis performed in the M1T1 Mga-regulated PscpA and Psic promoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner. PMID:22773785

  7. Absolute cross sections for electronic excitations of cytosine by low energy electron impact

    PubMed Central

    Bazin, M.; Michaud, M.; Sanche, L.

    2013-01-01

    The absolute cross sections (CS) for electronic excitations of cytosine by electron impact between 5 and 18 eV were measured by electron-energy loss (EEL) spectroscopy of the molecule deposited at low coverage on an inert Ar substrate. The lowest EEL features found at 3.55 and 4.02 eV are ascribed to transitions from the ground state to the two lowest triplet 1 3A′(π→π*) and 2 3A′(π→π*) valence states of the molecule. Their energy dependent CS exhibit essentially a common maximum at about 6 eV with a value of 1.84 × 10−17 cm2 for the former and 4.94 × 10−17 cm2 for the latter. In contrast, the CS for the next EEL feature at 4.65 eV, which is ascribed to the optically allowed transition to the 2 1A′(π→π*) valence state, shows only a steep rise to about 1.04 × 10−16 cm2 followed by a monotonous decrease with the incident electron energy. The higher EEL features at 5.39, 6.18, 6.83, and 7.55 eV are assigned to the excitations of the 3 3, 1A′(π→π*), 4 1A′(π→π*), 5 1A′(π→π*), and 6 1A′(π→π*) valence states, respectively. The CS for the 3 3, 1A′ and 4 1A′ states exhibit a common enhancement at about 10 eV superimposed on a more or less a steep rise, reaching respectively a maximum of 1.27 and 1.79 × 10−16 cm2, followed by a monotonous decrease. This latter enhancement and the maximum seen at about 6 eV in the lowest triplet states correspond to the core-excited electron resonances that have been found by dissociative electron attachment experiments with cytosine in the gas phase. The weak EEL feature found at 5.01 eV with a maximum CS of 3.8 × 10−18 cm2 near its excitation threshold is attributed to transitions from the ground state to the 1 3, 1A″(n→π*) states. The monotonous rise of the EEL signal above 8 eV is attributed to the ionization of the molecule. It is partitioned into four excitation energy regions at about 8.55, 9.21, 9.83, and 11.53 eV, which correspond closely to the ionization energies of

  8. Quantum chemical MP2 results on some hydrates of cytosine: binding sites, energies and the first hydration shell.

    PubMed

    Fogarasi, Géza; Szalay, Péter G

    2015-11-28

    A detailed quantum chemical investigation was undertaken to obtain the structure and energetics of cytosine hydrates Cyt·nH2O, with n = 1 to 7. The MP2(fc)/aug-cc-pVDZ level was used as the standard, with some DFT (B3LYP) and coupled cluster calculations, as well as calculations with the aug-cc-pVTZ basis set added for comparison. In a systematic search for microhydrated forms of cytosine, we have found that several structures have not yet been reported in the literature. The energies of different isomers, as well as binding energies are compared. When predicting the stability of a complex, we suggest using a scheme where the water molecules are extracted from a finite model of bulk water. Finally, based on energetic data, we suggest a rational definition of the first hydration shell; with this definition, it contains just six water molecules. PMID:26487481

  9. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process.

    NASA Technical Reports Server (NTRS)

    Yang, C. C.; Oro, J.

    1971-01-01

    Study of the formation of purines, pyrimidines, and other bases from CO, H2, and NH3 under conditions similar to those used in the Fischer-Tropsch process. It is found that industrial nickel/iron alloy catalyzes the synthesis of adenine, guanine, cytosine, and other nitrogenous compounds from mixtures of CO, H2, and NH3 at temperatures of about 600 C. Sufficient sample was accumulated to isolate as solid products adenine, guanine, and cytosine, which were identified by infrared spectrophotometry. In the absence of nickel/iron catalyst, at 650 C, or in the presence of this catalyst, at 450 C, no purines or pyrimidines were synthesized. These results confirm and extend some of the work reported by Kayatsu et al. (1968).

  10. Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage.

    PubMed

    Shih, I H; Been, M D

    2001-02-13

    Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76 Delta) ribozymes in D(2)O and H(2)O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76 Delta mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under k(cat)/K(M) conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (beta) of 0.51 was determined for the base-rescued reaction of C76 Delta. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage. PMID:11171978

  11. Genome-Wide Identification and Comparative Analysis of Cytosine-5 DNA Methyltransferase and Demethylase Families in Wild and Cultivated Peanut.

    PubMed

    Wang, Pengfei; Gao, Chao; Bian, Xiaotong; Zhao, Shuzhen; Zhao, Chuanzhi; Xia, Han; Song, Hui; Hou, Lei; Wan, Shubo; Wang, Xingjun

    2016-01-01

    DNA methylation plays important roles in genome protection, regulation of gene expression and is associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferase and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequenced, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in MET, CMT, and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 members didn't contain UBA domain which was different from other plants such as Arabidopsis, maize and soybean. Five DNA demethylase encoding genes were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTase genes mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferase and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or PEG stress could influence the expression level of C5-MTase and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut in the future. PMID:26870046

  12. Various cytosine/adenine permease homologues are involved in the toxicity of 5-fluorocytosine in Saccharomyces cerevisiae.

    PubMed

    Paluszynski, John P; Klassen, Roland; Rohe, Matthias; Meinhardt, Friedhelm

    2006-07-15

    5-Fluorocytosine (5-FC), a medically applied antifungal agent (Ancotil), is also active against the model organism Saccharomyces cerevisiae. 5-FC uptake in S. cerevisiae was considered to be mediated by the FCY2-encoded cytosine/adenine permease. By applying a highly sensitive assay, a low-level but dose-dependent toxicity of 5-FC in fcy2 mutants was detected, whereas cells deficient in the cytosine deaminase (encoded by FCY1), which is essential for intracellular conversion of 5-FC to 5-fluorouracil, display strong dose-independent resistance. Thus, an alternative, Fcy2-independent access pathway for 5-FC exists in S. cerevisiae. A genome-wide search for cytosine permease homologues identified two uncharacterized candidate genes, designated FCY21 and FCY22, both of which exhibit highest similarity to FCY2. Disruption of either FCY21 or FCY22 resulted in strains displaying low-level resistance, indicating the functional involvement of both gene products in 5-FC toxicity. When mutations in FCY21 or FCY22 were combined with the FCY2 disruption, both double mutants displayed stronger resistance when compared to the FCY2 mutant alone. Disruptions in all three permease genes consequently conferred the highest degree of resistance, not only towards 5-FC but also to the toxic adenine analogon 8-azaadenine. As residual 5-FC sensitivity was, however, even detectable in the fcy2 fcy21 fcy22 mutant, we analysed the relevance of other FCY2 homologues, i.e. TPN1, FUR4, DAL4, FUI1 and yOR071c, for 5-FC toxicity. Among these, Tpn1, Fur4 and the one encoded by yOR071c were found to contribute significantly to 5-FC toxicity, thus revealing alternative entry routes for 5-FC via other cytosine/adenine permease homologues. PMID:16845689

  13. Genome-Wide Identification and Comparative Analysis of Cytosine-5 DNA Methyltransferase and Demethylase Families in Wild and Cultivated Peanut

    PubMed Central

    Wang, Pengfei; Gao, Chao; Bian, Xiaotong; Zhao, Shuzhen; Zhao, Chuanzhi; Xia, Han; Song, Hui; Hou, Lei; Wan, Shubo; Wang, Xingjun

    2016-01-01

    DNA methylation plays important roles in genome protection, regulation of gene expression and is associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferase and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequenced, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in MET, CMT, and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 members didn't contain UBA domain which was different from other plants such as Arabidopsis, maize and soybean. Five DNA demethylase encoding genes were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTase genes mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferase and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or PEG stress could influence the expression level of C5-MTase and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut in the future. PMID:26870046

  14. Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair.

    PubMed

    Dumat, Blaise; Larsen, Anders Foller; Wilhelmsson, L Marcus

    2016-06-20

    Herein, we report on the use of a tricyclic cytosine FRET pair, incorporated into DNA with different base pair separations, to study Z-DNA and B-Z DNA junctions. With its position inside the DNA structure, the FRET pair responds to a B- to Z-DNA transition with a distinct change in FRET efficiency for each donor/acceptor configuration allowing reliable structural probing. Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA. This nucleobase analogue FRET pair represents a valuable addition to the limited number of fluorescence methods available to study Z-DNA and we suggest it will facilitate, for example, deciphering the B- to Z-DNA transition mechanism and investigating the interaction of DNA with Z-DNA binding proteins. PMID:26896804

  15. Retinoic acid alone and in combination with cytosine arabinoside induces differentiation of human myelomonocytic and monoblastic leukaemic cells.

    PubMed

    Hassan, H T; Rees, J K

    1988-01-01

    The effect of retinoic acid (RA) alone and in combination with cytosine arabinoside (Ara-C) on differentiation of fresh human myeloid leukaemic cells from patients with AML was studied. Cells from six patients: three with acute myelomonocytic leukaemia AMMoL and three with acute monoblastic leukaemia AMoL with a percentage of blasts greater than 70, were treated in an in vitro primary suspension culture with retinoic acid (10(-7) M), cytosine arabinoside (100 ng/ml) or both in combination. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in RPMI 1640 culture medium supplemented with 20 per cent fetal bovine serum and 10 per cent (PHA-LCM) phytohaemagglutinin leucocyte conditioned medium and incubated for 6 days at 37 degrees C in a humidified incubator containing 5 per cent CO2 in air. Morphological and functional differentiation into terminal mature elements was induced in all leukaemia cells of the six patients following exposure to the combination of both agents. These results suggest the potential usefulness of the combination of a differentiating agent (retinoic acid) and an antileukaemic drug (cytosine arabinoside) in the treatment of acute myeloid leukaemias: AMMoL and AMoL. This combination warrants a clinical trial. PMID:3422632

  16. Reduced genomic cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein.

    PubMed

    Carlone, Diana L; Lee, Jeong-Heon; Young, Suzanne R L; Dobrota, Erika; Butler, Jill Sergesketter; Ruiz, Joseph; Skalnik, David G

    2005-06-01

    Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP(-)(/)(-) cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP(-)(/)(-) embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP(-)(/)(-) cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP(-)(/)(-) cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP(-)(/)(-) phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development. PMID:15923607

  17. New roles for DNA cytosine modification, eRNA, anchors, and superanchors in developing B cell progenitors.

    PubMed

    Benner, Christopher; Isoda, Takeshi; Murre, Cornelis

    2015-10-13

    B-cell fate is orchestrated by a series of well-characterized developmental regulators. Here, we found that the onset of B-cell development was accompanied by large-scale changes in DNA cytosine modifications associated with promoters, enhancers, and anchors. These changes were tightly linked to alterations in transcription factor occupancy and nascent RNA (eRNA) transcription. We found that the prepro-B to the pro-B-cell transition was associated with a global exchange of DNA cytosine modifications for polycomb-mediated repression at CpG islands. Hypomethylated regions were found exclusively in the active/permissive compartment of the nucleus and were predominantly associated with regulatory elements or anchors that orchestrate the folding patterns of the genome. We identified superanchors, characterized by clusters of hypomethylated CCCTC-binding factor (CTCF)-bound elements, which were predominantly located at boundaries that define topological associated domains. A particularly prominent hypomethylated superanchor was positioned down-stream of the Ig heavy chain (Igh) locus. Analysis of global formaldehyde-cross-linking studies indicated that the Igh locus superanchor interacts with the VH region repertoire across vast genomic distances. We propose that the Igh locus superanchor sequesters the VH and DHJH regions into a spatial confined geometric environment to promote rapid first-passage times. Collectively, these studies demonstrate how, in developing B cells, DNA cytosine modifications associated with regulatory and architectural elements affect patterns of gene expression, folding patterns of the genome, and antigen receptor assembly. PMID:26417104

  18. Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair

    PubMed Central

    Dumat, Blaise; Larsen, Anders Foller; Wilhelmsson, L. Marcus

    2016-01-01

    Herein, we report on the use of a tricyclic cytosine FRET pair, incorporated into DNA with different base pair separations, to study Z-DNA and B-Z DNA junctions. With its position inside the DNA structure, the FRET pair responds to a B- to Z-DNA transition with a distinct change in FRET efficiency for each donor/acceptor configuration allowing reliable structural probing. Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA. This nucleobase analogue FRET pair represents a valuable addition to the limited number of fluorescence methods available to study Z-DNA and we suggest it will facilitate, for example, deciphering the B- to Z-DNA transition mechanism and investigating the interaction of DNA with Z-DNA binding proteins. PMID:26896804

  19. Endothelial Progenitor Cells Combined with Cytosine Deaminase-Endostatin for Suppression of Liver Carcinoma.

    PubMed

    Chen, Rong; Yu, Hui; An, Yan-Li; Chen, Hua-Jun; Jia, ZhenYu; Teng, Gao-Jun

    2016-06-01

    Transplantation of gene transfected endothelial progenitor cells (EPCs) provides a novel method for treatment of human tumors. To study treatment of hepatocellular carcinoma using cytosine deaminase (CD)- and endostatin (ES)-transfected endothelial progenitor cells (EPCs), mouse bone marrow-derived EPCs were cultured and transfected with Lenti6.3-CD-EGFP and Lenti6.3-ES-Monomer-DsRed labeled with superparamagnetic iron oxide (SPIO) nanoparticles. DiD (lipophilic fluorescent dye)-labeled EPCs were injected into normal mice and mice with liver carcinoma. The EPCs loaded with CD-ES were infused into the mice through caudal veins and tumor volumes were measured. The tumor volumes in the EPC + SPIO + CD/5-Fc + ES group were found to be smaller as a result and grew more slowly than those from the EPC + SPIO + LV (lentivirus, empty vector control) group. Survival times were also measured after infusion of the cells into the mice. The median survival time was found to be longer in the EPC + SPIO + CD/5-Fc + ES group than in the others. In conclusion, the EPCs transfected with CD-ES suppressed the liver carcinoma cells in vitro, migrated primarily to the carcinoma, inhibited tumor growth, and also extended the median survival time for the mice with liver carcinoma. PMID:27319212

  20. How Does Guanine-Cytosine Base Pair Affect Excess-Electron Transfer in DNA?

    PubMed

    Lin, Shih-Hsun; Fujitsuka, Mamoru; Majima, Tetsuro

    2015-06-25

    Charge transfer and proton transfer in DNA have attracted wide attention due to their relevance in biological processes and so on. Especially, excess-electron transfer (EET) in DNA has strong relation to DNA repair. However, our understanding on EET in DNA still remains limited. Herein, by using a strongly electron-donating photosensitizer, trimer of 3,4-ethylenedioxythiophene (3E), and an electron acceptor, diphenylacetylene (DPA), two series of functionalized DNA oligomers were synthesized for investigation of EET dynamics in DNA. The transient absorption measurements during femtosecond laser flash photolysis showed that guanine:cytosine (G:C) base pair affects EET dynamics in DNA by two possible mechanisms: the excess-electron quenching by proton transfer with the complementary G after formation of C(•-) and the EET hindrance by inserting a G:C base pair as a potential barrier in consecutive thymines (T's). In the present paper, we provided useful information based on the direct kinetic measurements, which allowed us to discuss EET through oligonucleotides for the investigation of DNA damage/repair. PMID:26042867

  1. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  2. ESI-MS studies of palladium (II) complexes with 1-(p-toluenesulfonyl)cytosine/cytosinato ligands.

    PubMed

    Kobetić, Renata; Gembarovski, Dubravka; Visnjevac, Aleksandar; Zinić, Biserka; Gabelica-Marković, Vesna

    2010-01-01

    The mononuclear complex Pd(1-TosC-N3)(2)Cl(2) (2) containing 1-(p-toluenesulfonyl)cytosine (1) as a ligand, as well as dinuclear complexes Pd(2)(1-TosC(-)-N3,N4)(4) (3) and Pd(2)(1-TosC(-)-N3,N4)(2)DMSO(2)Cl(2) (4) containing the ligand anion (1-TosC(-)), was mass analyzed by electrospray ionization ion trap MS/MS and high resolution MS. Complexes 3 and 4 were obtained by recrystallization of 2 from DMF and DMSO, respectively. The behavior of complex 2 in different solutions was monitored by electrospray ionization mass spectrometry (ESI-MS). Under the applied ESI-MS conditions, complex 2 in methanol reorganized itself dominantly as new complex 3 and the solvent did not coordinate the formed species. In H(2)O/DMSO, CH(3)CN/DMSO and CH(3)OH/DMSO solutions, complex 2 formed several new species with solvent molecules involved in their structure, e.g. complex 4 was formed as the major product. The newly formed species were also examined by LC-MS-DAD, confirming the solvent induced reorganization and the solution instability of complex 2. PMID:19882593

  3. [Recent development of antitumor antimetabolites in Japan--cytosine arabinoside analogues].

    PubMed

    Tsukagoshi, S

    1997-05-01

    Since there have been relatively high incidence of cancer of the digestive organs in Japan, many 5-fluorouracil analogues have been studied as the drugs to treat such cancers. Beside these fluoropyrimine compounds, cytosine arabinoside (ara-C) analogues have also been studied, and some of them have shown appreciable clinical activities against human malignancies. In this paper, as such analogues, experimental and clinical studies of gemcitabine (dFdC). DMDC and cytarabine ocfosfate were reviewed. Among these drugs, gemcitabine (Eli Lilly, Japan) showed more than 20% response rate against non-small cell lung cancer in the late phase II study in Japan. Unfortunately, clinical study of DMDC (Yoshitomi) is currently suspended because of the lack of the hint of clinical activity, but the author believes that this might show some clinical activities by changing the treatment regimens in the future. Cytarabine ocfosfate (Nippon Kayaku) has already put on market as the first drug to be active against ANLL and MDS by giving orally. PMID:9170512

  4. Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside

    SciTech Connect

    Ponzoni, M.; Lanciotti, M.; Melodia, A.; Casalaro, A.; Cornaglia-Ferraris, P. )

    1989-03-01

    The effects of cytosine-arabinoside (ARA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. Low doses of ARA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 hours after a dose of ARA-C as low as 0.1 {mu}g/ml. The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in ({sup 3}H)thymidine incorporation was also observed within 24 hours and the cell growth was completely inhibited on the sixth day. Moreover, ARA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. At the same time ARA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ARA-C do promote the differentiation of GI-ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.

  5. The pyrimidin analogue cyclopentenyl cytosine induces alloantigen-specific non-responsiveness of human T lymphocytes

    PubMed Central

    Nikolaeva, N; Bemelman, F J; Yong, S-L; Verschuur, A; van Lier, R A W; ten Berge, I J M

    2008-01-01

    Cyclopentenyl cytosine (CPEC) has been shown to induce apoptosis in human T lymphoblastic cell lines and T cells from leukaemia patients. In this study we have addressed the question of whether CPEC is able to decrease proliferation and effector functions of human alloresponsive T lymphocytes and induce T cell anergy. The proliferative capacity of human peripheral blood mononuclear cells in response to allogeneic stimulation was measured by 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester staining. Flow cytometric analysis was performed using surface CD4, CD8, CD25, CD103 and intracellular perforin, granzyme A, granzyme B, caspase-3 and forkhead box P3 (FoxP3) markers. The in vivo immunosuppressive capacity was tested in a murine skin graft model. Addition of CPEC at a concentration of 20 nM strongly decreased the expansion and cytotoxicity of alloreactive T cells. Specific restimulation in the absence of CPEC showed that the cells became anergic. The drug induced caspase-dependent apoptosis of alloreactive T lymphocytes. Finally, CPEC increased the percentage of CD25high FoxP3+ CD4+ and CD103+ CD8+ T cells, and potentiated the effect of rapamycin in increasing the numbers of alloreactive regulatory T cells. Treatment with CPEC of CBA/CA mice transplanted with B10/Br skin grafts significantly prolonged graft survival. We conclude that CPEC inhibits proliferation and cytotoxicity of human alloreactive T cells and induces alloantigen non-responsiveness in vitro. PMID:18062797

  6. Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells.

    PubMed

    Park, Jin Sung; Chang, Da-Young; Kim, Ji-Hoi; Jung, Jin Hwa; Park, JoonSeong; Kim, Se-Hyuk; Lee, Young-Don; Kim, Sung-Soo; Suh-Kim, Haeyoung

    2013-01-01

    Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation. PMID:23429359

  7. Variation in cytosine methylation patterns during ploidy level conversions in Eragrostis curvula.

    PubMed

    Ochogavía, Ana C; Cervigni, Gerardo; Selva, Juan P; Echenique, Viviana C; Pessino, Silvina C

    2009-05-01

    In many species polyploidization involves rearrangements of the progenitor genomes, at both genetic and epigenetic levels. We analyzed the cytosine methylation status in a 'tetraploid-diploid-tetraploid' series of Eragrostis curvula with a common genetic background by using the MSAP (Methylation-sensitive Amplified Polymorphism) technique. Considerable levels of polymorphisms were detected during ploidy conversions. The total level of methylation observed was lower in the diploid genotype compared to the tetraploid ones. A significant proportion of the epigenetic modifications occurring during the tetraploid-diploid conversion reverted during the diploid-tetraploid one. Genetic and expression data from previous work were used to analyze correlation with methylation variation. All genetic, epigenetic and gene expression variation data correlated significantly when compared by pairs in simple Mantel tests. Dendrograms reflecting genetic, epigenetic and expression distances as well as principal coordinate analysis suggested that plants of identical ploidy levels present similar sets of data. Twelve (12) different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated, sequenced and characterized. PMID:19160057

  8. The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters*

    PubMed Central

    Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

    2014-01-01

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. PMID:25320081

  9. A quantum chemical insight to intermolecular hydrogen bonding interaction between cytosine and nitrosamine: Structural and energetic investigations

    NASA Astrophysics Data System (ADS)

    Khalili, Behzad

    2016-03-01

    Hydrogen bond interactions which are formed during complex formation between cytosine and nitrosamine have been fully investigated using B3LYP, B3PW91 and MP2 methods in conjunction with various basis sets including 6-311++G (d,p), 6-311++G (2d,2p), 6-311++G (df,pd) and AUG-cc-pVDZ. Three regions around the most stable conformer of cytosine in the gas phase with six possible double H-bonded interactions were considered. Two intermolecular hydrogen bonds of type NC-N-HNA and O-H(N-H)C-ONA were found on the potential energy surface in a cyclic system with 8-member in CN1, CN3, CN5 and 7-member in CN2, CN4, CN6 systems. Results of binding energy calculation at all applied methods reveal that the CN1 structure is the most stable one which is formed by interaction of nitrosamine with cytosine in S1 region. The BSSE-corrected binding energy for six complex system is ranging from -23.8 to -43.6 kJ/mol at MP2/6-311++G (df,pd) level and the stability order is as CN1 > CN2 > CN3 > CN4 > CN5 > CN6 in all studied levels of theories. The NBO results reveal that the charge transfer occurred from cytosine to nitrosamine in CN1, CN3, CN5 and CN6 whereas this matter in the case of CN2 and CN4 was reversed. The relationship between BEs with red shift of H-bond involved bonds vibrational frequencies, charge transfer energies during complex formation and electron densities at H-bond BCPs were discussed. In addition activation energetic properties related to the proton transfer process between cytosine and nitrosamine have been calculated at MP2/6-311++G (df,pd) level. AIM results imply that H-bond interactions are electrostatic with partially covalent characteristic in nature.

  10. Tissue culture-induced transpositional activity of mPing is correlated with cytosine methylation in rice

    PubMed Central

    Ngezahayo, Frédéric; Xu, Chunming; Wang, Hongyan; Jiang, Lily; Pang, Jinsong; Liu, Bao

    2009-01-01

    Background mPing is an endogenous MITE in the rice genome, which is quiescent under normal conditions but can be induced towards mobilization under various stresses. The cellular mechanism responsible for modulating the activity of mPing remains unknown. Cytosine methylation is a major epigenetic modification in most eukaryotes, and the primary function of which is to serve as a genome defense system including taming activity of transposable elements (TEs). Given that tissue-culture is capable of inducing both methylation alteration and mPing transposition in certain rice genotypes, it provides a tractable system to investigate the possible relationship between the two phenomena. Results mPing transposition and cytosine methylation alteration were measured in callus and regenerated plants in three rice (ssp. indica) genotypes, V14, V27 and R09. All three genotypes showed transposition of mPing, though at various frequencies. Cytosine methylation alteration occurred both at the mPing-flanks and at random loci sampled globally in callus and regenerated plants of all three genotypes. However, a sharp difference in the changing patterns was noted between the mPing-flanks and random genomic loci, with a particular type of methylation modification, i.e., CNG hypermethylation, occurred predominantly at the mPing-flanks. Pearson's test on pairwise correlations indicated that mPing activity is positively correlated with specific patterns of methylation alteration at random genomic loci, while the element's immobility is positively correlated with methylation levels of the mPing's 5'-flanks. Bisulfite sequencing of two mPing-containing loci showed that whereas for the immobile locus loss of CG methylation in the 5'-flank was accompanied by an increase in CHG methylation, together with an overall increase in methylation of all three types (CG, CHG and CHH) in the mPing-body region, for the active locus erasure of CG methylation in the 5'-flank was not followed by such a

  11. Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase

    PubMed Central

    Jarchow-Choy, Sarah K.; Krueger, Andrew T.; Liu, Haibo; Gao, Jianmin; Kool, Eric T.

    2011-01-01

    Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3′-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson–Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)n polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)n, fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA. PMID:20947563

  12. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  13. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  14. Mosaic organization of DNA nucleotides

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Havlin, S.; Simons, M.; Stanley, H. E.; Goldberger, A. L.

    1994-01-01

    Long-range power-law correlations have been reported recently for DNA sequences containing noncoding regions. We address the question of whether such correlations may be a trivial consequence of the known mosaic structure ("patchiness") of DNA. We analyze two classes of controls consisting of patchy nucleotide sequences generated by different algorithms--one without and one with long-range power-law correlations. Although both types of sequences are highly heterogenous, they are quantitatively distinguishable by an alternative fluctuation analysis method that differentiates local patchiness from long-range correlations. Application of this analysis to selected DNA sequences demonstrates that patchiness is not sufficient to account for long-range correlation properties.

  15. Nucleotide excision repair in humans.

    PubMed

    Spivak, Graciela

    2015-12-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429

  16. Clinical results and pharmacokinetics of high-dose cytosine arabinoside (HD ARA-C).

    PubMed

    Breithaupt, H; Pralle, H; Eckhardt, T; von Hattingberg, M; Schick, J; Löffler, H

    1982-10-01

    Four patients with acute nonlymphoblastic leukemia and one malignant teratoma refractory to conventional chemotherapy were treated with high doses of cytosine arabinoside (HD ARA-C). They received up to 12 cycles of 1.8 to 3 g/m2 every 12 hours applied by 2-hour infusions. A total of 55 HD ARA-C infusions was performed. All leukemic patients responded. A complete clearance of blasts from the bone marrow was observed in two patients following 8-12 cycles of 3 g/m2. However, relapses occurred after three and seven weeks, in one case with resistance to HD ARA-C. The patient with malignant teratoma did not respond. No severe toxicity emerged even after repeated applications. Adverse reactions included moderate nausea and vomiting (4 patients), diarrhea (2 patients), hepatic dysfunction (1 patient), bone pain (1 patient), blurred vision (1 patient), conjunctivitis (1 patient), and exanthema with partial epidermiolysis (1 patient). Granulocytopenia occurring between 3-8 days after having started the therapy, subsided within 4-25 days. Plasma levels of ARA-C and the metabolite uracil arabinoside (ARA-U) were monitored. At steady state plasma concentrations of ARA-C were 32-97 microM (8-24 micrograms/ml). ARA-C disappeared from the plasma mono- or biphasic with a terminal half-life (t50%) of 7.8-12.6 minutes. The total clearance (Cl) of ARA-C varied between 1.7 and 2.9 liters/kg . h, and the distribution volume (Vss) between 0.44 and 0.86 liters/kg. Cerebrospinal fluid (CSF) levels of ARA-C reached 10-15% of steady state concentrations in plasma. PMID:7104969

  17. Vacuum-Ultraviolet photoionization studies of the microhydrationof DNA bases (Guanine, Cytosine, Adenine and Thymine)

    SciTech Connect

    Belau, L.; Wilson, K.R.; Leone, S.R.; Musahid, Ahmed

    2007-01-22

    In this work, we report on a photoionization study of the microhydration of the four DNA bases. Gas-phase clusters of water with DNA bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] are generated via thermal vaporization of the bases and expansion of the resultant vapor in a continuous supersonic jet expansion of water seeded in Ar. The resulting clusters are investigated by single-photon ionization with tunable vacuum-ultraviolet synchrotron radiation and mass analyzed using reflectron mass spectrometry. Photoionization efficiency (PIE) curves are recorded for the DNA bases and the following water (W) clusters: G, GW{sub n} (n = 1-3); C, CW{sub n} (n = 1-3); A, AW{sub n} (n = 1,2); and T, TW{sub n} (n = 1-3). Appearance energies (AE) are derived from the onset of these PIE curves (all energies in eV): G (8.1 {+-} 0.1), GW (8.0 {+-} 0.1), GW{sub 2} (8.0 {+-} 0.1), and GW{sub 3} (8.0); C (8.65 {+-} 0.05), CW (8.45 {+-} 0.05), CW{sub 2} (8.4 {+-} 0.1), and CW{sub 3} (8.3 {+-} 0.1); A (8.30 {+-} 0.05), AW (8.20 {+-} 0.05), and AW{sub 2} (8.1 {+-} 0.1); T (8.90 {+-} 0.05); and TW (8.75 {+-} 0.05), TW{sub 2} (8.6 {+-} 0.1), and TW{sub 3} (8.6 {+-} 0.1). The AEs of the DNA bases decrease slightly with the addition of water molecules (up to three) but do not converge to values found for photoinduced electron removal from DNA bases in solution.

  18. Nonadditive changes to cytosine methylation as a consequence of hybridization and genome duplication in Senecio (Asteraceae).

    PubMed

    Hegarty, Matthew J; Batstone, Tom; Barker, Gary L; Edwards, Keith J; Abbott, Richard J; Hiscock, Simon J

    2011-01-01

    The merger of two or more divergent genomes within an allopolyploid nucleus can facilitate speciation and adaptive evolution in flowering plants. Widespread changes to gene expression have been shown to result from interspecific hybridisation and polyploidy in a number of plant species, and attention has now shifted to determining the epigenetic processes that drive these changes. We present here an analysis of cytosine methylation patterns in triploid F(1) Senecio (ragwort) hybrids and their allohexaploid derivatives. We observe that, in common with similar studies in Arabidopsis, Spartina and Triticum, a small but significant proportion of loci display nonadditive methylation in the hybrids, largely resulting from interspecific hybridisation. Despite this, genome duplication results in a secondary effect on methylation, with reversion to additivity at some loci and novel methylation status at others. We also observe differences in methylation state between different allopolyploid generations, predominantly in cases of additive methylation with regard to which parental methylation state is dominant. These changes to methylation state in both F(1) triploids and their allohexaploid derivatives largely mirror the overall patterns of nonadditive gene expression observed in our previous microarray analyses and may play a causative role in generating those expression changes. These similar global changes to DNA methylation resulting from hybridisation and genome duplication may serve as a source of epigenetic variation in natural populations, facilitating adaptive evolution. Our observations that methylation state can also vary between different generations of polyploid hybrids suggests that newly formed allopolyploid species may display a high degree of epigenetic diversity upon which natural selection can act. PMID:21073590

  19. Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

    PubMed

    Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

    2015-07-01

    We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. PMID:26211587

  20. Cytosine Deaminase/5-Fluorocytosine Exposure Induces Bystander and Radiosensitization Effects in Hypoxic Glioblastoma Cells in vitro

    SciTech Connect

    Chen, Jennifer K.; Hu, Lily J.; Wang Dongfang; Lamborn, Kathleen R.; Deen, Dennis F. . E-mail: dennisdeen@juno.com

    2007-04-01

    Purpose: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. Methods and Materials: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. Results: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. Conclusion: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.

  1. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

    PubMed Central

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide

  2. Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers.

    PubMed

    Vyhlidal, Carrie A; Bi, Chengpeng; Ye, Shui Qing; Leeder, J Steven

    2016-07-01

    Members of the human CYP3A family of metabolizing enzymes exhibit developmental changes in expression whereby CYP3A7 is expressed in fetal tissues, followed by a transition to expression of CYP3A4 in the first months of life. Despite knowledge about the general pattern of CYP3A activity in human development, the mechanisms that regulate developmental expression remain poorly understood. Epigenetic changes, including cytosine methylation, have been suggested to play a role in the regulation of CYP3A expression. The objective of this study was to investigate changes in cytosine methylation of the CYP3A4 and CYP3A7 genes in human pediatric and prenatal livers. The methylation status of cytosine-phospho-guanine dinucleotides was determined in 16 pediatric liver samples using methyl-seq and confirmed by bisulfite sequencing of 48 pediatric and 34 prenatal liver samples. Samples were separated by age into five groups (prenatal, < 1 year of age, 1.8-6 years, 7-11 years, and 12-17 years). Methyl-seq anaylsis revealed that cytosines in the proximal promoter of CYP3A7 are hypomethylated in neonates compared with adolescents (P < 0.001). In contrast, a cytosine 383 base pair upstream of CYP3A4 is hypermethylated in liver samples from neonates compared with adolescents (P = 0.00001). Developmental changes in methylation of cytosines in the proximal promoters of CYP3A4 and CYP3A7 in pediatric livers were confirmed by bisulfite sequencing. In addition, the methylation status of cytosine in the CYP3A4 and CYP3A7 proximal promoters correlated with changes in developmental expression of mRNA for the two enzymes. PMID:26772622

  3. Effect of C5-Methylation of Cytosine on the UV-Induced Reactivity of Duplex DNA: Conformational and Electronic Factors.

    PubMed

    Banyasz, Akos; Esposito, Luciana; Douki, Thierry; Perron, Marion; Lepori, Clément; Improta, Roberto; Markovitsi, Dimitra

    2016-05-12

    C5-methylation of cytosines is strongly correlated with UV-induced mutations detected in skin cancers. Mutational hot-spots appearing at TCG sites are due to the formation of pyrimidine cyclobutane dimers (CPDs). The present study, performed for the model DNA duplex (TCGTA)3·(TACGA)3 and the constitutive single strands, examines the factors underlying the effect of C5-methylation on pyrimidine dimerization at TCG sites. This effect is quantified for the first time by quantum yields ϕ. They were determined following irradiation at 255, 267, and 282 nm and subsequent photoproduct analysis using HPLC coupled to mass spectrometry. C5-methylation leads to an increase of the CPD quantum yield up to 80% with concomitant decrease of that of pyrimidine(6-4) pyrimidone adducts (64PPs) by at least a factor of 3. The obtained ϕ values cannot be explained only by the change of the cytosine absorption spectrum upon C5-methylation. The conformational and electronic factors that may affect the dimerization reaction are discussed in light of results obtained by fluorescence spectroscopy, molecular dynamics simulations, and quantum mechanical calculations. Thus, it appears that the presence of an extra methyl on cytosine affects the sugar puckering, thereby enhancing conformations of the TC step that are prone to CPD formation but less favorable to 64PPs. In addition, C5-methylation diminishes the amplitude of conformational motions in duplexes; in the resulting stiffer structure, ππ* excitations may be transferred from initially populated exciton states to reactive pyrimidines giving rise to CPDs. PMID:27075054

  4. Long-range correlations in nucleotide sequences

    NASA Astrophysics Data System (ADS)

    Peng, C.-K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-03-01

    DNA SEQUENCES have been analysed using models, such as an it-step Markov chain, that incorporate the possibility of short-range nucleotide correlations1. We propose here a method for studying the stochastic properties of nucleotide sequences by constructing a 1:1 map of the nucleotide sequence onto a walk, which we term a 'DNA walk'. We then use the mapping to provide a quantitative measure of the correlation between nucleotides over long distances along the DNA chain. Thus we uncover in the nucleotide sequence a remarkably long-range power law correlation that implies a new scale-invariant property of DNA. We find such long-range correlations in intron-containing genes and in nontranscribed regulatory DNA sequences, but not in complementary DNA sequences or intron-less genes.

  5. Hybrid Coupled Cluster and Molecular Dynamics Approach: Application to the Excitation Spectrum of Cytosine in the Native DNA Environment

    SciTech Connect

    Valiev, Marat; Kowalski, Karol

    2006-12-07

    Evolution of the excited state energies of cytosine base in the native DNA environment was investigated using hybrid coupled cluster and classical molecular dynamics approach. The time averaged excitation energies obtained with the variant of the completely renormalized equation-of-motion with singles, doubles, and non-iterative triples approach that includes a bulk of the correlation effects for excited states, are compared with the analogous calculations in the gas phase. Significant blue shifts for the two lowest singlet excitation energies can be observed as a result of interaction of the quantum system with surrounding environment.

  6. Investigation of the mechanisms of photo-induced formation of cyclobutane dimers of cytosine and 2,4-diaminopyrimidine.

    PubMed

    Kancheva, Pavlina B; Delchev, Vassil B

    2016-09-01

    The mechanisms of the formation of cyclobutane dimers (CBD) of cytosine and 2,4-diaminopyrimidine were studied at the CC2 theoretical level and cc-pVDZ basis functions. Four orientations of the two monomers are explored: cys-syn, cis-anti, trans-syn, and trans-anti. The research revealed that in all cases the cyclobutane structures are formed along the (1)ππ* excited-state reaction paths of the stacked aggregates. We localized the S1/S0 conical intersections mediating those transformations. The results obtained agree well with the previously reported investigations on the cis-syn cyclodimer formations of other pyrimidines. PMID:27572158

  7. The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts.

    PubMed

    Waters, R; Crocombe, K; Mirzayans, R

    1982-05-01

    Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of Dna polymerase alpha, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase alpha, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells. PMID:6808389

  8. Automated Identification of Nucleotide Sequences

    NASA Technical Reports Server (NTRS)

    Osman, Shariff; Venkateswaran, Kasthuri; Fox, George; Zhu, Dian-Hui

    2007-01-01

    STITCH is a computer program that processes raw nucleotide-sequence data to automatically remove unwanted vector information, perform reverse-complement comparison, stitch shorter sequences together to make longer ones to which the shorter ones presumably belong, and search against the user s choice of private and Internet-accessible public 16S rRNA databases. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] In STITCH, a template 16S rRNA sequence is used to position forward and reverse reads. STITCH then automatically searches known 16S rRNA sequences in the user s chosen database(s) to find the sequence most similar to (the sequence that lies at the smallest edit distance from) each spliced sequence. The result of processing by STITCH is the identification of the most similar well-described bacterium. Whereas previously commercially available software for analyzing genetic sequences operates on one sequence at a time, STITCH can manipulate multiple sequences simultaneously to perform the aforementioned operations. A typical analysis of several dozen sequences (length of the order of 103 base pairs) by use of STITCH is completed in a few minutes, whereas such an analysis performed by use of prior software takes hours or days.

  9. Intersystem crossing rates of S1 state keto-amino cytosine at low excess energy.

    PubMed

    Lobsiger, Simon; Etinski, Mihajlo; Blaser, Susan; Frey, Hans-Martin; Marian, Christel; Leutwyler, Samuel

    2015-12-21

    The amino-keto tautomer of supersonic jet-cooled cytosine undergoes intersystem crossing (ISC) from the v = 0 and low-lying vibronic levels of its S1((1)ππ(∗)) state. We investigate these ISC rates experimentally and theoretically as a function of S1 state vibrational excess energy Eexc. The S1 vibronic levels are pumped with a ∼5 ns UV laser, the S1 and triplet state ion signals are separated by prompt or delayed ionization with a second UV laser pulse. After correcting the raw ISC yields for the relative S1 and T1 ionization cross sections, we obtain energy dependent ISC quantum yields QISC (corr)=1%-5%. These are combined with previously measured vibronic state-specific decay rates, giving ISC rates kISC = 0.4-1.5 ⋅ 10(9) s(-1), the corresponding S1⇝S0 internal conversion (IC) rates are 30-100 times larger. Theoretical ISC rates are computed using SCS-CC2 methods, which predict rapid ISC from the S1; v = 0 state with kISC = 3 ⋅ 10(9) s(-1) to the T1((3)ππ(∗)) triplet state. The surprisingly high rate of this El Sayed-forbidden transition is caused by a substantial admixture of (1)nOπ(∗) character into the S1((1)ππ(∗)) wave function at its non-planar minimum geometry. The combination of experiment and theory implies that (1) below Eexc = 550 cm(-1) in the S1 state, S1⇝S0 internal conversion dominates the nonradiative decay with kIC ≥ 2 ⋅ 10(10) s(-1), (2) the calculated S1⇝T1 ((1)ππ(∗)⇝(3)ππ(∗)) ISC rate is in good agreement with experiment, (3) being El-Sayed forbidden, the S1⇝T1 ISC is moderately fast (kISC = 3 ⋅ 10(9) s(-1)), and not ultrafast, as claimed by other calculations, and (4) at Eexc ∼ 550 cm(-1) the IC rate increases by ∼50 times, probably by accessing the lowest conical intersection (the C5-twist CI) and thereby effectively switching off the ISC decay channels. PMID:26696056

  10. Intersystem crossing rates of S1 state keto-amino cytosine at low excess energy

    NASA Astrophysics Data System (ADS)

    Lobsiger, Simon; Etinski, Mihajlo; Blaser, Susan; Frey, Hans-Martin; Marian, Christel; Leutwyler, Samuel

    2015-12-01

    The amino-keto tautomer of supersonic jet-cooled cytosine undergoes intersystem crossing (ISC) from the v = 0 and low-lying vibronic levels of its S1(1ππ∗) state. We investigate these ISC rates experimentally and theoretically as a function of S1 state vibrational excess energy Eexc. The S1 vibronic levels are pumped with a ˜5 ns UV laser, the S1 and triplet state ion signals are separated by prompt or delayed ionization with a second UV laser pulse. After correcting the raw ISC yields for the relative S1 and T1 ionization cross sections, we obtain energy dependent ISC quantum yields QISC corr = 1 % -5%. These are combined with previously measured vibronic state-specific decay rates, giving ISC rates kISC = 0.4-1.5 ṡ 109 s-1, the corresponding S1⇝S0 internal conversion (IC) rates are 30-100 times larger. Theoretical ISC rates are computed using SCS-CC2 methods, which predict rapid ISC from the S1; v = 0 state with kISC = 3 ṡ 109 s-1 to the T1(3ππ∗) triplet state. The surprisingly high rate of this El Sayed-forbidden transition is caused by a substantial admixture of 1nOπ∗ character into the S1(1ππ∗) wave function at its non-planar minimum geometry. The combination of experiment and theory implies that (1) below Eexc = 550 cm-1 in the S1 state, S1⇝S0 internal conversion dominates the nonradiative decay with kIC ≥ 2 ṡ 1010 s-1, (2) the calculated S1⇝T1 (1ππ∗⇝3ππ∗) ISC rate is in good agreement with experiment, (3) being El-Sayed forbidden, the S1⇝T1 ISC is moderately fast (kISC = 3 ṡ 109 s-1), and not ultrafast, as claimed by other calculations, and (4) at Eexc ˜ 550 cm-1 the IC rate increases by ˜50 times, probably by accessing the lowest conical intersection (the C5-twist CI) and thereby effectively switching off the ISC decay channels.

  11. Pulsed magnetic field from video display terminals enhances teratogenic effects of cytosine arabinoside in mice

    SciTech Connect

    Chiang, H.; Wu, R.Y.; Shao, B.J.; Fu, Y.D.; Yao, G.D.; Lu, D.J.

    1995-05-01

    Eighty-nine Swiss Webster mice were randomly divided into four groups: a control group, a pulsed magnetic field (PMF) group, a cytosine arabinoside (ara-C, a teratogen) group, and a combined PMF + ara-C group. Mice in the PMF and PMF + ara-C groups were irradiated with a PMF (a sawtooth waveform with 52 {mu}s rise time, 12{mu}s decay time, and 15.6 kHz frequency) at a peak magnetic flux density of 40 {mu}T for 4 hours daily on days 6-17 of gestation. The mice in the ara-C and the PMF + ara-C groups were injected intraperitoneally on day 9 of gestation with 10 mg/kg of ara-C. The incidence of resorption and dead fetuses was not affected by PMF but was increased by ara-C injection. The malformation incidence of cleft palate (CP) and/or cleft lip (CL) was significantly higher in all three of the treated groups than in the control group (P < 0.05). If, however, statistical analyses had been done on litters rather than on individual fetuses, they would show that the incidence of CP and/or CL in the PMF group is not significantly greater than that in the control group. A significantly higher incidence of CP and/or CL was found in the PMF + ara-C group (49%) than the ara-C alone group (26.1%). These data suggest that PMF might enhance the development of ara-C-induced CP and/or CL. The incidence of minor variations in skeletal development, including reduction of skeletal calcification and loss of skeleton, was not statistically significant in the PMF group. However, it was higher in the two ara-C-treated groups, and there was no significant difference between the ara-C alone group and the ara-C + PMF group. From these results it is concluded that the very weak embryotoxic effects of PMF exposure may be revealed and enhanced in combination with a teratogenic agent.

  12. Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

    PubMed Central

    Fasullo, Michael; Endres, Lauren

    2015-01-01

    Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases. PMID:25923076

  13. The effects of tautomerization and protonation on the adenine-cytosine mismatches: a density functional theory study.

    PubMed

    Masoodi, Hamid Reza; Bagheri, Sotoodeh; Abareghi, Mahsa

    2016-06-01

    In the present work, we demonstrate the results of a theoretical study concerned with the question how tautomerization and protonation of adenine affect the various properties of adenine-cytosine mismatches. The calculations, in gas phase and in water, are performed at B3LYP/6-311++G(d,p) level. In gas phase, it is observed that any tautomeric form of investigated mismatches is more stabilized when adenine is protonated. As for the neutral mismatches, the mismatches containing amino form of cytosine and imino form of protonated adenine are more stable. The role of aromaticity on the stability of tautomeric forms of mismatches is investigated by NICS(1)ZZ index. The stability of mispairs decreases by going from gas phase to water. It can be explained using dipole moment parameter. The influence of hydrogen bonds on the stability of mismatches is examined by atoms in molecules and natural bond orbital analyses. In addition to geometrical parameters and binding energies, the study of the topological properties of electron charge density aids in better understanding of these mispairs. PMID:26198186

  14. Characterizing radiation-induced oxidation of DNA by way of the monohydrated guanine-cytosine radical cation.

    PubMed

    Jaeger, Heather M; Schaefer, Henry F

    2009-06-11

    The interaction of one water molecule with the guanine-cytosine radical cation has been studied with ab initio and density functional methods in order to help elucidate the nature of oxidized aqueous DNA. The theoretical spin density of [GC]*(+) reveals that the radical center is localized on guanine. The adiabatic ionization potential lowers from 7.63 to 6.71 eV in concurrence with the formation of the Watson-Crick base pair and hydration by one water molecule. A natural bond orbital analysis of partial charges shows that approximately 80% of the positive charge persists on guanine upon hydration and formation of the Watson-Crick base pair with cytosine. Hydration energies were computed with second-order Z-averaged perturbation theory (ZAPT2) using the aug-cc-pVDZ basis set at 11 stationary points on the B3LYP/DZP++ potential energy surface. The hydration energy at the global minimum is 14.2 kcal mol(-1). The lowest energy structures correspond to hydration near the glycosidic bond sites. Structural changes in the Watson-Crick base pair are predominantly seen for monohydration in the groove regions of double-helix DNA. PMID:19445496

  15. DNA cytosine and methylcytosine deamination by APOBEC3B: enhancing methylcytosine deamination by engineering APOBEC3B

    PubMed Central

    Fu, Yang; Ito, Fumiaki; Zhang, Gewen; Fernandez, Braulio; Yang, Hanjing; Chen, Xiaojiang S.

    2015-01-01

    APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) is a family of enzymes that deaminates cytosine (C) to uracil (U) on nucleic acid. APOBEC3B (A3B) functions in innate immunity against intrinsic and invading retroelements and viruses. A3B can also induce genomic DNA mutations to cause cancer. A3B contains two cytosine deaminase domains (CD1, CD2), and there are conflicting reports about whether both domains are active. Here we demonstrate that only CD2 of A3B (A3BCD2) has C deamination activity. We also reveal that both A3B and A3BCD2 can deaminate methylcytosine (mC). Guided by structural and functional analysis, we successfully engineered A3BCD2 to gain over two orders of magnitude higher activity for mC deamination. Important determinants that contribute to the activity and selectivity for mC deamination have been identified, which reveals that multiple elements, rather than single ones, contribute to the mC deamination activity and selectivity in A3BCD2 and possibly other APOBECs. PMID:26195824

  16. Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat.

    PubMed

    Shaked, H; Kashkush, K; Ozkan, H; Feldman, M; Levy, A A

    2001-08-01

    Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes, although little is known about the type of changes that occur, how rapidly they occur, and the type of sequences involved. To address these matters, we have surveyed F1 hybrids between diploid species from the wheat (Aegilops and Triticum) group and their derived allotetraploids by screening a large number of loci using amplified fragment length polymorphism and DNA gel blot analysis and by assaying the extent of cytosine methylation. We found that sequence elimination is one of the major and immediate responses of the wheat genome to wide hybridization or allopolyploidy, that it affects a large fraction of the genome, and that it is reproducible. In one cross between AE: sharonensis x AE: umbellulata, 14% of the loci from AE: sharonensis were eliminated compared with only 0.5% from AE: umbellulata, with most changes occurring in the F1 hybrid. In contrast, crosses between AE: longissima x T. urartu showed that sequence elimination was more frequent after chromosome doubling. Alterations in cytosine methylation occurred in approximately 13% of the loci, either in the F1 hybrid or in the allopolyploid. For eight of nine bands that were isolated, the sequences that underwent elimination corresponded to low-copy DNA, whereas alterations in methylation patterns affected both repetitive DNA sequences, such as retrotransposons, and low-copy DNA in approximately equal proportions. PMID:11487690

  17. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    SciTech Connect

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-04-04

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  18. Mutagenic effects induced by the attack of NO2 radical to the guanine-cytosine base pair

    PubMed Central

    Cerón-Carrasco, José P.; Requena, Alberto; Zúñiga, José; Jacquemin, Denis

    2015-01-01

    We investigate the attack of the nitrogen dioxide radical (NO•2) to the guanine—cytosine (GC) base pair and the subsequent tautomeric reactions able to induce mutations, by means of density functional theory (DFT) calculations. The conducted simulations allow us to identify the most reactive sites of the GC base pair. Indeed, the computed relative energies demonstrate that the addition of the NO•2 radical to the C8 position of the guanine base forms to the most stable adduct. Although the initial adducts might evolve to non-canonical structures via inter-base hydrogen bonds rearrangements, the probability for the proton exchange to occur lies in the same range as that observed for undamaged DNA. As a result, tautomeric errors in NO2-attacked DNA arises at the same rate as in canonical DNA, with no macroscopic impact on the overall stability of DNA. The potential mutagenic effects of the GC–NO•2 radical adducts likely involve side reactions, e.g., the GC deprotonation to the solvent, rather than proton exchange between guanine and cytosine basis. PMID:25798437

  19. Spontaneous Oligomerization of Nucleotide Alternatives in Aqueous Solutions

    NASA Astrophysics Data System (ADS)

    Smith, Karen E.; House, Christopher H.; Dworkin, Jason P.; Callahan, Michael P.

    2016-03-01

    On early Earth, a primitive polymer that could spontaneously form from likely available precursors may have preceded both RNA and DNA as the first genetic material. Here, we report that heated aqueous solutions containing 5-hydroxymethyluracil (HMU) result in oligomers of uracil, heated solutions containing 5-hydroxymethylcytosine (HMC) result in oligomers of cytosine, and heated solutions containing both HMU and HMC result in mixed oligomers of uracil and cytosine. Oligomerization of hydroxymethylated pyrimidines, which may have been abundant on the primitive Earth, might have been important in the development of simple informational polymers.

  20. Single Nucleotide Polymorphisms and Osteoarthritis

    PubMed Central

    Wang, Ting; Liang, Yuting; Li, Hong; Li, Haibo; He, Quanze; Xue, Ying; Shen, Cong; Zhang, Chunhua; Xiang, Jingjing; Ding, Jie; Qiao, Longwei; Zheng, Qiping

    2016-01-01

    Abstract Osteoarthritis (OA) is a complex disorder characterized by degenerative articular cartilage and is largely attributed to genetic risk factors. Single nucleotide polymorphisms (SNPs) are common DNA variants that have shown promising and efficiency, compared with positional cloning, to map candidate genes of complex diseases, including OA. In this study, we aim to provide an overview of multiple SNPs from a number of genes that have recently been linked to OA susceptibility. We also performed a comprehensive meta-analysis to evaluate the association of SNP rs7639618 of double von Willebrand factor A domains (DVWA) gene with OA susceptibility. A systematic search of studies on the association of SNPs with susceptibility to OA was conducted in PubMed and Google scholar. Studies subjected to meta-analysis include human and case-control studies that met the Hardy–Weinberg equilibrium model and provide sufficient data to calculate an odds ratio (OR). A total of 9500 OA cases and 9365 controls in 7 case-control studies relating to SNP rs7639618 were included in this study and the ORs with 95% confidence intervals (CIs) were calculated. Over 50 SNPs from different genes have been shown to be associated with either hip (23), or knee (20), or both (13) OA. The ORs of these SNPs for OA and the subtypes are not consistent. As to SNP rs7639618 of DVWA, increased knee OA risk was observed in all genetic models analyzed. Specifically, people from Asian with G-allele showed significantly increased risk of knee OA (A versus G: OR = 1.28, 95% CI 1.13–1.46; AA versus GG: OR = 1.60, 95% CI 1.25–2.05; GA versus GG: OR = 1.31, 95% CI 1.18–1.44; AA versus GA+GG: OR = 1.34, 95% CI 1.12–1.61; AA+GA versus GG: OR = 1.40, 95% CI 1.19–1.64), but not in Caucasians or with hip OA. Our results suggest that multiple SNPs play different roles in the pathogenesis of OA and its subtypes; SNP rs7639618 of DVWA gene is associated with a significantly increased

  1. Advances in targeting cyclic nucleotide phosphodiesterases

    PubMed Central

    Maurice, Donald H.; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C.

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

  2. Advances in targeting cyclic nucleotide phosphodiesterases.

    PubMed

    Maurice, Donald H; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C

    2014-04-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

  3. A working hypothesis on the interdependent genesis of nucleotide bases, protein amino acids, and primitive genetic code

    NASA Astrophysics Data System (ADS)

    Egami, Fujio

    1981-09-01

    In the course of experimental approach to the chemical evolution in the primeval sea, we have found that the main products from formaldehyde and hydroxylamine are glycine, alanine, serine, aspartic acid etc., and the products from glycine and formaldehyde are serine and aspartic acid. Guanine is found in the two-letter genetic codons of all these amino acids. Based upon the finding and taking into consideration the probable synthetic pathways of nucleotide bases and protein amino acids in the course of chemical evolution and a correlation between the two-letter codons and the number of carbon atoms in the carbon skeleton of amino acids, 1 have been led to a working hypothesis on the interdependent genesis of nucleotide bases, protein amino acids, and primitive genetic code as shown in Table I. Protein amino acids can be classified into two groups: Purine Group amino acids and Pyrimidine Group amino acids. Purine bases and Pyrimidine bases are predominant in two-letter codons of amino acids belonging to the former and the latter group respectively. Guanine, adenine, and amino acids of the Purine Group may be regarded as synthesized from C1 and C2 compounds and N1 compounds (including C1N1 compunds such as HCN), probably through glycine, in the early stage of chemical evolution. Uracil, cytosine, and amino acids of the Pyrimidine Group may be regarded as synthesized directly or indirectly from three-carbon chain compounds. This synthesis became possible after the accumulation of three-carbon chain compounds and their derivatives in the primeval sea. The Purine Group can be further classified into a Guanine or (Gly+nC1) Subgroup and an Adenine or (Gly+nC2) Subgroup or simply nC2 Subgroup. The Pyrimidine Group can be further classified into a Uracil or C3C6C9 Subgroup and a Cytosine or C5-chain Subgroup (Table I). It is suggested that the primitive genetic code was established by a specific interaction between amino acids and their respective nucleotide bases. The

  4. Palindromic-nucleotide substitutions (PNS) of hepatitis C virus genotypes 1 and 5a from South Africa.

    PubMed

    Prabdial-Sing, N; Giangaspero, M; Puren, A J; Mahlangu, J; Barrow, P; Bowyer, S M

    2011-08-01

    The HCV stem-loop subdomains III-a, -b and -c have been shown to reflect the characteristics of the virus and identify isolates by genus, genotype and subtype. The aim of this study was to investigate the genotype-specific PNS within the 5'UTR of prevalent HCV genotypes (1 and 5a) found in South Africa. The genotype 5a (N = 35) and genotype 1 sequences (N=20) were from patients presenting with liver disease or haemophilia, respectively. PNS HCV typing characteristics, defined previously, were observed. The PNS method differentiated subtypes 1a and 1c from subtype 1b by the base change at nucleotide position 243. A lack of structural data from the variable loci V1 of the 5'UTR did not allow us to further differentiate the subtypes of 1. A nucleotide change from a thymine (T) to a cytosine (C) at position 183 was found among genotype 5a sequences. This mutation changed the stable U-AA bond to a Y AA bulge at base-pair position 32. There was an insertion of a single adenine (A) at position 207. At present PNS analysis is labour intensive but, with development of further software to aid the computer analysis, it has the potential to provide a rapid, reliable alternative to phylogenetic analysis. PMID:21600241

  5. In vitro incorporation of LNA nucleotides.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    An LNA modified nucleoside triphosphate 1 was synthesized in order to investigate its potential to act as substrate for DNA strand synthesis by polymerases. Primer extension assays for the incorporation experiments revealed that Phusion High Fidelity DNA polymerase is an efficient enzyme for incorporation of the LNA nucleotide and for extending strand to full length. It was also observed that pfu DNA polymerase could incorporate the LNA nucleotide but it failed to extend the strand to a full length product. PMID:18058567

  6. 6-Substituted 2-Aminopurine-2'-deoxyribonucleoside 5'-Triphosphates that Trace Cytosine Methylation.

    PubMed

    von Watzdorf, Janina; Marx, Andreas

    2016-08-17

    Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2'-deoxy 5-methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O(6) -alkylated 2'-deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6-substituted 2-aminopurine-2'-deoxyribonucleoside 5'-triphosphates modified at position 6 with various functionalities. We found that sensing of 5-methylation by this class of nucleotides is more general, not being restricted to O(6) -alkyl modification of dGTP but also applying to other functionalities. PMID:27253512

  7. Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes.

    PubMed Central

    Fournier, M; Haumont, E; de Henau, S; Gangloff, J; Grosjean, H

    1983-01-01

    An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not. Images PMID:6300762

  8. Regulation of mammalian nucleotide metabolism and biosynthesis

    PubMed Central

    Lane, Andrew N.; Fan, Teresa W.-M.

    2015-01-01

    Nucleotides are required for a wide variety of biological processes and are constantly synthesized de novo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer. PMID:25628363

  9. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    SciTech Connect

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; Grimwood, Jane; Grigoriev, Igor V.; Van Etten, James L.; Keeling, Patrick J.

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.

  10. Repair of a Dimeric Azetidine Related to the Thymine-Cytosine (6-4) Photoproduct by Electron Transfer Photoreduction.

    PubMed

    Fraga-Timiraos, Ana B; Lhiaubet-Vallet, Virginie; Miranda, Miguel A

    2016-05-10

    Photolyases are intriguing enzymes that take advantage of sunlight to restore lesions like cyclobutane pyrimidine dimers or (6-4) photoproducts. This work focused on the photoreductive process responsible for splitting of the azetidine ring proposed to occur during (6-4) photoproduct repair at a thymine-cytosine sequence. A model compound formed by photocycloaddition between thymine and 6-azauracil has been designed to mimic the elusive azetidine intermediate. The photoinduced electron transfer process has been investigated by means of steady-state and time-resolved fluorescence using photosensitizers with oxidation potentials in the singlet excited state ranging from -3.3 to -2.1 V vs. SCE. Azetidine ring splitting and recovery of "repaired" bases were proven by HPLC analysis. PMID:27061458

  11. Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine.

    PubMed

    Morla-Folch, Judit; Xie, Hai-nan; Gisbert-Quilis, Patricia; Gómez-de Pedro, Sara; Pazos-Perez, Nicolas; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2015-11-01

    Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. PMID:26447808

  12. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    PubMed

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. PMID:26999445

  13. Functionalized Tricyclic Cytosine Analogues Provide Nucleoside Fluorophores with Improved Photophysical Properties and a Range of Solvent Sensitivities

    PubMed Central

    Rodgers, Brittney J.; Elsharif, Nada A.; Vashisht, Nisha; Mingus, Macy M.; Mulvahill, Mark A.; Stengel, Gudrun; Kuchta, Robert D.

    2014-01-01

    Tricyclic cytosines (tC and tCO frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron donating and electron withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tCO and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment. PMID:24311229

  14. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field

    PubMed Central

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds’ vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. PMID:26999445

  15. Transcriptional similarity in couples reveals the impact of shared environment and lifestyle on gene regulation through modified cytosines.

    PubMed

    Tang, Ke; Zhang, Wei

    2016-01-01

    Gene expression is a complex and quantitative trait that is influenced by both genetic and non-genetic regulators including environmental factors. Evaluating the contribution of environment to gene expression regulation and identifying which genes are more likely to be influenced by environmental factors are important for understanding human complex traits. We hypothesize that by living together as couples, there can be commonly co-regulated genes that may reflect the shared living environment (e.g., diet, indoor air pollutants, behavioral lifestyle). The lymphoblastoid cell lines (LCLs) derived from unrelated couples of African ancestry (YRI, Yoruba people from Ibadan, Nigeria) from the International HapMap Project provided a unique model for us to characterize gene expression pattern in couples by comparing gene expression levels between husbands and wives. Strikingly, 778 genes were found to show much smaller variances in couples than random pairs of individuals at a false discovery rate (FDR) of 5%. Since genetic variation between unrelated family members in a general population is expected to be the same assuming a random-mating society, non-genetic factors (e.g., epigenetic systems) are more likely to be the mediators for the observed transcriptional similarity in couples. We thus evaluated the contribution of modified cytosines to those genes showing transcriptional similarity in couples as well as the relationships these CpG sites with other gene regulatory elements, such as transcription factor binding sites (TFBS). Our findings suggested that transcriptional similarity in couples likely reflected shared common environment partially mediated through cytosine modifications. PMID:27326381

  16. Transcriptional similarity in couples reveals the impact of shared environment and lifestyle on gene regulation through modified cytosines

    PubMed Central

    Tang, Ke

    2016-01-01

    Gene expression is a complex and quantitative trait that is influenced by both genetic and non-genetic regulators including environmental factors. Evaluating the contribution of environment to gene expression regulation and identifying which genes are more likely to be influenced by environmental factors are important for understanding human complex traits. We hypothesize that by living together as couples, there can be commonly co-regulated genes that may reflect the shared living environment (e.g., diet, indoor air pollutants, behavioral lifestyle). The lymphoblastoid cell lines (LCLs) derived from unrelated couples of African ancestry (YRI, Yoruba people from Ibadan, Nigeria) from the International HapMap Project provided a unique model for us to characterize gene expression pattern in couples by comparing gene expression levels between husbands and wives. Strikingly, 778 genes were found to show much smaller variances in couples than random pairs of individuals at a false discovery rate (FDR) of 5%. Since genetic variation between unrelated family members in a general population is expected to be the same assuming a random-mating society, non-genetic factors (e.g., epigenetic systems) are more likely to be the mediators for the observed transcriptional similarity in couples. We thus evaluated the contribution of modified cytosines to those genes showing transcriptional similarity in couples as well as the relationships these CpG sites with other gene regulatory elements, such as transcription factor binding sites (TFBS). Our findings suggested that transcriptional similarity in couples likely reflected shared common environment partially mediated through cytosine modifications. PMID:27326381

  17. Photoinitiator Nucleotide for Quantifying Nucleic Acid Hybridization

    PubMed Central

    Johnson, Leah M.; Hansen, Ryan R.; Urban, Milan; Kuchta, Robert D.; Bowman, Christopher N.

    2010-01-01

    This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sensitive, rapid and visual detection of DNA hybridization events. This approach holds potential for various DNA labeling schemes and for applications benefiting from selective DNA-based polymerization initiators. Here, we demonstrate covalent, enzymatic incorporation of an eosin-photoinitiator 2′-deoxyuridine-5′-triphosphate (EITC-dUTP) conjugate into surface-immobilized DNA hybrids. Subsequent radical chain photoinitiation from these sites using an acrylamide/bis-acrylamide formulation yields a dynamic detection range between 500pM and 50nM of DNA target. Increasing EITC-nucleotide surface densities leads to an increase in surface-based polymer film heights until achieving a film height plateau of 280nm ±20nm at 610 ±70 EITC-nucleotides/μm2. Film heights of 10–20 nm were obtained from eosin surface densities of approximately 20 EITC-nucleotides/μm2 while below the detection limit of ~10 EITC-nucleotides/μm2, no detectable films were formed. This unique threshold behavior is utilized for instrument-free, visual quantification of target DNA concentration ranges. PMID:20337438

  18. Proofreading of misincorporated nucleotides in DNA transcription.

    PubMed

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighboring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction. PMID:22643861

  19. Proofreading of misincorporated nucleotides in DNA transcription.

    PubMed

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighbouring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction. PMID:22551978

  20. Measurements of single nucleotide electronic states as nanoelectronic fingerprints for identification of DNA nucleobases, their protonated and unprotonated states, isomers, and tautomers.

    PubMed

    Ribot, Josep Casamada; Chatterjee, Anushree; Nagpal, Prashant

    2015-04-16

    Several nanoelectronic techniques have been explored to distinguish the sequence of nucleic acids in DNA macromolecules. Identification of unique electronic signatures using nanopore conductance, tunneling spectroscopy, or other nanoelectronic techniques depends on electronic states of the DNA nucleotides. While several experimental and computational studies have focused on interaction of nucleobases with different substrates, the effect of nucleic acid biochemistry on its electronic properties has been largely unexplored. Here, we present correlated measurements of frontier molecular orbitals and higher-order electronic states for four DNA nucleobases (adenine, cytosine, thymine, and guanine), and first-principle quantum chemical density functional theoretical (DFT) computations. Using different pH conditions in our experiments, we show that small changes in the biochemical state of these nucleic acids strongly affect the intrinsic electronic structure, measured using scanning tunneling spectroscopy (STS). In our experimental measurements and computations, significant differences were observed between the position of frontier orbitals and higher-energy states between protonated and unprotonated nucleic acids, isomers, and different keto-enol tautomer's formed in these nucleotides, leading to their facile identification. Furthermore, we show unique "electronic fingerprints" for all nucleotides (A, G, T, C) using STS, with most distinct states identified at acidic pH. These results can have important implications for identification of nucleic acid sequences in DNA molecules using a high-throughput nanoelectronic identification technique. PMID:25793310

  1. Hybridization chain reaction modulated DNA-hosted silver nanoclusters for fluorescent identification of single nucleotide polymorphisms in the let-7 miRNA family.

    PubMed

    Qiu, Xue; Wang, Pei; Cao, Zhijuan

    2014-10-15

    A simple microRNA (miRNA) detection system based on hybridization chain reaction (HCR) has been developed using highly fluorescent DNA-hosted silver (Ag) nanoclusters. In this assay, a new type of hairpin DNA probe (MB1) containing a poly-cytosine nucleotide loop is designed and used as one of the HCR monomers, which is also demonstrated to be an ideal template for in situ synthesis of highly fluorescent Ag nanoclusters. Correspondingly, another HCR monomer (MB2) contains a poly-guanine nucleotide sticky end. Two monomers are stable to coexist in solution until the introduction of the initiator strand (let-7a) triggers a cascade of hybridization events that yields nicked double helices analogous. By taking advantage of HCR, a small amount of let-7a leads to the conformational change of a large amount of MB1, which results in the decrease of fluorescent signal greatly. Overall, this label-free, enzyme-free method allows the sensitive detection of let-7a with high specificity towards single nucleotide polymorphisms in the let-7 miRNA family. In addition, the simple "mix and measure" assay can be extended to detect other types of targets upon slight modification, and thus provides a tool for the early diagnosis and risk assessment of malignancy. PMID:24836018

  2. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    PubMed Central

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  3. Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence.

    PubMed

    Gladyshev, Eugene; Kleckner, Nancy

    2016-05-01

    Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous

  4. Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence

    PubMed Central

    Kleckner, Nancy

    2016-01-01

    Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous

  5. The International Nucleotide Sequence Database Collaboration.

    PubMed

    Cochrane, Guy; Karsch-Mizrachi, Ilene; Takagi, Toshihisa

    2016-01-01

    The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org) comprises three global partners committed to capturing, preserving and providing comprehensive public-domain nucleotide sequence information. The INSDC establishes standards, formats and protocols for data and metadata to make it easier for individuals and organisations to submit their nucleotide data reliably to public archives. This work enables the continuous, global exchange of information about living things. Here we present an update of the INSDC in 2015, including data growth and diversification, new standards and requirements by publishers for authors to submit their data to the public archives. The INSDC serves as a model for data sharing in the life sciences. PMID:26657633

  6. Nucleotide `maps' of digests of deoxyribonucleic acid

    PubMed Central

    Murray, K.

    1970-01-01

    Various digests of 32P-labelled DNA were examined by two-dimensional ionophoresis on cellulose acetate and DEAE-cellulose paper. The products from digestion with pancreatic deoxyribonuclease and Neurospora crassa endonuclease were qualitatively closely similar, but very complex, and were used to investigate the mapping behaviour of nucleotides in various ionophoretic systems. Ionophoresis on DEAE-cellulose paper in triethylamine carbonate, pH 9.7, followed by ionophoresis in the second dimension at pH1.9 gave high resolution of nucleotides in very complex mixtures and permitted the fractionation of larger quantities than is possible on cellulose acetate. High resolution of nucleotides in compact spots was obtained with two-dimensional ionophoresis on cellulose acetate and AE-cellulose paper, a system that is a useful supplement to those based on DEAE-cellulose paper. ImagesPLATE 7PLATE 1PLATE 2PLATE 3PLATE 4PLATE 5PLATE 6 PMID:5476726

  7. Regulation of Ion Channels by Pyridine Nucleotides

    PubMed Central

    Kilfoil, Peter J.; Tipparaju, Srinivas M.; Barski, Oleg A.; Bhatnagar, Aruni

    2014-01-01

    Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion–transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide–binding β-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP+ to Kvβ removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K+ transporters. These nucleotides also have been shown to modify the activity of the plasma membrane KATP channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit—the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP+ metabolite, NAADP+, regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias. PMID:23410881

  8. Nucleotide chloramines and neutrophil-mediated cytotoxicity.

    PubMed

    Bernofsky, C

    1991-03-01

    Hypochlorite is a reactive oxidant formed as an end product of the respiratory burst in activated neutrophils. It is responsible for killing bacteria and has been implicated in neutrophil-mediated tissue injury associated with the inflammatory process. Although hypochlorite is a potent cytotoxic agent, the primary mechanism by which it exerts its effect is unclear. This review examines evidence that the primary event in hypochlorite cytotoxicity is the loss of adenine nucleotides from the target cell. This loss appears to be mediated by the formation of adenine nucleotide chloramines which are reactive intermediates with a free radical character and are capable of forming stable ligands with proteins and nucleic acids. PMID:1848195

  9. Nucleotide sequence of bacteriophage fd DNA.

    PubMed Central

    Beck, E; Sommer, R; Auerswald, E A; Kurz, C; Zink, B; Osterburg, G; Schaller, H; Sugimoto, K; Sugisaki, H; Okamoto, T; Takanami, M

    1978-01-01

    The sequence of the 6,408 nucleotides of bacteriophage fd DNA has been determined. This allows to deduce the exact organisation of the filamentous phage genome and provides easy access to DNA segments of known structure and function. PMID:745987

  10. Nucleotide-metabolizing enzymes in Chlamydomonas flagella.

    PubMed

    Watanabe, T; Flavin, M

    1976-01-10

    Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts. PMID:397

  11. Cyclic nucleotide phosphodiesterase 1 and vascular aging.

    PubMed

    Yan, Chen

    2015-12-01

    VSMCs (vascular smooth muscle cells) play critical roles in arterial remodelling with aging, hypertension and atherosclerosis. VSMCs exist in diverse phenotypes and exhibit phenotypic plasticity, e.g. changing from a quiescent/contractile phenotype to an active myofibroblast-like, often called 'synthetic', phenotype. Synthetic VSMCs are able to proliferate, migrate and secrete ECM (extracellular matrix) proteinases and ECM proteins. In addition, they produce pro-inflammatory molecules, providing an inflammatory microenvironment for leucocyte penetration, accumulation and activation. The aging VSMCs have also shown changes in cellular phenotype, responsiveness to contracting and relaxing mediators, replicating potential, matrix synthesis, inflammatory mediators and intracellular signalling. VSMC dysfunction plays a key role in age-associated vascular remodelling. Cyclic nucleotide PDEs (phosphodiesterases), by catalysing cyclic nucleotide hydrolysis, play a critical role in regulating the amplitude, duration and compartmentalization of cyclic nucleotide signalling. Abnormal alterations of PDEs and subsequent changes in cyclic nucleotide homoeostasis have been implicated in a number of different diseases. In the study published in the latest issue of Clinical Science, Bautista Niño and colleagues have shown that, in cultured senescent human VSMCs, PDE1A and PDE1C mRNA levels are significantly up-regulated and inhibition of PDE1 activity with vinpocetine reduced cellular senescent makers in senescent VSMCs. Moreover, in the premature aging mice with genomic instability (Ercc1(d/-)), impaired aortic ring relaxation in response to SNP (sodium nitroprusside), an NO (nitric oxide) donor, was also largely improved by vinpocetine. More interestingly, using data from human GWAS (genome-wide association studies), it has been found that PDE1A single nucleotide polymorphisms is significantly associated with diastolic blood pressure and carotid intima-media thickening, two

  12. Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation.

    PubMed

    Lavania, Umesh C; Srivastava, Sarita; Lavania, Seshu; Basu, Surochita; Misra, Nandeesh Kumar; Mukai, Yasuhiko

    2012-08-01

    Whole genome duplication leads to autopolyploidy and brings about an increase in cell size, concentration of secondary metabolites and enhanced cytosine methylation. The increased cell size offers a positive advantage to polyploids for cell-surface-related activities, but there is a differential response to change in body size across species and taxonomic groups. Although polyploidy has been very extensively studied, having genetic, ecological and evolutionary implications, there is no report that underscores the significance of native secondary metabolites vis-à-vis body size with ploidy change. To address this problem we targeted unique diploid-autotetraploid paired sets of eight diverse clones of six species of Cymbopogon- a species complex of aromatic grasses that accumulate qualitatively different monoterpene essential oils (secondary metabolite) in their vegetative biomass. Based on the qualitative composition of essential oils and the plant body size relationship between the diploid versus autotetraploid paired sets, we show that polyploidy brings about enhanced accumulation of secondary metabolites in all cases, but exerts differential effects on body size in various species. It is observed that the accumulation of alcohol-type metabolites (e.g. geraniol) does not inhibit increase in body size with ploidy change from 2× to 4× (r = 0.854, P < 0.01), but aldehyde-type metabolites (e.g. citral) appear to drastically impede body development (r = -0.895). Such a differential response may be correlated to the metabolic steps involved in the synthesis of essential oil components. When changed to tetraploidy, the progenitor diploids requiring longer metabolic steps in production of their secondary metabolites are stressed, and those having shorter metabolite routes better utilize their resources for growth and vigour. In situ immunodetection of 5-methylcytosine sites reveals enhanced DNA methylation in autopolyploids. It is underpinned that the qualitative

  13. Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Shanak, Siba; Helms, Volkhard

    2014-12-01

    Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

  14. Comparative Transcriptomics of H. pylori Strains AM5, SS1 and Their hpyAVIBM Deletion Mutants: Possible Roles of Cytosine Methylation

    PubMed Central

    Kumar, Ritesh; Mukhopadhyay, Asish K.; Ghosh, Prachetash; Rao, Desirazu N.

    2012-01-01

    Helicobacter pylori is an important human pathogen and one of the most successful chronic colonizers of the human body. H. pylori uses diverse mechanisms to modulate its interaction with the host in order to promote chronic infection and overcome host immune response. Restriction-modification genes are a major part of strain-specific genes present in H. pylori. The role of N6 - adenine methylation in bacterial gene regulation and virulence is well established but not much is known about the effect of C5 -cytosine methylation on gene expression in prokaryotes. In this study, it was observed by microarray analysis and RT-PCR, that deletion of an orphan C5 -cytosine methyltransferase, hpyAVIBM in H. pylori strains AM5and SS1 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. AM5ΔhpyAVIBM mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. hpyAVIBM from strain 26695 is able to complement mutant SS1 and AM5 strains. This study highlights a possible significance of cytosine methylation in the physiology of H. pylori. PMID:22879937

  15. Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine.

    PubMed

    Fox, B A; Belperron, A A; Bzik, D J

    1999-01-01

    To improve genetic models available for the analysis of apicomplexan protozoan parasites, bacterial sequences encoding the 427 amino acid cytosine deaminase (CD) gene were fused, in-frame, to an engineered linker domain of the high level pyrimethamine resistant form of the parasite bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Toxoplasma gondii was transformed with the plasmid containing the fused pyrimethamine resistant dihydrofolate reductase-cytosine deaminase-thymidylate synthase (DHFRm2m3-CD-TS) gene and parasites were selected in a high level of pyrimethamine. Transfected parasites that acquired resistance to pyrimethamine were cloned and evaluated for expression of the CD genetic marker. CD transgenic parasites acquired a high sensitivity to 5-fluorocytosine due to the intraparasitic conversion of this non-toxic prodrug to the cytotoxic compound 5-fluorouracil. Exogenously supplied cytosine or uracil rescued the growth of CD transgenic T. gondii parasites that were cultured in the presence of cytotoxic concentrations of 5-fluorouracil or 5-fluorocytosine. Bacterial CD fused to the pyrimethamine resistant DHFR-TS marker provides a novel genetic tool for new positive and negative genetic selection strategies in several protozoan parasites. An advantage of the CD genetic marker is that it is derived from a bacterial gene and can therefore be used in nearly any parasite genetic background for negative selection. This novel system should facilitate new approaches for the development of improved model genetic systems for the biological investigation of apicomplexan parasites. PMID:10029312

  16. A single nucleotide polymorphism in NEUROD1 is associated with production traits in Nelore beef cattle.

    PubMed

    de Oliveira, P S N; Tizioto, P C; Malago, W; do Nascimento, M L; Cesar, A S M; Diniz, W J S; de Souza, M M; Lanna, D P D; Tullio, R R; Mourão, G B; de A Mudadu, M; Coutinho, L L; de A Regitano, L C

    2016-01-01

    Feed efficiency and carcass characteristics are late-measured traits. The detection of molecular markers associated with them can help breeding programs to select animals early in life, and to predict breeding values with high accuracy. The objective of this study was to identify polymorphisms in the functional and positional candidate gene NEUROD1 (neurogenic differentiation 1), and investigate their associations with production traits in reference families of Nelore cattle. A total of 585 steers were used, from 34 sires chosen to represent the variability of this breed. By sequencing 14 animals with extreme residual feed intake (RFI) values, seven single nucleotide polymorphisms (SNPs) in NEUROD1 were identified. The investigation of marker effects on the target traits RFI, backfat thickness (BFT), ribeye area (REA), average body weight (ABW), and metabolic body weight (MBW) was performed with a mixed model using the restricted maximum likelihood method. SNP1062, which changes cytosine for guanine, had no significant association with RFI or REA. However, we found an additive effect on ABW (P ≤ 0.05) and MBW (P ≤ 0.05), with an estimated allele substitution effect of -1.59 and -0.93 kg0.75, respectively. A dominant effect of this SNP for BFT was also found (P ≤ 0.010). Our results are the first that identify NEUROD1 as a candidate that affects BFT, ABW, and MBW. Once confirmed, the inclusion of this SNP in dense panels may improve the accuracy of genomic selection for these traits in Nelore beef cattle as this SNP is not currently represented on SNP chips. PMID:27420997

  17. Formation of Amino Acids and Nucleotide Bases in a Titan Atmosphere Simulation Experiment

    PubMed Central

    Yelle, R.V.; Buch, A.; Carrasco, N.; Cernogora, G.; Dutuit, O.; Quirico, E.; Sciamma-O'Brien, E.; Smith, M.A.; Somogyi, Á.; Szopa, C.; Thissen, R.; Vuitton, V.

    2012-01-01

    Abstract The discovery of large (>100 u) molecules in Titan's upper atmosphere has heightened astrobiological interest in this unique satellite. In particular, complex organic aerosols produced in atmospheres containing C, N, O, and H, like that of Titan, could be a source of prebiotic molecules. In this work, aerosols produced in a Titan atmosphere simulation experiment with enhanced CO (N2/CH4/CO gas mixtures of 96.2%/2.0%/1.8% and 93.2%/5.0%/1.8%) were found to contain 18 molecules with molecular formulae that correspond to biological amino acids and nucleotide bases. Very high-resolution mass spectrometry of isotopically labeled samples confirmed that C4H5N3O, C4H4N2O2, C5H6N2O2, C5H5N5, and C6H9N3O2 are produced by chemistry in the simulation chamber. Gas chromatography–mass spectrometry (GC-MS) analyses of the non-isotopic samples confirmed the presence of cytosine (C4H5N3O), uracil (C5H4N2O2), thymine (C5H6N2O2), guanine (C5H5N5O), glycine (C2H5NO2), and alanine (C3H7NO2). Adenine (C5H5N5) was detected by GC-MS in isotopically labeled samples. The remaining prebiotic molecules were detected in unlabeled samples only and may have been affected by contamination in the chamber. These results demonstrate that prebiotic molecules can be formed by the high-energy chemistry similar to that which occurs in planetary upper atmospheres and therefore identifies a new source of prebiotic material, potentially increasing the range of planets where life could begin. Key Words: Astrochemistry—Planetary atmospheres—Titan—Astrobiology. Astrobiology 12, 809–817. PMID:22917035

  18. [Mesenchymal stem cells expressing cytosine deaminase inhibit growth of murine melanoma B16F10 in vivo].

    PubMed

    Krasikova, L S; Karshieva, S S; Cheglakov, I B; Belyavsky, A V

    2015-01-01

    The aim of this study was to estimate the efficacy of mesenchymal stem cell-based suicide gene therapy in mice bearing murine melanoma B16F10. Adipose mesenchymal stem cells (MSCs) were transfected with plasmid constructs expressing cytosine deaminase fused with uracil phosphoribosyltransferase (CDA/UPRT) or CDA/UPRT fused with HSV-1 tegument protein VP22 (CDA/UPRT/VP22). In this study, we demonstrate that direct intratumoral transplantation of MSCs expressing CDA/UPRT or CDA/UPRT/VP22 followed by systemic administration of 5-fluorocytosine (5-FC) results in a significant inhibition of tumor growth. There was a 53% reduction in tumor volume in mice treated with CDA/UPRT-MSCs and 58% reduction in tumor volume in mice treated with CDA/UPRT/VP22-MSCs as compared with control animals transplanted with B16F10 melanoma alone. Injection of CDA/UPRT-MSC and CDA/UPRT/VP22-MSC prolonged the life span of mice bearing B16F10 melanoma by 15 and 26%, respectively. The data indicate that in murine B16F10 melanoma model, MSCs encoding CDA/UPRT suicide gene have a significant antitumor effect. PMID:26710783

  19. Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

    SciTech Connect

    Zhang, Jufeng; Wang, Zhanli; Wei, Fang; Qiu, Wei; Zhang, Liangren; Huang, Qian . E-mail: qhuang@sjtu.edu.cn

    2007-08-17

    Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.

  20. Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons.

    PubMed Central

    Ishitani, R; Chuang, D M

    1996-01-01

    Cytosine arabinonucleoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis and, importantly, is also an inducer of apoptosis. We recently reported that age-induced apoptotic cell death of cultured cerebellar neurons is directly associated with an over-expression of a particulate 38-kDa protein, identified by us as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). We now show that the AraC-induced neuronal death of immature cerebellar granule cells in culture is effectively delayed by actinomycin-D, cycloheximide, or aurintricarboxylic acid (a DNase inhibitor). Furthermore, two GAPDH antisense, but not their corresponding sense, oligodeoxyribonucleotides markedly arrested AraC-induced apoptosis. This protection was more effective than that induced by the above-mentioned classical inhibitors of apoptosis. Prior to AraC-induced neuronal death, GAPDH mRNA levels increased by approximately 2.5-fold, and this mRNA accumulation was blocked by actinomycin-D and the GAPDH antisense (but not sense) oligonucleotide. Like actinomycin-D, a GAPDH antisense oligonucleotide also suppressed the AraC-induced over-expression of the 38-kDa particulate protein (i.e., GAPDH), while the corresponding sense oligonucleotide was totally ineffective. Thus, the present results show that GAPDH over-expression is involved in AraC-induced apoptosis of cultured cerebellar granule cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:8790435

  1. (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, a potent and selective inhibitor of human cytomegalovirus replication.

    PubMed Central

    Snoeck, R; Sakuma, T; De Clercq, E; Rosenberg, I; Holy, A

    1988-01-01

    From a series of phosphonylmethoxyalkylpurine and -pyrimidine derivatives, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC] emerged as a particularly potent and selective inhibitor of the replication of human cytomegalovirus (CMV). Its potency against CMV was similar to that of the structurally related adenine derivative (S)-HPMPA but higher than that of the reference compounds phosphonoformate and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG). The minimum concentrations of phosphonoformate, DHPG, (S)-HPMPA, and (S)-HPMPC required to inhibit CMV plaque formation by 50% were 15, 0.7, 0.1, and 0.07 microgram/ml, respectively. The selectivity indices of phosphonoformate, DHPG, (S)-HPMPA, and (S)-HPMPC, as determined by the ratio of the 50% inhibitory concentration for cell growth to the 50% inhibitory concentration for plaque formation for CMV (AD-169 strain), were 14, 150, 200 and 1,500, respectively. Corresponding values for the CMV Davis strain were 20, 200, 100, and 1,000, respectively. (S)-HPMPC was inhibitory to CMV plaque formation even when added to the cells at 24 or 48 h postinfection. When (S)-HPMPC was added immediately postinfection, a 24- or 48-h incubation time sufficed to obtain a marked inhibitory effect on CMV replication. Such limited incubation time was insufficient for DHPG to achieve any protection against CMV. PMID:2854454

  2. Genomic Change, Retrotransposon Mobilization and Extensive Cytosine Methylation Alteration in Brassica napus Introgressions from Two Intertribal Hybridizations

    PubMed Central

    Zhang, Xueli; Ge, Xianhong; Shao, Yujiao; Sun, Genlou; Li, Zaiyun

    2013-01-01

    Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4–39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed. PMID:23468861

  3. The efficacy and adverse event profile of dexamethasone, melphalan, actinomycin D, and cytosine arabinoside (DMAC) chemotherapy in relapsed canine lymphoma

    PubMed Central

    Parsons-Doherty, Melissa; Poirier, Valerie J.; Monteith, Gabrielle

    2014-01-01

    In this retrospective study, a chemotherapy protocol using dexamethasone, melphalan, actinomycin D, and cytosine arabinoside (DMAC) was evaluated for efficacy and adverse event profile as a first line rescue protocol in 86 client-owned dogs previously treated with a CHOP-based protocol. Forty-three dogs (43%) achieved remission (16% complete remission, 27% partial remission), and 57% were non-responders. The median overall progression-free survival (PFS) was 24 days. Adverse events included thrombocytopenia in 41% of dogs, neutropenia in 17% of dogs, and gastrointestinal toxicity in 13% of dogs. Overall, 16% (13/79) dogs experienced grade III to IV thrombocytopenia, 8% (6/74) dogs grade III to IV neutropenia and 1% (1/79) dogs grade III to IV gastrointestinal toxicity. The efficacy of the DMAC protocol is similar to that of other rescue protocols in dogs with relapsed lymphoma but is associated with shorter PFS. The main toxicity is thrombocytopenia, which may limit treatment. PMID:24489398

  4. Cytosine Arabinoside and Mitoxantrone Followed by Second Allogeneic Transplant for the Treatment of Children With Refractory Juvenile Myelomonocytic Leukemia

    PubMed Central

    Patel, Sachit A.; Grovas, Alfred C.; Gordon, Bruce G.; Harper, James L.; Warkentin, Phyllis I.; Wisecarver, James L.; Sanger, Warren G.; Coccia, Peter F.

    2014-01-01

    Hematopoietic stem cell transplantation (HSCT) remains the only curative option for most patients with juvenile myelomonocytic leukemia (JMML). However, persistent disease and relapse rates after transplant range from 26% to 58%. We report the successful use of second HSCT after preparation with mitoxantrone and cytosine arabinoside (Ara-C) for patients with refractory or recurrent disease. Between 1993 and 2006, 5 children who underwent HSCT at our institution as initial therapy for JMML had persistent disease or relapsed. Pre-HSCT conditioning varied and donors were either HLA-matched siblings (n=2) or matched unrelated donors (n=3). After initial HSCT, they subsequently received high-dose Ara-C (3 g/m2 IV) every 12 hours on days −8 through −3 and mitoxantrone (10 mg/m2/d IV) on days −8, −7, −6 followed by second HSCT from their original donors. All 5 patients are alive at 88, 179, 199, 234, and 246 months with no evidence of JMML, no significant toxicity, and 100% donor chimera as determined by PCR short-tandem repeat analysis. Our experience supports second transplant utilizing high-dose Ara-C and mitoxantrone in children with JMML who do not respond or relapse after first transplant. PMID:24322499

  5. Bivalent Regions of Cytosine Methylation and H3K27 Acetylation Suggest an Active Role for DNA Methylation at Enhancers.

    PubMed

    Charlet, Jessica; Duymich, Christopher E; Lay, Fides D; Mundbjerg, Kamilla; Dalsgaard Sørensen, Karina; Liang, Gangning; Jones, Peter A

    2016-05-01

    The role of cytosine methylation in the structure and function of enhancers is not well understood. In this study, we investigate the role of DNA methylation at enhancers by comparing the epigenomes of the HCT116 cell line and its highly demethylated derivative, DKO1. Unlike promoters, a portion of regular and super- or stretch enhancers show active H3K27ac marks co-existing with extensive DNA methylation, demonstrating the unexpected presence of bivalent chromatin in both cultured and uncultured cells. Furthermore, our findings also show that bivalent regions have fewer nucleosome-depleted regions and transcription factor-binding sites than monovalent regions. Reduction of DNA methylation genetically or pharmacologically leads to a decrease of the H3K27ac mark. Thus, DNA methylation plays an unexpected dual role at enhancer regions, being anti-correlated focally at transcription factor-binding sites but positively correlated globally with the active H3K27ac mark to ensure structural enhancer integrity. PMID:27153539

  6. The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase.

    PubMed

    Chang, Da-Young; Yoo, Seung-Wan; Hong, Youngtae; Kim, Sujeong; Kim, Se Joong; Yoon, Sung-Hwa; Cho, Kyung-Gi; Paek, Sun Ha; Lee, Young-Don; Kim, Sung-Soo; Suh-Kim, Haeyoung

    2010-10-15

    Suicide genes have recently emerged as an attractive alternative therapy for the treatment of various types of intractable cancers. The efficacy of suicide gene therapy relies on efficient gene delivery to target tissues and the localized concentration of final gene products. Here, we showed a potential ex vivo therapy that used mesenchymal stem cells (MSCs) as cellular vehicles to deliver a bacterial suicide gene, cytosine deaminase (CD) to brain tumors. MSCs were engineered to produce CD enzymes at various levels using different promoters. When co-cultured, CD-expressing MSCs had a bystander, anti-cancer effect on neighboring C6 glioma cells in proportion to the levels of CD enzymes that could convert a nontoxic prodrug, 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) in vitro. Consistent with the in vitro results, for early stage brain tumors induced by intracranial inoculation of C6 cells, transplantation of CD-expressing MSCs reduced tumor mass in proportion to 5-FC dosages. However, for later stage, established tumors, a single treatment was insufficient, but only multiple transplantations were able to successfully repress tumor growth. Our findings indicate that the level of total CD enzyme activity is a critical parameter that is likely to affect the clinical efficacy for CD gene therapy. Our results also highlight the potential advantages of autograftable MSCs compared with other types of allogeneic stem cells for the treatment of recurrent glioblastomas through repetitive treatments. PMID:20473873

  7. How the CCA-Adding Enzyme Selects Adenine over Cytosine at Position 76 of tRNA

    SciTech Connect

    B Pan; Y Xiong; T Steitz

    2011-12-31

    CCA-adding enzymes [ATP(CTP):tRNA nucleotidyltransferases] add CCA onto the 3' end of transfer RNA (tRNA) precursors without using a nucleic acid template. Although the mechanism by which cytosine (C) is selected at position 75 of tRNA has been established, the mechanism by which adenine (A) is selected at position 76 remains elusive. Here, we report five cocrystal structures of the enzyme complexed with both a tRNA mimic and nucleoside triphosphates under catalytically active conditions. These structures suggest that adenosine 5'-monophosphate is incorporated onto the A76 position of the tRNA via a carboxylate-assisted, one-metal-ion mechanism with aspartate 110 functioning as a general base. The discrimination against incorporation of cytidine 5'-triphosphate (CTP) at position 76 arises from improper placement of the {alpha} phosphate of the incoming CTP, which results from the interaction of C with arginine 224 and prevents the nucleophilic attack by the 3' hydroxyl group of cytidine75.

  8. Thermodynamic Potential for the Abiotic Synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in Hydrothermal Systems

    NASA Astrophysics Data System (ADS)

    Larowe, Douglas E.; Regnier, Pierre

    2008-10-01

    The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine, and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics of biomolecule synthesis were computed using the concentrations of aqueous N2, CO, CO2 and H2 reported in the modern Rainbow hydrothermal system. The concentrations of precursor molecules that can be synthesized are strongly dependent on temperature with larger concentrations prevailing at lower temperatures. Similarly, the thermodynamic drive to synthesize nucleobases, ribose and deoxyribose varies considerably as a function of temperature: all of the biomolecules considered in this study are thermodynamically favored to be synthesized throughout the temperature range from 0°C to between 150°C and 250°C, depending on the biomolecule. Furthermore, activity diagrams have been generated to illustrate that activities in the range of 10-2- 10-6 for nucleobases, ribose and deoxyribose can be in equilibrium with a range of precursor molecule activities at 150°C and 500 bars. The results presented here support the notion that hydrothermal systems could have played a fundamental role in the origin of life, and can be used to plan and constrain experimental investigation of the abiotic synthesis of nucleic-acid related biomolecules.

  9. Grid selection of models of nucleotide substitution

    PubMed Central

    Loureiro, Marta; Pan, Miguel; Rodríguez-Pascual, Manuel; Posada, David; Mayo, Rafael

    2016-01-01

    jModelTest is a Java program for the statistical selection of models of nucleotide substitution with thousands of users around the world. For large data sets, the calculations carried out by this program can be too expensive for many users. Here we describe the port of the jModeltest code for Grid computing using DRMAA. This work should facilitate the use of jModelTest on a broad scale. PMID:20543444

  10. The multiple codes of nucleotide sequences.

    PubMed

    Trifonov, E N

    1989-01-01

    Nucleotide sequences carry genetic information of many different kinds, not just instructions for protein synthesis (triplet code). Several codes of nucleotide sequences are discussed including: (1) the translation framing code, responsible for correct triplet counting by the ribosome during protein synthesis; (2) the chromatin code, which provides instructions on appropriate placement of nucleosomes along the DNA molecules and their spatial arrangement; (3) a putative loop code for single-stranded RNA-protein interactions. The codes are degenerate and corresponding messages are not only interspersed but actually overlap, so that some nucleotides belong to several messages simultaneously. Tandemly repeated sequences frequently considered as functionless "junk" are found to be grouped into certain classes of repeat unit lengths. This indicates some functional involvement of these sequences. A hypothesis is formulated according to which the tandem repeats are given the role of weak enhancer-silencers that modulate, in a copy number-dependent way, the expression of proximal genes. Fast amplification and elimination of the repeats provides an attractive mechanism of species adaptation to a rapidly changing environment. PMID:2673451

  11. Vacuum ultraviolet photoionization of carbohydrates and nucleotides.

    PubMed

    Shin, Joong-Won; Bernstein, Elliot R

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5(')-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C-C and C-O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results. PMID:25669546

  12. Evolution of functional six-nucleotide DNA.

    PubMed

    Zhang, Liqin; Yang, Zunyi; Sefah, Kwame; Bradley, Kevin M; Hoshika, Shuichi; Kim, Myong-Jung; Kim, Hyo-Joong; Zhu, Guizhi; Jiménez, Elizabeth; Cansiz, Sena; Teng, I-Ting; Champanhac, Carole; McLendon, Christopher; Liu, Chen; Zhang, Wen; Gerloff, Dietlind L; Huang, Zhen; Tan, Weihong; Benner, Steven A

    2015-06-01

    Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries. PMID:25966323

  13. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    SciTech Connect

    Shin, Joong-Won; Bernstein, Elliot R.

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  14. Visualization of cyclic nucleotide dynamics in neurons

    PubMed Central

    Gorshkov, Kirill; Zhang, Jin

    2014-01-01

    The second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) transduce many neuromodulatory signals from hormones and neurotransmitters into specific functional outputs. Their production, degradation and signaling are spatiotemporally regulated to achieve high specificity in signal transduction. The development of genetically encodable fluorescent biosensors has provided researchers with useful tools to study these versatile second messengers and their downstream effectors with unparalleled spatial and temporal resolution in cultured cells and living animals. In this review, we introduce the general design of these fluorescent biosensors and describe several of them in more detail. Then we discuss a few examples of using cyclic nucleotide fluorescent biosensors to study regulation of neuronal function and finish with a discussion of advances in the field. Although there has been significant progress made in understanding how the specific signaling of cyclic nucleotide second messengers is achieved, the mechanistic details in complex cell types like neurons are only just beginning to surface. Current and future fluorescent protein reporters will be essential to elucidate the role of cyclic nucleotide signaling dynamics in the functions of individual neurons and their networks. PMID:25538560

  15. Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS).

    PubMed

    Kim, Yun-Gon; Park, Hyung-Yeon; Yoo, Dongwon; Sung, Changmin; Song, Eunjung; Lee, Jae-Hun; Choi, Yun-Hui; Kim, Yong-Hyun; Lee, Chang-Soo; Park, Kyungmoon; Kim, Byung-Gee; Yang, Yung-Hun

    2012-04-15

    Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases. PMID:22459405

  16. Evolution of Functional Six-Nucleotide DNA

    PubMed Central

    Zhang, Liqin; Yang, Zunyi; Sefah, Kwame; Bradley, Kevin M.; Hoshika, Shuichi; Kim, Myong-Jung; Kim, Hyo-Joong; Zhu1, Guizhi; Jiménez, Elizabeth; Cansiz, Sena; Teng, I-Ting; Champanhac, Carole; McLendon, Christopher; Liu, Chen; Zhang, Wen; Gerloff, Dietlind L.

    2015-01-01

    Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add additional functional groups, not found in natural DNA but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via strict Watson-Crick geometry. These were added to lies in a ibrarlaboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection systems, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Z’s and P’s. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system, and that GACTZP libraries are richer reservoir of functionality than standard libraries. PMID:25966323

  17. Effect of a constant rate infusion of cytosine arabinoside on mortality in dogs with meningoencephalitis of unknown origin.

    PubMed

    Lowrie, M; Thomson, S; Smith, P; Garosi, L

    2016-07-01

    Administration of cytosine arabinoside (CA) by continuous rate infusion (CRI) has pharmacokinetic and pharmacodynamic advantages over traditional intermittent dosing. Whether these advantages translate into clinical efficacy remains unknown. The aim of this study was to assess the efficacy and safety of CRI of CA in dogs with meningoencephalitis of unknown origin (MUO) and to compare outcomes with a group of historical control dogs treated with conventional intermittent subcutaneous (SC) administration of CA; both groups received adjunctive prednisolone. It was hypothesised that a CRI of CA for 24 h at 100 mg/m(2) would improve survival and lesion resolution compared with conventional SC delivery of 50 mg/m(2) every 12 h for 48 h. Eighty dogs with suspected MUO were recruited from consecutive dogs presenting with suspected MUO from 2006 to 2015. All dogs underwent routine clinical evaluation, magnetic resonance imaging of the brain and cerebrospinal fluid analysis. There were 39 dogs in the SC group and 41 dogs in the CRI group; baseline characteristics were similar in both groups. Survival at 3 months was 22/39 (44%) with SC delivery versus 37/41 (90%) with CRI. No dose-limiting toxicities were noted for either group. The resolution rate of magnetic resonance imaging and cerebrospinal fluid abnormalities at the 3 month re-examination were substantially improved in the CRI group versus the SC group. The CRI regimen produced a survival advantage over the SC route of administration without clinically significant toxicity. These data supports the routine use of CRI at first presentation for the treatment of MUO in dogs. PMID:27240905

  18. Comparison of the Antiviral Effects of 5-Methoxymethyl-deoxyuridine with 5-Iododeoxyuridine, Cytosine Arabinoside, and Adenine Arabinoside

    PubMed Central

    Babiuk, Lorne A.; Meldrum, Blair; Gupta, V. Sagar; Rouse, Barry T.

    1975-01-01

    The antiviral activity of 5-methoxymethyl-2′-deoxyuridine (MMUdR) was compared with that of 5-iodo-2′-deoxyuridine (IUdR), cytosine arabinoside (Ara-C), and adenine arabinoside (Ara-A). At concentrations of 2 to 4 μg/ml, MMUdR was inhibitory to herpes simplex virus type 1, but concentrations as high as 128 μg/ml were not inhibitory to three other herpesviruses tested (equine rhinopneumonitis virus, murine cytomegalovirus, and feline rhinopneumonitis virus) or to vaccinia virus. The other nucleosides, in contrast, were inhibitory at similar concentrations (1 to 8 μg/ml) against all viruses tested. The inhibition of HSV-1 by MMUdR appeared to be the result of interference with virus replication rather than the result of drug toxicity to host cells. The drug was not toxic to host cells at 100 times the antiviral concentrations, and pretreatment of host cells with high concentrations of MMUdR had no effect on subsequent virus replication. Combination of MMUdR with either IUdR, Ara-A, or Ara-C gave an enhanced antiviral effect, suggesting that the mechanism of action of MMUdR is different from that of the other three drugs. Antiviral indexes were calculated for each compound and were found to be >250, 80, 40, and 8 for MMUdR, IUdR, Ara-A, and Ara-C, respectively. These were defined as the minimum dose at which toxicity was observed microscopically divided by the dose which reduced plaque numbers by 50%. PMID:1239978

  19. Theoretical study of the protonation of the one-electron-reduced guanine-cytosine base pair by water.

    PubMed

    Hsu, Sodio C N; Wang, Tzu-Pin; Kao, Chai-Lin; Chen, Hui-Fen; Yang, Po-Yu; Chen, Hsing-Yin

    2013-02-21

    Prototropic equilibria in ionized DNA play an important role in charge transport and radiation damage of DNA and, therefore, continue to attract considerable attention. Although it is well-established that electron attachment will induce an interbase proton transfer from N1 of guanine (G) to N3 of cytosine (C), the question of whether the surrounding water in the major and minor grooves can protonate the one-electron-reduced G:C base pair still remains open. In this work, density functional theory (DFT) calculations were employed to investigate the energetics and mechanism for the protonation of the one-electron-reduced G:C base pair by water. Through the calculations of thermochemical cycles, the protonation free energies were estimated to be in the range of 11.6-14.2 kcal/mol. The calculations for the models of C(•-)(H(2)O)(8) and G(-H1)(-)(H(2)O)(16), which were used to simulate the detailed processes of protonation by water before and after the interbase proton transfer, respectively, revealed that the protonation proceeds through a concerted double proton transfer involving the water molecules in the first and second hydration shells. Comparing the present results with the rates of interbase proton transfer and charge transfer along DNA suggests that protonation on the C(•-) moiety is not competitive with interbase proton transfer, but the possibility of protonation on the G(-H1)(-) moiety after interbase proton transfer cannot be excluded. Electronic-excited-state calculations were also carried out by the time-dependent DFT approach. This information is valuable for experimental identification in the future. PMID:23363248

  20. Cyclopentenyl cytosine induces senescence in breast cancer cells through the nucleolar stress response and activation of p53.

    PubMed

    Huang, Min; Whang, Patrick; Lewicki, Patrick; Mitchell, Beverly S

    2011-07-01

    The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated β-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G(2)- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G(1) phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death. PMID:21464199

  1. Phase 1 and pharmacokinetic study of bolus-infusion flavopiridol followed by cytosine arabinoside and mitoxantrone for acute leukemias

    PubMed Central

    Smith, B. Douglas; Resar, Linda S.; Greer, Jacqueline M.; Blackford, Amanda; Zhao, Ming; Moton-Nelson, Dwella; Alino, Katrina; Levis, Mark J.; Gore, Steven D.; Joseph, Biju; Carraway, Hetty; McDevitt, Michael A.; Bagain, Lorena; Mackey, Karen; Briel, Janet; Doyle, L. Austin; Wright, John J.; Rudek, Michelle A.

    2011-01-01

    Flavopiridol is a protein bound, cytotoxic, cyclin-dependent kinase inhibitor. Flavopiridol given by 1-hour bolus at 50 mg/m2 daily 3 times followed by cytosine arabinoside and mitoxantrone (FLAM) is active in adults with poor-risk acute leukemias. A pharmacologically derived “hybrid” schedule (30-minute bolus followed by 4-hour infusion) of flavopiridol was more effective than bolus administration in refractory chronic lymphocytic leukemia. Our phase 1 trial “hybrid FLAM” in 55 adults with relapsed/refractory acute leukemias began at a total flavopiridol dose of 50 mg/m2 per day 3 times (20-mg/m2 bolus, 30-mg/m2 infusion). Dose-limiting toxicity occurred at level 6 (30-mg/m2 bolus, 70-mg/m2 infusion) with tumor lysis, hyperbilirubinemia, and mucositis. Death occurred in 5 patients (9%). Complete remission occurred in 22 (40%) across all doses. Overall and disease-free survivals for complete remission patients are more than 60% at more than 2 years. Pharmacokinetics demonstrated a dose-response for total and unbound plasma flavopiridol unrelated to total protein, albumin, peripheral blast count, or toxicity. Pharmacodynamically, flavopiridol inhibited mRNAs of multiple cell cycle regulators, but with uniform increases in bcl-2. “Hybrid FLAM” is active in relapsed/refractory acute leukemias, with a recommended “hybrid” dose of bolus 30 mg/m2 followed by infusion of 60 mg/m2 daily for 3 days. This clinical trial is registered at www.clinicaltrials.gov as #NCT00470197. PMID:21239698

  2. Infection with a Virulent Strain of Wolbachia Disrupts Genome Wide-Patterns of Cytosine Methylation in the Mosquito Aedes aegypti

    PubMed Central

    Ye, Yixin H.; Woolfit, Megan; Huttley, Gavin A.; Rancès, Edwige; Caragata, Eric P.; Popovici, Jean; O'Neill, Scott L.; McGraw, Elizabeth A.

    2013-01-01

    Background Cytosine methylation is one of several reversible epigenetic modifications of DNA that allow a greater flexibility in the relationship between genotype and phenotype. Methylation in the simplest models dampens gene expression by modifying regions of DNA critical for transcription factor binding. The capacity to methylate DNA is variable in the insects due to diverse histories of gene loss and duplication of DNA methylases. Mosquitoes like Drosophila melanogaster possess only a single methylase, DNMT2. Description Here we characterise the methylome of the mosquito Aedes aegypti and examine its relationship to transcription and test the effects of infection with a virulent strain of the endosymbiont Wolbachia on the stability of methylation patterns. Conclusion We see that methylation in the A. aegypti genome is associated with reduced transcription and is most common in the promoters of genes relating to regulation of transcription and metabolism. Similar gene classes are also methylated in aphids and honeybees, suggesting either conservation or convergence of methylation patterns. In addition to this evidence of evolutionary stability, we also show that infection with the virulent wMelPop Wolbachia strain induces additional methylation and demethylation events in the genome. While most of these changes seem random with respect to gene function and have no detected effect on transcription, there does appear to be enrichment of genes associated with membrane function. Given that Wolbachia lives within a membrane-bound vacuole of host origin and retains a large number of genes for transporting host amino acids, inorganic ions and ATP despite a severely reduced genome, these changes might represent an evolved strategy for manipulating the host environments for its own gain. Testing for a direct link between these methylation changes and expression, however, will require study across a broader range of developmental stages and tissues with methods that detect

  3. Cyclopentenyl Cytosine Induces Senescence in Breast Cancer Cells through the Nucleolar Stress Response and Activation of p53S⃞

    PubMed Central

    Huang, Min; Whang, Patrick; Lewicki, Patrick

    2011-01-01

    The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated β-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G2- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G1 phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death. PMID:21464199

  4. Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside

    SciTech Connect

    Yang, G.S.; Wang, C.; Minkin, S.; Minden, M.D.; McCulloch, E.A. )

    1991-07-01

    The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. The authors report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) suicide technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR.

  5. Hypoxia imaging predicts success of hypoxia-induced cytosine deaminase/5-fluorocytosine gene therapy in a murine lung tumor model.

    PubMed

    Lee, B-F; Lee, C-H; Chiu, N-T; Hsia, C-C; Shen, L-H; Shiau, A-L

    2012-04-01

    Tc-99m-HL91 is a hypoxia imaging biomarker. The aim of this study was to investigate the value of Tc-99m-HL91 imaging for hypoxia-induced cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy in a murine lung tumor model. C57BL/6 mice were implanted with Lewis lung carcinoma cells transduced with the hypoxia-inducible promoter-driven CD gene (LL2/CD) or luciferase gene (LL2/Luc) serving as the control. When tumor volumes reached 100 mm(3), pretreatment images were acquired after injection of Tc-99m-HL91. The mice were divided into low and high hypoxic groups based on the tumor-to-non-tumor ratio of Tc-99m-HL91. They were injected daily with 5-FC (500 mg kg(-1)) or the vehicle for 1 week. When tumor volumes reached 1000 mm(3), autoradiography and histological examinations were performed. Treatment with 5-FC delayed tumor growth and enhanced the survival of mice bearing high hypoxic LL2/CD tumors. The therapeutic effect of hypoxia-induced CD/5-FC gene therapy was more pronounced in high hypoxic tumors than in low hypoxic tumors. This study provides the first evidence that Tc-99m-HL91 can serve as an imaging biomarker for predicting the treatment responses of hypoxia-regulated CD/5-FC gene therapy in animal tumor models. Our results suggest that hypoxia imaging using Tc-99m-HL91 has the predictive value for the success of hypoxia-directed treatment regimens. PMID:22281757

  6. Complex formation of cadmium with sugar residues, nucleobases, phosphates, nucleotides, and nucleic acids.

    PubMed

    Sigel, Roland K O; Skilandat, Miriam; Sigel, Astrid; Operschall, Bert P; Sigel, Helmut

    2013-01-01

    Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromatic-nitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono- to the di- and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono- or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand

  7. Navigating the Nucleotide Excision Repair Threshold

    PubMed Central

    Liu, Liren; Lee, Jennifer; Zhou, Pengbo

    2010-01-01

    Nucleotide excision repair (NER) is the primary DNA repair pathway that removes helix-distorting DNA strand damage induced by ultraviolet light (UV) irradiation or chemical carcinogens to ensure genome integrity. While the core NER proteins that carry out damage recognition, excision and repair reactions have been identified and extensively characterized, and the NER pathway has been reconstituted in vitro, the regulatory pathways that govern the threshold levels of NER have not been fully elucidated. This mini-review focuses on recently discovered transcriptional and post-translational mechanisms that specify the capacity of NER, and suggests the potential implications of modulating NER activity in cancer prevention and therapeutic intervention. PMID:20458729

  8. Facing growth in the European Nucleotide Archive

    PubMed Central

    Cochrane, Guy; Alako, Blaise; Amid, Clara; Bower, Lawrence; Cerdeño-Tárraga, Ana; Cleland, Iain; Gibson, Richard; Goodgame, Neil; Jang, Mikyung; Kay, Simon; Leinonen, Rasko; Lin, Xiu; Lopez, Rodrigo; McWilliam, Hamish; Oisel, Arnaud; Pakseresht, Nima; Pallreddy, Swapna; Park, Youngmi; Plaister, Sheila; Radhakrishnan, Rajesh; Rivière, Stephane; Rossello, Marc; Senf, Alexander; Silvester, Nicole; Smirnov, Dmitriy; ten Hoopen, Petra; Toribio, Ana; Vaughan, Daniel; Zalunin, Vadim

    2013-01-01

    The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/) collects, maintains and presents comprehensive nucleic acid sequence and related information as part of the permanent public scientific record. Here, we provide brief updates on ENA content developments and major service enhancements in 2012 and describe in more detail two important areas of development and policy that are driven by ongoing growth in sequencing technologies. First, we describe the ENA data warehouse, a resource for which we provide a programmatic entry point to integrated content across the breadth of ENA. Second, we detail our plans for the deployment of CRAM data compression technology in ENA. PMID:23203883

  9. Complete Nucleotide Sequence of Tn10

    PubMed Central

    Chalmers, Ronald; Sewitz, Sven; Lipkow, Karen; Crellin, Paul

    2000-01-01

    The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals. PMID:10781570

  10. NADH peroxidase: kinetic mechanism and nucleotide specificity

    SciTech Connect

    Stoll, V.S.; Blanchard, J.S.

    1987-05-01

    NADH peroxidase is a flavoprotein reductase isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide dependent reduction of hydrogen peroxide to water. Initial velocity, product and dead-end inhibition studies have been performed and all support a ping-pong kinetic mechanism. Further support for the ping-pong nature of the kinetic mechanism are the hydrogen peroxide independent transhydrogenase activity of the enzyme, measured either with thio-NAD or with radiolabeled NAD (isotope exchange studies). Kinetic parameters will be presented for a number of reduced pyridine nucleotide analogs. Analogs which have been modified in the adenine ring exhibit much higher K/sub m/'s relative to their adenine analogs. NADH peroxidase catalyzes the stereo-specific removal of the 4S hydrogen of NADH and primary deuterium kinetic isotope effects have been determined for a number of these substrates with 4S-deuterated molecules. There is a strong correlation between their steady-state K/sub m/ and /sup D/V/K. Small values for /sup D/V are interpreted as supporting rate-limitation in the oxidative half-reaction. These data will be discussed in terms of a kinetic and chemical mechanism proposed for NADH peroxidase.

  11. (Biological applications of nucleosides and nucleotides)

    SciTech Connect

    Srivastava, P.C.

    1990-08-20

    The traveler was invited to visit The Meditech Group, VTT Technology, Inc., Reactor Laboratory, VTT Technical Research Center of Finland (VTT), Otakaari, Espoo, Finland. The Meditech Group commands a 70 percent market share of Finland's radiopharmaceutical business and plans to expand its activities to other Scandinavian countries as well as in the Leningrad area of USSR. Meditech has plans to separate itself from Technical Research Center of Finland and its subsidiary VTT Technology, Inc., to become a private radiopharmaceutical company in the near future. As a private company, Meditech could expand its activities to encompass radiopharmaceutical research and development and may require foreign technical experts to support its research endeavors. The traveler also attended the Ninth International Round Table Conference on Nucleosides, Nucleotides, and Their Biological Applications held at the Biomedical Center, University of Uppsala, Uppsala, Sweden. The meeting focused on the chemistry and biology of RNA and DNA and their building blocks, nucleosides and nucleotides. The traveler also presented an invited paper entitled Design, Synthesis and Tumor Specificity of Azomycin Ribo- and Acyclonucleosides,'' describing his recent work at Oak Ridge National Laboratory.

  12. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  13. Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg²⁺, Nucleotides, and RNA.

    PubMed

    Frankel, Erica A; Bevilacqua, Philip C; Keating, Christine D

    2016-03-01

    Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid-liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5'-mono-, di-, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg(2+) and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg(2+) concentration of 10 mM had 1-5 M Mg(2+) in the coacervates, and RNA random sequence (N54) partitioned ∼10,000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg(2+), and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg(2+) concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments. PMID:26844692

  14. Adsorption of adenine, cytosine, thymine, and uracil on sulfide-modified montmorillonite: FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry studies.

    PubMed

    Carneiro, Cristine E A; Berndt, Graciele; de Souza Junior, Ivan G; de Souza, Cláudio M D; Paesano, Andrea; da Costa, Antonio C S; di Mauro, Eduardo; de Santana, Henrique; Zaia, Cássia T B V; Zaia, Dimas A M

    2011-10-01

    In the present work the interactions of nucleic acid bases with and adsorption on clays were studied at two pHs (2.00, 7.00) using different techniques. As shown by Mössbauer and EPR spectroscopies and X-ray diffractometry, the most important finding of this work is that nucleic acid bases penetrate into the interlayer of the clays and oxidize Fe(2+) to Fe(3+), thus, this interaction cannot be regarded as a simple physical adsorption. For the two pHs the order of the adsorption of nucleic acid bases on the clays was: adenine ≈ cytosine > thymine > uracil. The adsorption of adenine and cytosine on clays increased with decreasing of the pH. For unaltered montmorillonite this result could be explained by electrostatic forces between adenine/cytosine positively charged and clay negatively charged. However for montmorillonite modified with Na(2)S, probably van der Waals forces also play an important role since both adenine/cytosine and clay were positively charged. FT-IR spectra showed that the interaction between nucleic acid bases and clays was through NH(+) or NH (2) (+) groups. X-ray diffractograms showed that nucleic acid bases adsorbed on clays were distributed into the interlayer surface, edge sites and external surface functional groups (aluminol, silanol) EPR spectra showed that the intensity of the line g ≈ 2 increased probably because the oxidation of Fe(2+) to Fe(3+) by nucleic acid bases and intensity of the line g = 4.1 increased due to the interaction of Fe(3+) with nucleic acid bases. Mössbauer spectra showed a large decreased on the Fe(2+) doublet area of the clays due to the reaction of nucleic acid bases with Fe(2+). PMID:21717172

  15. Biochemical Basis of Immunological and Retroviral Responses to DNA-targeted Cytosine Deamination by Activation-induced Cytidine Deaminase and APOBEC3G*

    PubMed Central

    Chelico, Linda; Pham, Phuong; Petruska, John; Goodman, Myron F.

    2009-01-01

    Activation-induced cytidine deaminase (AID) and APOBEC3G catalyze deamination of cytosine to uracil on single-stranded DNA, thereby setting in motion a regulated hypermutagenic process essential for human well-being. However, if regulation fails, havoc ensues. AID plays a central role in the synthesis of high affinity antibodies, and APOBEC3G inactivates human immunodeficiency virus-1. This minireview highlights biochemical and structural properties of AID and APOBEC3G, showing how studies using the purified enzymes provide valuable insight into the considerably more complex biology governing antibody generation and human immunodeficiency virus inactivation. PMID:19684020

  16. Structural Basis for Nucleotide Exchange in Heterotrimeric G Proteins

    PubMed Central

    Dror, Ron O.; Mildorf, Thomas J.; Hilger, Daniel; Manglik, Aashish; Borhani, David W.; Arlow, Daniel H.; Philippsen, Ansgar; Villanueva, Nicolas; Yang, Zhongyu; Lerch, Michael T.; Hubbell, Wayne L.; Kobilka, Brian K.; Sunahara, Roger K.; Shaw, David E.

    2016-01-01

    G protein–coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains—previously observed to separate widely upon receptor binding to expose the nucleotide-binding site—separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism. PMID:26089515

  17. Targeting the Immune System to Fight Cancer Using Chemical Receptor Homing Vectors Carrying Polyinosine/Cytosine (PolyIC).

    PubMed

    Levitzki, Alexander

    2012-01-01

    Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, polyinosine/cytosine (polyIC), into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA-binding proteins, such as dsRNA dependent protein kinase (PKR), Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-1), and melanoma differentiation-associated gene 5 (MDA5). The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1) recruitment of the immune system is localized to the tumor. (2) The response is rapid, leading to fast tumor eradication. (3) The bystander effects lead to the eradication of tumor cells not harboring the target. (4) The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which can be a small molecule, a single chain antibody, or an affibody

  18. Targeting the Immune System to Fight Cancer Using Chemical Receptor Homing Vectors Carrying Polyinosine/Cytosine (PolyIC)

    PubMed Central

    Levitzki, Alexander

    2012-01-01

    Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, polyinosine/cytosine (polyIC), into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA-binding proteins, such as dsRNA dependent protein kinase (PKR), Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-1), and melanoma differentiation-associated gene 5 (MDA5). The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1) recruitment of the immune system is localized to the tumor. (2) The response is rapid, leading to fast tumor eradication. (3) The bystander effects lead to the eradication of tumor cells not harboring the target. (4) The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which can be a small molecule, a single chain antibody, or an affibody

  19. Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside.

    PubMed

    Yang, G S; Wang, C; Minkin, S; Minden, M D; McCulloch, E A

    1991-07-01

    The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) "suicide" technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR. The hydrocortisone effect was dose and time related; protection from ara-C increased from 10(-8) to 10(-5) M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara

  20. Signal transduction by guanine nucleotide binding proteins.

    PubMed

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  1. Nicotinamide nucleotide synthesis in regenerating rat liver

    PubMed Central

    Ferris, G. M.; Clark, J. B.

    1971-01-01

    1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors. PMID:4398891

  2. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

    PubMed Central

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  3. The Levels of Soluble Nucleotides in Wheat Aleurone Tissue 1

    PubMed Central

    Collins, G. G.; Jenner, C. F.; Paleg, L. G.

    1972-01-01

    The content of soluble nucleotides in aleurone layers isolated from mature wheat (Triticum aestivum var. Olympic) grain was investigated. The most abundant nucleotides were adenosine triphosphate, uridine triphosphate, and uridine diphosphoglucose. Smaller amounts of guanosine triphosphate, cytidine triphosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide were also identified. The levels of some of these nucleotides were increased after incubation of the tissue under certain conditions. Nucleotide levels were measured at intervals during incubation of aleurone layers in water. The changes observed are discussed in relation to a response by the tissue to wounding. PMID:16657969

  4. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  5. Pyridine nucleotide coenzymes: Chemical, biological, and medical aspects. Vol. 2, Pt. A

    SciTech Connect

    Dolphin, D.; Poulson, R.; Avramovic, O.

    1987-01-01

    This text contains the following: History of the Pyridine Nucleotides Nomenclature; Evolution of Pyridine Nucleotide; Relationship Between Biosynthesis and Evolution; Crystal Structure; Coenzyme Conformations; Protein Interactions; Optical Spectroscopy of the Pyridine Nucleotides; Excited States of Pyridine Nucleotide Coenzymes; Fluorescence and Phosphorescence; Nuclear Magnetic Resonance Spectroscopy of Pyridine Nucleotides; Mass Spectrometry of Pyridine Nucleotides; Mechanism of Action of the Pyridine Nucleotides; Chemical Stability and Reactivity of Pyridine Nucleotide Coenzymes; Stereochemistry of Fatty Acid Biosynthesis and Metabolism; Kinetics of Pyridine Nucleotide-Utilizing Enzymes; Preparation and Properties of NAD and NADP Analogs; Model Studies and Biological Activity of Analogs; and Spin-Labeled Pyridine Nucleotide Derivatives.

  6. Vesicular nucleotide transport: a brief history and the vesicular nucleotide transporter as a target for drug development.

    PubMed

    Hiasa, Miki; Togawa, Natsuko; Moriyama, Yoshinori

    2014-01-01

    Neurons and neuroendocrine cells store nucleotides in vesicles and release them upon stimulation, leading to intercellular purinergic signaling. The molecular machinery responsible for the vesicular storage of nucleotides was a long standing enigma, however, recently the transporter involving in the process was identified. This article summarizes the history of vesicular storage of nucleotides and the identification of the vesicular nucleotide transporter (VNUT) responsible for the process. The significance of VNUT as a drug target to control purinergic chemical transmission is also discussed. PMID:23886392

  7. Effect of D-valine and cytosine arabinoside on (/sup 3/H)thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    SciTech Connect

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by (/sup 3/H)thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.

  8. Nanodosimetry of Auger electrons: A case study from the decay of 125I and 0–18-eV electron stopping cross sections of cytosine

    PubMed Central

    Michaud, M.; Bazin, M.; Sanche, L.

    2013-01-01

    Radiopharmaceuticals emitting Auger electrons are often injected into patients undergoing cancer treatment with targeted radionuclide therapy (TRT). In this type of radiotherapy, the radiation source is radial and most of the emitted primary particles are low-energy electrons (LEEs) having kinetic energies distributed mostly from zero to a few hundred electron volts with very short ranges in biological media. These LEEs generate a high density of energy deposits and clustered damage, thus offering a relative biological effectiveness comparable to that of alpha particles. In this paper, we present a simple model and corresponding measurements to assess the energy deposited near the site of the radiopharmaceuticals in TRT. As an example, a calculation is performed for the decay of a single 125I radionuclide surrounded by a 1-nm-radius spherical shell of cytosine molecules using the energy spectrum of LEEs emitted by 125I along with their stopping cross sections between 0 and 18 eV. The dose absorbed by the cytosine shell, which occupies a volume of 4 nm3, is extremely high. It amounts to 79 kGy per decay of which 3%, 39%, and 58% is attributed to vibrational excitations, electronic excitations, and ionization processes, respectively. PMID:24976798

  9. Nanodosimetry of Auger electrons: A case study from the decay of (125)I and 0-18-eV electron stopping cross sections of cytosine.

    PubMed

    Michaud, M; Bazin, M; Sanche, L

    2013-03-01

    Radiopharmaceuticals emitting Auger electrons are often injected into patients undergoing cancer treatment with targeted radionuclide therapy (TRT). In this type of radiotherapy, the radiation source is radial and most of the emitted primary particles are low-energy electrons (LEEs) having kinetic energies distributed mostly from zero to a few hundred electron volts with very short ranges in biological media. These LEEs generate a high density of energy deposits and clustered damage, thus offering a relative biological effectiveness comparable to that of alpha particles. In this paper, we present a simple model and corresponding measurements to assess the energy deposited near the site of the radiopharmaceuticals in TRT. As an example, a calculation is performed for the decay of a single (125)I radionuclide surrounded by a 1-nm-radius spherical shell of cytosine molecules using the energy spectrum of LEEs emitted by (125)I along with their stopping cross sections between 0 and 18 eV. The dose absorbed by the cytosine shell, which occupies a volume of 4 nm(3), is extremely high. It amounts to 79 kGy per decay of which 3%, 39%, and 58% is attributed to vibrational excitations, electronic excitations, and ionization processes, respectively. PMID:24976798

  10. A novel radiochemical approach to 1-(2'-deoxy-2'-[(18) F]fluoro-β-d-arabinofuranosyl)cytosine ((18) F-FAC).

    PubMed

    Meyer, Jan-Philip; Probst, Katrin C; Trist, Iuni M L; McGuigan, Christopher; Westwell, Andrew D

    2014-09-01

    (18) F-FAC (1-(2'-deoxy-2'-[(18) F]fluoro-β-D-arabinofuranosyl)-cytosine) is an important 2'-fluoro-nucleoside-based positron emission tomography (PET) tracer that has been used for in vivo prediction of response to the widely used cancer chemotherapy drug gemcitabine. Previously reported synthetic routes to (18) F-FAC have relied on early introduction of the (18) F radiolabel prior to attachment to protected cytosine base. Considering the (18) F radiochemical half-life (110 min) and the technical challenges of multi-step syntheses on PET radiochemistry modular systems, late-stage radiofluorination is preferred for reproducible and reliable radiosynthesis with in vivo applications. Herein, we report the first late-stage radiosynthesis of (18) F-FAC. Cytidine derivatives with leaving groups at the 2'-position are particularly prone to undergo anhydro side-product formation upon heating because of their electron density at the 2-carbonyl pyrimidone oxygen. Our rationally developed fluorination precursor showed an improved reactivity-to-stability ratio at elevated temperatures. (18) F-FAC was obtained in radiochemical yields of 4.3-5.5% (n = 8, decay-corrected from end of bombardment), with purities ≥98% and specific activities ≥63 GBq/µmol. The synthesis time was 168 min. PMID:25257474

  11. Nanodosimetry of Auger electrons: A case study from the decay of 125I and 0-18-eV electron stopping cross sections of cytosine

    NASA Astrophysics Data System (ADS)

    Michaud, M.; Bazin, M.; Sanche, L.

    2013-03-01

    Radiopharmaceuticals emitting Auger electrons are often injected into patients undergoing cancer treatment with targeted radionuclide therapy (TRT). In this type of radiotherapy, the radiation source is radial and most of the emitted primary particles are low-energy electrons (LEEs) having kinetic energies distributed mostly from zero to a few hundred electron volts with very short ranges in biological media. These LEEs generate a high density of energy deposits and clustered damage, thus offering a relative biological effectiveness comparable to that of alpha particles. In this paper, we present a simple model and corresponding measurements to assess the energy deposited near the site of the radiopharmaceuticals in TRT. As an example, a calculation is performed for the decay of a single 125I radionuclide surrounded by a 1-nm-radius spherical shell of cytosine molecules using the energy spectrum of LEEs emitted by 125I along with their stopping cross sections between 0 and 18 eV. The dose absorbed by the cytosine shell, which occupies a volume of 4 nm3, is extremely high. It amounts to 79 kGy per decay of which 3%, 39%, and 58% is attributed to vibrational excitations, electronic excitations, and ionization processes, respectively.

  12. Microbial metabolism of thiopurines: A method to measure thioguanine nucleotides.

    PubMed

    Movva, Ramya; Lobb, Michael; Ó Cuív, Páraic; Florin, Timothy H J; Duley, John A; Oancea, Iulia

    2016-09-01

    Thiopurines are anti-inflammatory prodrugs. We hypothesised that bacteria may contribute to conversion to active drug. Escherichia coli strain DH5α was evaluated to determine whether it could metabolise the thiopurine drugs, thioguanine or mercaptopurine, to thioguanine nucleotides. A rapid and reliable high performance liquid chromatography (ultraviolet detection) method was developed to quantify indirectly thioguanine nucleotides, by measuring thioguanine nucleoside. PMID:27444548

  13. From Single Nucleotide Polymorphism to Transcriptional Mechanism

    PubMed Central

    Martini, Sebastian; Nair, Viji; Patel, Sanjeevkumar R.; Eichinger, Felix; Nelson, Robert G.; Weil, E. Jennifer; Pezzolesi, Marcus G.; Krolewski, Andrzej S.; Randolph, Ann; Keller, Benjamin J.; Werner, Thomas; Kretzler, Matthias

    2013-01-01

    Genome-wide association studies have proven to be highly effective at defining relationships between single nucleotide polymorphisms (SNPs) and clinical phenotypes in complex diseases. Establishing a mechanistic link between a noncoding SNP and the clinical outcome is a significant hurdle in translating associations into biological insight. We demonstrate an approach to assess the functional context of a diabetic nephropathy (DN)-associated SNP located in the promoter region of the gene FRMD3. The approach integrates pathway analyses with transcriptional regulatory pattern-based promoter modeling and allows the identification of a transcriptional framework affected by the DN-associated SNP in the FRMD3 promoter. This framework provides a testable hypothesis for mechanisms of genomic variation and transcriptional regulation in the context of DN. Our model proposes a possible transcriptional link through which the polymorphism in the FRMD3 promoter could influence transcriptional regulation within the bone morphogenetic protein (BMP)-signaling pathway. These findings provide the rationale to interrogate the biological link between FRMD3 and the BMP pathway and serve as an example of functional genomics-based hypothesis generation. PMID:23434934

  14. Nucleotide-binding mechanisms in pseudokinases

    PubMed Central

    Hammarén, Henrik M.; Virtanen, Anniina T.; Silvennoinen, Olli

    2015-01-01

    Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10% of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades. Furthermore, several pseudokinases have been linked to human diseases, particularly cancer, which is raising interest for therapeutic approaches towards these proteins. The ATP-binding pocket is a well-established drug target and elucidation of the mechanism and properties of nt binding in pseudokinases is of significant interest and importance. Recent studies have demonstrated that members of the pseudokinase family are very diverse in structure as well as in their ability and mechanism to bind nts or perform phosphoryl transfer reactions. This diversity also precludes prediction of pseudokinase function, or the importance of nt binding for said function, based on primary sequence alone. Currently available data indicate that ∼40% of pseudokinases are able to bind nts, whereas only few are able to catalyse occasional phosphoryl transfer. Pseudokinases employ diverse mechanisms to bind nts, which usually occurs at low, but physiological, affinity. ATP binding serves often a structural role but in most cases the functional roles are not precisely known. In the present review, we discuss the various mechanisms that pseudokinases employ for nt binding and how this often low-affinity binding can be accurately analysed. PMID:26589967

  15. Nucleotide Phosphohydrolase in Purified Vaccinia Virus

    PubMed Central

    Munyon, William; Paoletti, Enzo; Ospina, Julio; Grace, James T.

    1968-01-01

    Purified infectious vaccinia virus has been shown to contain an enzyme or enzymes that remove the terminal phosphate group from adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The Km for ATP of this enzyme is 5.5 × 10−4m, and the relative rates of the reaction with ATP, GTP, UTP, and CTP are 1.00, 0.34, 0.15, and 0.29, respectively. The virus enzyme does not react with any of the diphosphates. The rate of the reaction is proportional to the amount of virus added and is linear for 130 min. The virus nucleotide phosphohydrolase activity is greatly stimulated by Mg++ and very slightly stimulated by Ca++. The small residual activity observed in the absence of divalent cations is completely inhibited by ethylenediaminetetraacetic acid. Neither Na+ nor K+ ions, nor any mixture of these, was found to stimulate the reaction significantly, and ouabain, at 10−4m, inhibited the reaction by only 27%. The response of the vaccinia enzyme to mono- and divalent cations and to ouabain indicates that the vaccinia enzyme has different properties from those associated with microsomes and mitochondria. PMID:4986904

  16. Exploiting Nucleotide Composition to Engineer Promoters

    PubMed Central

    Mauceli, Evan; Ernst, Wolfgang; Baumann, Martina; Biagi, Tara; Swofford, Ross; Russell, Pamela; Zody, Michael C.; Di Palma, Federica; Lindblad-Toh, Kerstin; Grabherr, Reingard M.

    2011-01-01

    The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a “proof-of-concept” for custom-designing promoters that are suitable for biotechnological and medical applications. PMID:21625601

  17. Oxidative DNA Damage and Nucleotide Excision Repair

    PubMed Central

    Melis, Joost P.M.; Luijten, Mirjam

    2013-01-01

    Abstract Significance: Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. Recent Advances: NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. Critical Issues: The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. Future Directions: Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues. Antioxid. Redox Signal. 18, 2409–2419. PMID:23216312

  18. Cardiac Na+ Current Regulation by Pyridine Nucleotides

    PubMed Central

    Liu, Man; Sanyal, Shamarendra; Gao, Ge; Gurung, Iman S.; Zhu, Xiaodong; Gaconnet, Georgia; Kerchner, Laurie J.; Shang, Lijuan L.; Huang, Christopher L-H.; Grace, Andrew; London, Barry; Dudley, Samuel C.

    2009-01-01

    Rationale Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein reduce cardiac Na+ current (INa) and cause Brugada Syndrome (BrS). GPD1-L has >80% amino acid homology with glycerol-3-phosphate dehydrogenase, which is involved in nicotinamide adenine dinucleotide (NAD)-dependent energy metabolism. Objective Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Nav1.5). Methods and Results HEK293 cells stably expressing Nav1.5 and rat neonatal cardiomyocytes were used. The influence of NADH/NAD+ on arrhythmic risk was evaluated in wild-type or SCN5A+/− mouse heart. A280V GPD1-L caused a 2.48 ± 0.17-fold increase in intracellular NADH level (P<0.001). NADH application or co-transfection with A280V GPD1-L resulted in decreased INa (0.48 ± 0.09 or 0.19 ±0.04 of control group, respectively; P<0.01), which was reversed by NAD+, chelerythrine, or superoxide dismutase (SOD). NAD+ antagonism of the Na+ channel downregulation by A280V GPD1-L or NADH was prevented by a protein kinase A (PKA) inhibitor, PKAI6–22. The effects of NADH and NAD+ were mimicked by a phorbol ester and forskolin, respectively. Increasing intracellular NADH was associated with an increased risk of ventricular tachycardia (VT) in wild-type mouse hearts. Extracellular application of NAD+ to SCN5A+/− mouse hearts ameliorated the risk of VT. Conclusions Our results show that Nav1.5 is regulated by pyridine nucleotides, suggesting a link between metabolism and INa. This effect required protein kinase C (PKC) activation and was mediated by oxidative stress. NAD+ could prevent this effect by activating PKA. Mutations of GPD1-L may downregulate Nav1.5 by altering the oxidized to reduced NAD(H) balance. PMID:19745168

  19. Synthetic Nucleotides as Probes of DNA Polymerase Specificity

    PubMed Central

    Walsh, Jason M.; Beuning, Penny J.

    2012-01-01

    The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example, Escherichia coli DNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides. PMID:22720133

  20. The Kinesin-1 tail conformationally restricts the nucleotide pocket.

    PubMed

    Wong, Yao Liang; Dietrich, Kristen A; Naber, Nariman; Cooke, Roger; Rice, Sarah E

    2009-04-01

    We have used electron paramagnetic resonance and fluorescence spectroscopy to study the interaction between the kinesin-1 head and its regulatory tail domain. The interaction between the tails and the enzymatically active heads has been shown to inhibit intrinsic and microtubule-stimulated ADP release. Here, we demonstrate that the probe mobility of two different spin-labeled nucleotide analogs in the kinesin-1 nucleotide pocket is restricted upon binding of the tail domain to kinesin-1 heads. This conformational restriction is distinct from the microtubule-induced changes in the nucleotide pocket. Unlike myosin V, this tail-induced restriction occurs independent of nucleotide state. We find that the head-tail interaction that causes the restriction only weakly stabilizes Mg(2+) in the nucleotide pocket. The conformational restriction also occurs when a tail construct containing a K922A point mutation is used. This mutation eliminates the tail's ability to inhibit ADP release, indicating that the tail does not inhibit nucleotide ejection from the pocket by simple steric hindrance. Together, our data suggest that the observed head-tail interaction serves as a scaffold to position K922 to exert its inhibitory effect, possibly by interacting with the nucleotide alpha/beta-phosphates in a manner analogous to the arginine finger regulators of some G proteins. PMID:19348763

  1. The Kinesin-1 Tail Conformationally Restricts the Nucleotide Pocket

    PubMed Central

    Wong, Yao Liang; Dietrich, Kristen A.; Naber, Nariman; Cooke, Roger; Rice, Sarah E.

    2009-01-01

    We have used electron paramagnetic resonance and fluorescence spectroscopy to study the interaction between the kinesin-1 head and its regulatory tail domain. The interaction between the tails and the enzymatically active heads has been shown to inhibit intrinsic and microtubule-stimulated ADP release. Here, we demonstrate that the probe mobility of two different spin-labeled nucleotide analogs in the kinesin-1 nucleotide pocket is restricted upon binding of the tail domain to kinesin-1 heads. This conformational restriction is distinct from the microtubule-induced changes in the nucleotide pocket. Unlike myosin V, this tail-induced restriction occurs independent of nucleotide state. We find that the head-tail interaction that causes the restriction only weakly stabilizes Mg2+ in the nucleotide pocket. The conformational restriction also occurs when a tail construct containing a K922A point mutation is used. This mutation eliminates the tail's ability to inhibit ADP release, indicating that the tail does not inhibit nucleotide ejection from the pocket by simple steric hindrance. Together, our data suggest that the observed head-tail interaction serves as a scaffold to position K922 to exert its inhibitory effect, possibly by interacting with the nucleotide α/β-phosphates in a manner analogous to the arginine finger regulators of some G proteins. PMID:19348763

  2. Nucleic acid analysis using terminal-phosphate-labeled nucleotides

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2008-04-22

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  3. Identifying 2'-O-methylationation sites by integrating nucleotide chemical properties and nucleotide compositions.

    PubMed

    Chen, Wei; Feng, Pengmian; Tang, Hua; Ding, Hui; Lin, Hao

    2016-06-01

    2'-O-methylationation is an important post-transcriptional modification and plays important roles in many biological processes. Although experimental technologies have been proposed to detect 2'-O-methylationation sites, they are cost-ineffective. As complements to experimental techniques, computational methods will facilitate the identification of 2'-O-methylationation sites. In the present study, we proposed a support vector machine-based method to identify 2'-O-methylationation sites. In this method, RNA sequences were formulated by nucleotide chemical properties and nucleotide compositions. In the jackknife cross-validation test, the proposed method obtained an accuracy of 95.58% for identifying 2'-O-methylationation sites in the human genome. Moreover, the model was also validated by identifying 2'-O-methylation sites in the Mus musculus and Saccharomyces cerevisiae genomes, and the obtained accuracies are also satisfactory. These results indicate that the proposed method will become a useful tool for the research on 2'-O-methylation. PMID:27191866

  4. 5-Methylation of Cytosine in CG:CG Base-Pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir; Olson, Wilma; Li, Yun

    2014-03-01

    Van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile ``opening'' mode and two shear ``sliding'' and ``tearing'' modes in the orthogonal plane. The stacking interactions of the methyl groups are observed to globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. The results have implications for the epigenetic control of DNA mechanics.

  5. Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

    PubMed Central

    Buskiewicz, Iwona A.; Burke, John M.

    2012-01-01

    The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme–substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair. PMID:22274955

  6. 5-Methylation of Cytosine in CG:CG Base-pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

    PubMed Central

    Yusufaly, Tahir I.; Li, Yun; Olson, Wilma K.

    2014-01-01

    Van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile ‘opening’ mode and two shear ‘sliding’ and ‘tearing’ modes in the orthogonal plane. The stacking interactions of the methyl groups globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. Additionally, the indirect effects of the methyl groups on possible base-pair steps neighboring CG:CG are observed to be of comparable importance to their direct effects on CG:CG. The results have implications for the epigenetic control of DNA mechanics. PMID:24313757

  7. A possible prebiotic synthesis of purine, adenine, cytosine, and 4(3H)-pyrimidinone from formamide: implications for the origin of life.

    PubMed

    Saladino, R; Crestini, C; Costanzo, G; Negri, R; Di Mauro, E

    2001-05-01

    The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO(3) and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution. PMID:11377183

  8. Effects of temperature and isotopic substitution on electron attachment dynamics of guanine-cytosine base pair: Ring-polymer and classical molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Minoshima, Yusuke; Seki, Yusuke; Takayanagi, Toshiyuki; Shiga, Motoyuki

    2016-06-01

    The dynamical process of electron attachment to a guanine-cytosine pair in the normal (h-GC) and deuterated (d-GC) forms has been studied theoretically by semiclassical ring-polymer molecular dynamics (RPMD) simulations using the empirical valence bond model. The initially formed dipole-bound anion is converted rapidly to the valence-bound anion within about 0.1 ps in both h-GC and d-GC. However, the subsequent proton transfer in h-GC occurs with a rate five times greater than the deuteron transfer in d-GC. The change of rates with isotopic substitution and temperature variation in the RPMD simulations are quantitatively and qualitatively different from those in the classical molecular dynamics (MD) simulations, demonstrating the importance of nuclear quantum effects on the dynamics of this system.

  9. An Insight into the Environmental Effects of the Pocket of the Active Site of the Enzyme. Ab initio ONIOM-Molecular Dynamics (MD) Study on Cytosine Deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2008-02-01

    We applied the ONIOM-molecular dynamics (MD) method to cytosine deaminase to examine the environmental effects of the amino acid residues in the pocket of the active site on the substrate taking account of their thermal motion. The ab initio ONIOM-MD simulations show that the substrate uracil is strongly perturbed by the amino acid residue Ile33, which sandwiches the uracil with His62, through the steric contact due to the thermal motion. As a result, the magnitude of the thermal oscillation of the potential energy and structure of the substrate uracil significantly increases. TM and MA were partly supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

  10. [Nucleotide sequence determination of yeast mitochondrial phenylalanine-tRNA].

    PubMed

    Martin, R; Sibler, A P; Schneller, J M; Keith, G; Stahl, A J; Dirheimer, G

    1978-10-01

    The primary structure of mitochondrial tRNAPhe from Saccharomyces cerevisiae, purified by two-dimensional polyacrylamide gel electrophoresis, was determined using, standard procedures on in vivo 32P-labeled tRNA, as well as the new 5'-end postlabeling techniques. We propose a cloverleaf model which allows for tertiary interaction between cytosine in position 46 and guanine in position 15 and maximizes base pairing in the psi C stem, thus excluding the uracile in position 50 from base pairing in the psi C stem. Comparison of the primary structure of this tRNA with all other known procaryotic, chloroplastic or cytoplasmic tRNAsPhe sequences does not lead to any conclusion about the endosymbiotic theory of mitochondria evolution. PMID:103657

  11. Functional and structural characterization of DR_0079 from Deinococcus radiodurans, a novel Nudix hydrolase with a preference for cytosine (deoxy) ribonucleoside 5'-di- and triphosphates

    SciTech Connect

    Buchko, Garry W.; Litvinova, Olga; Robinson, Howard; Yakunin, Alexander F.; Kennedy, Michael A.

    2008-06-24

    The Deinococcus radiodurans Nudix hydrolase DR0079 was assayed for activity towards a wide variety of substrates and observed to have a marked specificity for cytosine ribonucleoside 5’-diphosphate (CDP) and cytosine ribonucleoside 5’-triphosphate (CTP) with a slight preference for CDP. The next most specific substrates, with a relative activity of <50%, were the corresponding deoxyribose nucleosides, dCDP and dCTP. Enzyme hydrolase activity at the site of the phosphodiester bond was corroborated using 31P NMR spectroscopy to follow the phosphorus resonances for two substrates, CDP and IDP, and the hydrolysis products, NMP and Pi. Optimum activity for CDP was determined to be at pH 9.0 – 9.5. The optimal divalent cation for CDP hydrolysis at this pH was Mg2+ followed by Mn2+ (~47%) and Co2+(~27%). The biochemical data is discussed with reference to the crystal structure for the D. radiodurans DR0079 that was determined in the apo-metal form at 1.9 Å resolution. The protein in the crystal structure contains nine β-strands, three α-helices, and two 3-10 helices that are organized into three subdomains; an N-terminal β-sheet, a central Nudix core, and a C-terminal helixturn- helix motif. As observed for all known structures of Nudix hydrolases, the α-helix of the ‘Nudix box’ is one of two helices that sandwich a ‘four-strand’ mixed β-sheet. Using 15N-labelled DR0079, NMR chemical shift mapping experiments were performed with the paramagnetic divalent cation Co2+ and the non-hydrolyzable substrate thymidine- 5’-O-(α,β-methylenediphosphate (TMP-CP). The results of the chemical shift perturbation experiments were mapped onto the crystal structure of DR0079 and a model for substrate binding proposed.

  12. DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line

    PubMed Central

    Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

    2010-01-01

    Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2′-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5′-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator TaxBLV decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267LTaxSN 5′-LTR compared with the L267 5′-LTR. Interestingly, DNA methylation inhibitors and TaxBLV synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the −154 or −129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at −129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5′-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency. PMID:20413592

  13. Pleiotropic phenotypes of the salt-tolerant and cytosine hypomethylated leafless inflorescence, evergreen dwarf and irregular leaf lamina mutants of Catharanthus roseus possessing Mendelian inheritance.

    PubMed

    Kumari, Renu; Sharma, Vishakha; Sharma, Vinay; Kumar, Sushil

    2013-12-01

    In Catharanthus roseus, three morphological cum salt-tolerant chemically induced mutants of Mendelian inheritance and their wild-type parent cv Nirmal were characterized for overall cytosine methylation at DNA repeats, expression of 119 protein coding and seven miRNA-coding genes and 50 quantitative traits. The mutants, named after their principal morphological feature(s), were leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill). The Southern-blot analysis of MspI digested DNAs of mutants probed with centromeric and 5S and 18S rDNA probes indicated that, in comparison to wild type, the mutants were extensively demethylated at cytosine sites. Among the 126 genes investigated for transcriptional expression, 85 were upregulated and 41 were downregulated in mutants. All of the five genes known to be stress responsive had increased expression in mutants. Several miRNA genes showed either increased or decreased expression in mutants. The C. roseus counterparts of CMT3, DRM2 and RDR2 were downregulated in mutants. Among the cell, organ and plant size, photosynthesis and metabolism related traits studied, 28 traits were similarly affected in mutants as compared to wild type. Each of the mutants also expressed some traits distinctively. The egd mutant possessed superior photosynthesis and water retention abilities. Biomass was hyperaccumulated in roots, stems, leaves and seeds of the lli mutant. The ill mutant was richest in the pharmaceutical alkaloids catharanthine, vindoline, vincristine and vinblastine. The nature of mutations, origins of mutant phenotypes and evolutionary importance of these mutants are discussed. PMID:24371160

  14. High-dose cytosine-arabinoside and cisplatin regimens as salvage therapy for refractory or relapsed AIDS-related non-Hodgkin's lymphoma.

    PubMed

    Bi, J; Espina, B M; Tulpule, A; Boswell, W; Levine, A M

    2001-12-15

    No effective salvage regimen has been defined for patients with AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) who do not respond to first-line chemotherapy that contains anthracycline. Combined dexamethasone, cytosine arabinoside, and cisplatin (DHAP) and etoposide, methylprednisolone, cytosine arabinoside, and cisplatin (ESHAP) have shown good response rates in HIV-negative patients with relapsed lymphomas. We retrospectively analyzed patients with refractory or relapsed AIDS-NHL who had been treated with either DHAP or ESHAP to evaluate the feasibility and efficacy of these regimens. Twenty-six patients with refractory or relapsed AIDS-NHL were treated between 1990 and 1999 either with DHAP ( n = 13) or with ESHAP ( n = 13). Only 1 patient from each group (8%) had achieved complete remission with any previous therapy, and most had progressive disease after the regimen immediately preceding DHAP or ESHAP. In the ESHAP group, 4 patients (31%) achieved complete remission (CR) and 3 patients (23%) attained partial remission (PR) for an overall response rate of 54%. The median survival was 7.1 months (range, 1-58.9+ months) from the time ESHAP was begun. Among the 3 patients with primary refractory lymphoma, there was 1 CR, 1 PR, and one patient with stable disease. In contrast, only 1 PR (7%) was observed with DHAP; the median survival was 3 months. Myelosuppression was the most significant toxicity with grade 4 neutropenia occurring in all who received ESHAP and in 54% of patients treated with DHAP. Neutropenic fever occurred in 8 (62%) ESHAP-treated and 6 (46%) DHAP-treated patients. Although hematologic toxicity is profound, ESHAP appears to be an active salvage regimen for patients with relapsed or refractory AIDS-NHL. PMID:11744828

  15. Anti-leukemic effect of sodium metaarsenite (KML001) in acute myeloid leukemia with breaking-down the resistance of cytosine arabinoside.

    PubMed

    Yoon, Jin Sun; Kim, Eun Shil; Park, Byeong Bae; Choi, Jung Hye; Won, Young Woong; Kim, Sujong; Lee, Young Yiul

    2015-05-01

    Sodium metaarsenite (NaAs2O3: code name KML001) is an orally bioavailable arsenic compound with potential anti-cancer activity. However, the effect of KML001 has not been studied in acute myeloid leukemia (AML). We investigated the anti-leukemic effect of KML001 in AML, and determined the mode of action of KML001. KML001 inhibited the cellular proliferation in all AML cell lines and primary AML blasts as well as HL-60R (cytosine arabinoside-resistant HL-60) cells, while As2O3 was not effective in primary AML blasts and AML cell lines including HL-60R cells. KML001 induced G1 arrest and apoptosis in HL-60 and HL-60R cells. KML001 inhibited the activation of STAT (signal transducer and activator of transcription) 1, 3, 5, NF-κB, AKT and PI3K, while phosphorylated PTEN was upregulated. In addition, activation of ERK, p38 and JNK was observed in KML001-induced growth inhibition of HL-60 and HL-60R cells. Furthermore, KML001 induced telomeric terminal restriction fragment (TRF) length shortening in a time-dependent manner in HL-60 and HL-60R cells. Real‑time PCR with RNA extracted from KML001-treated HL-60 and HL-60R cells showed a significant reduction of catalytic subunit of telomerase, hTERT, in a time-dependent manner. Additionally, γ-H2AX, a sensitive molecular marker of DNA damage, in HL-60 and HL-60R cells was induced by KML001. These results suggest that KML001 inhibits the proliferation of AML cells including cytosine arabinoside-resistant AML cells via various mechanisms such as cell cycle arrest, induction of apoptosis, inhibition of JAK/STAT and PI3K pathways, activation of MAPK pathway and telomere targeting. PMID:25695330

  16. DNAzyme-mediated catalysis with only guanosine and cytidine nucleotides

    PubMed Central

    Schlosser, Kenny; Li, Yingfu

    2009-01-01

    Single-stranded DNA molecules have the capacity to adopt catalytically active structures known as DNAzymes, although the fundamental limits of this ability have not been determined. Starting with a parent DNAzyme composed of all four types of standard nucleotides, we conducted a search of the surrounding sequence space to identify functional derivatives with catalytic cores composed of only three, and subsequently only two types of nucleotides. We provide the first report of a DNAzyme that contains only guanosine and cytidine deoxyribonucleotides in its catalytic domain, which consists of just 13 nucleotides. This DNAzyme catalyzes the Mn2+-dependent cleavage of an RNA phosphodiester bond ∼5300-fold faster than the corresponding uncatalyzed reaction, but ∼10 000-fold slower than the parent. The demonstration of a catalytic DNA molecule made from a binary nucleotide alphabet broadens our understanding of the fundamental limits of nucleic-acid-mediated catalysis. PMID:19050014

  17. A single-nucleotide exon found in Arabidopsis

    PubMed Central

    Guo, Lei; Liu, Chun-Ming

    2015-01-01

    The presence of introns in gene-coding regions is one of the most mysterious evolutionary inventions in eukaryotic organisms. It has been proposed that, although sequences involved in intron recognition and splicing are mainly located in introns, exonic sequences also contribute to intron splicing. The smallest constitutively spliced exon known so far has 6 nucleotides, and the smallest alternatively spliced exon has 3 nucleotides. Here we report that the Anaphase Promoting Complex subunit 11 (APC11) gene in Arabidopsis thaliana carries a constitutive single-nucleotide exon. In vivo transcription and translation assays performed using APC11-Green Fluorescence Protein (GFP) fusion constructs revealed that intron splicing surrounding the single-nucleotide exon is effective in both Arabidopsis and rice. This discovery warrants attention to genome annotations in the future. PMID:26657562

  18. A single-nucleotide exon found in Arabidopsis.

    PubMed

    Guo, Lei; Liu, Chun-Ming

    2015-01-01

    The presence of introns in gene-coding regions is one of the most mysterious evolutionary inventions in eukaryotic organisms. It has been proposed that, although sequences involved in intron recognition and splicing are mainly located in introns, exonic sequences also contribute to intron splicing. The smallest constitutively spliced exon known so far has 6 nucleotides, and the smallest alternatively spliced exon has 3 nucleotides. Here we report that the Anaphase Promoting Complex subunit 11 (APC11) gene in Arabidopsis thaliana carries a constitutive single-nucleotide exon. In vivo transcription and translation assays performed using APC11-Green Fluorescence Protein (GFP) fusion constructs revealed that intron splicing surrounding the single-nucleotide exon is effective in both Arabidopsis and rice. This discovery warrants attention to genome annotations in the future. PMID:26657562

  19. Single Nucleotide Polymorphisms for Pig Identification and Parentage Exclusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms have become an important type of marker for commercial diagnostic and parentage genotyping applications as automated genotyping systems have been developed that yield accurate genotypes. Unfortunately, a large number of highly informative public SNP markers tested in ...

  20. Supramolecular gels made from nucleobase, nucleoside and nucleotide analogs.

    PubMed

    Peters, Gretchen Marie; Davis, Jeffery T

    2016-06-01

    Supramolecular or molecular gels are attractive for various applications, including diagnostics, tissue scaffolding and targeted drug release. Gelators derived from natural products are of particular interest for biomedical purposes, as they are generally biocompatible and stimuli-responsive. The building blocks of nucleic acids (i.e. nucleobases, nucleosides, and nucleotides) are desirable candidates for supramolecular gelation as they readily engage in reversible, noncovalent interactions. In this review, we describe a number of organo- and hydrogels formed through the assembly of nucleosides, nucleotides, and their derivatives. While natural nucleosides and nucleotides generally require derivatization to induce gelation, guanosine and its corresponding nucleotides are well known gelators. This unique gelating ability is due to propensity of the guanine nucleobase to self-associate into stable higher-order assemblies, such as G-ribbons, G4-quartets, and G-quadruplexes. PMID:27146863

  1. A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein*

    PubMed Central

    Bartoszewski, Rafal A.; Jablonsky, Michael; Bartoszewska, Sylwia; Stevenson, Lauren; Dai, Qun; Kappes, John; Collawn, James F.; Bebok, Zsuzsa

    2010-01-01

    Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients. PMID:20628052

  2. A Pedigree with c.179 Cytosine to Threonine Missense Mutation of SLC12A3 Gene Presenting Gitelman's Syndrome

    PubMed Central

    Kim, Yaerim; Kang, Seong Sik; Park, Woo Yeong; Jin, Kyubok; Kim, Dae-Kwang

    2016-01-01

    A 42-year-old man came to the hospital presenting chest discomfort and general weakness. He had come to the hospital with the same symptoms 3 months ago and 12 years prior. His laboratory test showed hypokalemia, hypomagnesemia and hypocalciuria. The arterial blood gas analysis showed hypochloremic metabolic alkalosis. He had an ultrasonography guided renal biopsy, the result was normal at light microscopy and immunofluorescence microscopy. However, a special stain for Na-Cl cotransporter was weakly expressed compared with the control. The patient and his family underwent genetic sequencing about the SLC12A3 gene. He had a homozygous mutation in the 179th nucleotide of Exon 1 on the SLC12A3 gene (p.Thr60Met) and his parents and sisters were diagnosed as carrier state of Gitelman's syndrome (GS). GS is an inherited tubular disorder which presents mild hypokalemia, hypomagnesemia and hypocalciuria. Since the symptoms and laboratory results are not severe, it can go unnoticed by physicians. Herein we present a family with GS, diagnosed by genetic sequencing. PMID:27453715

  3. A 5′ cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

    SciTech Connect

    Zhu, Deyu; Stumpf, Craig R.; Krahn, Joseph M.; Wickens, Marvin; Tanaka Hall, Traci M.

    2010-11-03

    A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the 'core' PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

  4. Reducing nontemplated 3' nucleotide addition to polynucleotide transcripts

    DOEpatents

    Kao, C. Cheng

    2000-01-01

    Non-template 3' nucleotide addition to a transcript is reduced by transcribing a transcript from a template comprising an ultimate and/or penultimate 5' ribose having a C'2 substituent such as methoxy, which reduces non-template 3' nucleotide addition to the transcript. The methods are shown to be applicable to a wide variety of polymerases, including Taq, T7 RNA polymerase, etc.

  5. An introduction to recurrent nucleotide interactions in RNA.

    PubMed

    Sweeney, Blake A; Roy, Poorna; Leontis, Neocles B

    2015-01-01

    RNA secondary structure diagrams familiar to molecular biologists summarize at a glance the folding of RNA chains to form Watson–Crick paired double helices. However, they can be misleading: First of all, they imply that the nucleotides in loops and linker segments, which can amount to 35% to 50% of a structured RNA, do not significantly interact with other nucleotides. Secondly, they give the impression that RNA molecules are loosely organized in three-dimensional (3D) space. In fact, structured RNAs are compactly folded as a result of numerous long-range, sequence-specific interactions, many of which involve loop or linker nucleotides. Here, we provide an introduction for students and researchers of RNA on the types, prevalence, and sequence variations of inter-nucleotide interactions that structure and stabilize RNA 3D motifs and architectures, using Escherichia coli (E. coli) 16S ribosomal RNA as a concrete example. The picture that emerges is that almost all nucleotides in structured RNA molecules, including those in nominally single-stranded loop or linker regions, form specific interactions that stabilize functional structures or mediate interactions with other molecules. The small number of noninteracting, ‘looped-out’ nucleotides make it possible for the RNA chain to form sharp turns. Base-pairing is the most specific interaction in RNA as it involves edge-to-edge hydrogen bonding (H-bonding) of the bases. Non-Watson–Crick base pairs are a significant fraction (30% or more) of base pairs in structured RNAs. PMID:25664365

  6. Genome-wide screen of genes imprinted in sorghum endosperm, and the roles of allelic differential cytosine methylation.

    PubMed

    Zhang, Meishan; Li, Ning; He, Wenan; Zhang, Huakun; Yang, Wei; Liu, Bao

    2016-02-01

    Imprinting is an epigenetic phenomenon referring to allele-biased expression of certain genes depending on their parent of origin. Accumulated evidence suggests that, while imprinting is a conserved mechanism across kingdoms, the identities of the imprinted genes are largely species-specific. Using deep RNA sequencing of endosperm 14 days after pollination in sorghum, 5683 genes (29.27% of the total 19 418 expressed genes) were found to harbor diagnostic single nucleotide polymorphisms between two parental lines. The analysis of parent-of-origin expression patterns in the endosperm of a pair of reciprocal F1 hybrids between the two sorghum lines led to identification of 101 genes with ≥ fivefold allelic expression difference in both hybrids, including 85 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs). Thirty of these genes were previously identified as imprinted in endosperm of maize (Zea mays), rice (Oryza sativa) or Arabidopsis, while the remaining 71 genes are sorghum-specific imprinted genes relative to these three plant species. Allele-biased expression of virtually all of the 14 tested imprinted genes (nine MEGs and five PEGs) was validated by pyrosequencing using independent sources of RNA from various developmental stages and dissected parts of endosperm. Forty-six imprinted genes (30 MEGs and 16 PEGs) were assayed by quantitative RT-PCR, and the majority of them showed endosperm-specific or preferential expression relative to embryo and other tissues. DNA methylation analysis of the 5' upstream region and gene body for seven imprinted genes indicated that, while three of the four PEGs were associated with hypomethylation of maternal alleles, no MEG was associated with allele-differential methylation. PMID:26718755

  7. Targeting Cyclic Nucleotide Phosphodiesterase in the Heart: Therapeutic Implications

    PubMed Central

    Miller, Clint L.

    2010-01-01

    The second messengers, cAMP and cGMP, regulate a number of physiological processes in the myocardium, from acute contraction/relaxation to chronic gene expression and cardiac structural remodeling. Emerging evidence suggests that multiple spatiotemporally distinct pools of cyclic nucleotides can discriminate specific cellular functions from a given cyclic nucleotide-mediated signal. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing intracellular cyclic AMP and/or cyclic GMP, control the amplitude, duration, and compartmentation of cyclic nucleotide signaling. To date, more than 60 different isoforms have been described and grouped into 11 broad families (PDE1–PDE11) based on differences in their structure, kinetic and regulatory properties, as well as sensitivity to chemical inhibitors. In the heart, PDE isozymes from at least six families have been investigated. Studies using selective PDE inhibitors and/or genetically manipulated animals have demonstrated that individual PDE isozymes play distinct roles in the heart by regulating unique cyclic nucleotide signaling microdomains. Alterations of PDE activity and/or expression have also been observed in various cardiac disease models, which may contribute to disease progression. Several family-selective PDE inhibitors have been used clinically or pre-clinically for the treatment of cardiac or vascular-related diseases. In this review, we will highlight both recent advances and discrepancies relevant to cardiovascular PDE expression, pathophysiological function, and regulation. In particular, we will emphasize how these properties influence current and future development of PDE inhibitors for the treatment of pathological cardiac remodeling and dysfunction. PMID:20632220

  8. Moss Phylogeny Reconstruction Using Nucleotide Pangenome of Complete Mitogenome Sequences.

    PubMed

    Goryunov, D V; Nagaev, B E; Nikolaev, M Yu; Alexeevski, A V; Troitsky, A V

    2015-11-01

    Stability of composition and sequence of genes was shown earlier in 13 mitochondrial genomes of mosses (Rensing, S. A., et al. (2008) Science, 319, 64-69). It is of interest to study the evolution of mitochondrial genomes not only at the gene level, but also on the level of nucleotide sequences. To do this, we have constructed a "nucleotide pangenome" for mitochondrial genomes of 24 moss species. The nucleotide pangenome is a set of aligned nucleotide sequences of orthologous genome fragments covering the totality of all genomes. The nucleotide pangenome was constructed using specially developed new software, NPG-explorer (NPGe). The stable part of the mitochondrial genome (232 stable blocks) is shown to be, on average, 45% of its length. In the joint alignment of stable blocks, 82% of positions are conserved. The phylogenetic tree constructed with the NPGe program is in good correlation with other phylogenetic reconstructions. With the NPGe program, 30 blocks have been identified with repeats no shorter than 50 bp. The maximal length of a block with repeats is 140 bp. Duplications in the mitochondrial genomes of mosses are rare. On average, the genome contains about 500 bp in large duplications. The total length of insertions and deletions was determined in each genome. The losses and gains of DNA regions are rather active in mitochondrial genomes of mosses, and such rearrangements presumably can be used as additional markers in the reconstruction of phylogeny. PMID:26615445

  9. Targeted enzyme prodrug therapy for metastatic prostate cancer – a comparative study of L-methioninase, purine nucleoside phosphorylase, and cytosine deaminase

    PubMed Central

    2014-01-01

    Background Enzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3. Results All three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p < 0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV. Conclusions Strong binding of fusion proteins to the prostate cancer cells

  10. Getting it Right: How DNA Polymerases Select the Right Nucleotide.

    PubMed

    Ludmann, Samra; Marx, Andreas

    2016-01-01

    All living organisms are defined by their genetic code encrypted in their DNA. DNA polymerases are the enzymes that are responsible for all DNA syntheses occurring in nature. For DNA replication, repair and recombination these enzymes have to read the parental DNA and recognize the complementary nucleotide out of a pool of four structurally similar deoxynucleotide triphosphates (dNTPs) for a given template. The selection of the nucleotide is in accordance with the Watson-Crick rule. In this process the accuracy of DNA synthesis is crucial for the maintenance of the genome stability. However, to spur evolution a certain degree of freedom must be allowed. This brief review highlights the mechanistic basis for selecting the right nucleotide by DNA polymerases. PMID:27052761

  11. Fixed-Gap Tunnel Junction for Reading DNA Nucleotides

    PubMed Central

    2015-01-01

    Previous measurements of the electronic conductance of DNA nucleotides or amino acids have used tunnel junctions in which the gap is mechanically adjusted, such as scanning tunneling microscopes or mechanically controllable break junctions. Fixed-junction devices have, at best, detected the passage of whole DNA molecules without yielding chemical information. Here, we report on a layered tunnel junction in which the tunnel gap is defined by a dielectric layer, deposited by atomic layer deposition. Reactive ion etching is used to drill a hole through the layers so that the tunnel junction can be exposed to molecules in solution. When the metal electrodes are functionalized with recognition molecules that capture DNA nucleotides via hydrogen bonds, the identities of the individual nucleotides are revealed by characteristic features of the fluctuating tunnel current associated with single-molecule binding events. PMID:25380505

  12. Proton-translocating nicotinamide nucleotide transhydrogenase. Reconstitution of the extramembranous nucleotide-binding domains.

    PubMed

    Yamaguchi, M; Hatefi, Y

    1995-11-24

    The nicotinamide nucleotide transhydrogenase of bovine mitochondria is a homodimer of monomer M(r) = 109,065. The monomer is composed of three domains, an NH2-terminal 430-residue-long hydrophilic domain I that binds NAD(H), a central 400-residue-long hydrophobic domain II that is largely membrane intercalated and carries the enzyme's proton channel, and a COOH-terminal 200-residue-long hydrophilic domain III that binds NADP(H). Domains I and III protrude into the mitochondrial matrix, where they presumably come together to form the enzyme's catalytic site. The two-subunit transhydrogenase of Escherichia coli and the three-subunit transhydrogenase of Rhodospirillum rubrum have each the same overall tridomain hydropathy profile as the bovine enzyme. Domain I of the R. rubrum enzyme (the alpha 1 subunit) is water soluble and easily removed from the chromatophore membranes. We have isolated domain I of the bovine transhydrogenase after controlled trypsinolysis of the purified enzyme and have expressed in E. coli and purified therefrom domain III of this enzyme. This paper shows that an active bidomain transhydrogenase lacking domain II can be reconstituted by the combination of purified bovine domains I plus III or R. rubrum domain I plus bovine domain III. PMID:7499307

  13. Characterization of Nucleotide Misincorporation Patterns in the Iceman's Mitochondrial DNA

    PubMed Central

    Olivieri, Cristina; Ermini, Luca; Rizzi, Ermanno; Corti, Giorgio; Bonnal, Raoul; Luciani, Stefania; Marota, Isolina; De Bellis, Gianluca; Rollo, Franco

    2010-01-01

    Background The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation (“miscoding lesions”) has been the object of extensive investigations. Methodology/Principal Findings To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp) mitochondrial (mt) DNA sequences obtained from the 5,350–5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Ötzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp) mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85%) of the observed nucleotide misincorporations in ancient sequences. Conclusions/Significance This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine→thymine/guanine→adenine) transitions and

  14. Content discovery and retrieval services at the European Nucleotide Archive

    PubMed Central

    Silvester, Nicole; Alako, Blaise; Amid, Clara; Cerdeño-Tárraga, Ana; Cleland, Iain; Gibson, Richard; Goodgame, Neil; ten Hoopen, Petra; Kay, Simon; Leinonen, Rasko; Li, Weizhong; Liu, Xin; Lopez, Rodrigo; Pakseresht, Nima; Pallreddy, Swapna; Plaister, Sheila; Radhakrishnan, Rajesh; Rossello, Marc; Senf, Alexander; Smirnov, Dmitriy; Toribio, Ana Luisa; Vaughan, Daniel; Zalunin, Vadim; Cochrane, Guy

    2015-01-01

    The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe's primary resource for nucleotide sequence information. With the growing volume and diversity of public sequencing data comes the need for increased sophistication in data organisation, presentation and search services so as to maximise its discoverability and usability. In response to this, ENA has been introducing and improving checklists for use during submission and expanding its search facilities to provide targeted search results. Here, we give a brief update on ENA content and some major developments undertaken in data submission services during 2014. We then describe in more detail the services we offer for data discovery and retrieval. PMID:25404130

  15. Biocuration of functional annotation at the European nucleotide archive

    PubMed Central

    Gibson, Richard; Alako, Blaise; Amid, Clara; Cerdeño-Tárraga, Ana; Cleland, Iain; Goodgame, Neil; ten Hoopen, Petra; Jayathilaka, Suran; Kay, Simon; Leinonen, Rasko; Liu, Xin; Pallreddy, Swapna; Pakseresht, Nima; Rajan, Jeena; Rosselló, Marc; Silvester, Nicole; Smirnov, Dmitriy; Toribio, Ana Luisa; Vaughan, Daniel; Zalunin, Vadim; Cochrane, Guy

    2016-01-01

    The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is a repository for the submission, maintenance and presentation of nucleotide sequence data and related sample and experimental information. In this article we report on ENA in 2015 regarding general activity, notable published data sets and major achievements. This is followed by a focus on sustainable biocuration of functional annotation, an area which has particularly felt the pressure of sequencing growth. The importance of functional annotation, how it can be submitted and the shifting role of the biocurator in the context of increasing volumes of data are all discussed. PMID:26615190

  16. Biodegradation of DNA and nucleotides to nucleosides and free bases.

    PubMed

    Kruszewska, Hanna; Misicka, Aleksandra; Chmielowiec, Urszula

    2004-01-01

    Thirty-two different microorganisms were examined in order to check their ability to degrade an exogenous DNA. Bacteria from species: Stenotrophomonas maltophilia, Brevundimonas diminuta, Bacillus subtilis, Mycobacterium butyricum and fungus Fusarium moniliforme were capable to degrade DNA to nucleic bases or their derivatives. Degradation of DNA by S. maltophilia resulted in formation of free bases, such as hypoxanthine, thymine, uracil and xanthine. The optimum concentration of DNA seemed to be 3 mg ml(-1). The mode of degradation of DNA nucleotides depended on the type of nucleotide and its concentration, but nucleic bases or their derivatives were always formed at the end of the reaction process. PMID:14751311

  17. Biocuration of functional annotation at the European nucleotide archive.

    PubMed

    Gibson, Richard; Alako, Blaise; Amid, Clara; Cerdeño-Tárraga, Ana; Cleland, Iain; Goodgame, Neil; Ten Hoopen, Petra; Jayathilaka, Suran; Kay, Simon; Leinonen, Rasko; Liu, Xin; Pallreddy, Swapna; Pakseresht, Nima; Rajan, Jeena; Rosselló, Marc; Silvester, Nicole; Smirnov, Dmitriy; Toribio, Ana Luisa; Vaughan, Daniel; Zalunin, Vadim; Cochrane, Guy

    2016-01-01

    The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is a repository for the submission, maintenance and presentation of nucleotide sequence data and related sample and experimental information. In this article we report on ENA in 2015 regarding general activity, notable published data sets and major achievements. This is followed by a focus on sustainable biocuration of functional annotation, an area which has particularly felt the pressure of sequencing growth. The importance of functional annotation, how it can be submitted and the shifting role of the biocurator in the context of increasing volumes of data are all discussed. PMID:26615190

  18. Simplified computer programs for search of homology within nucleotide sequences.

    PubMed Central

    Kröger, M; Kröger-Block, A

    1984-01-01

    Four new computer programs for search of homology within nucleotide sequences are presented. The main scope of the program design is flexibility, independence of sequence length and the capability to be used by any molecular biologist without any prior computer experience. The programs offer a linear search, a search for maximal identity, an alignment along a given sequence and a search based on homology within the amino acid coding capacity of nucleotide sequences. The language is Fortran V. Copies are available on request. PMID:6546417

  19. Electrophoretic Transport of Single DNA Nucleotides through Nanoslits: A Molecular Dynamics Simulation Study.

    PubMed

    Xia, Kai; Novak, Brian R; Weerakoon-Ratnayake, Kumuditha M; Soper, Steven A; Nikitopoulos, Dimitris E; Moldovan, Dorel

    2015-09-01

    There is potential for flight time based DNA sequencing involving disassembly into individual nucleotides which would pass through a nanochannel with two or more detectors. We performed molecular dynamics simulations of electrophoretic motion of single DNA nucleotides through 3 nm wide hydrophobic slits with both smooth and rough walls. The electric field (E) varied from 0.0 to 0.6 V/nm. The nucleotides adsorb and desorb from walls multiple times during their transit through the slit. The nucleotide-wall interactions differed due to nucleotide hydrophobicities and wall roughness which determined duration and frequency of nucleotide adsorptions and their velocities while adsorbed. Transient association of nucleotides with one, two, or three sodium ions occurred, but the mean association numbers (ANs) were weak functions of nucleotide type. Nucleotide-wall interactions contributed more to separation of nucleotide flight time distributions than ion association and thus indicate that nucleotide-wall interactions play a defining role in successfully discriminating between nucleotides on the basis of their flight times through nanochannels/slits. With smooth walls, smaller nucleotides moved faster, but with rough walls larger nucleotides moved faster due to fewer favorable wall adsorption sites. This indicates that roughness, or surface patterning, might be exploited to achieve better time-of-flight based discrimination between nucleotides. PMID:26237155

  20. Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1

    PubMed Central

    Sharma, Sunny; Langhendries, Jean-Louis; Watzinger, Peter; Kötter, Peter; Entian, Karl-Dieter; Lafontaine, Denis L.J.

    2015-01-01

    The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson–Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function. PMID:25653167

  1. Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1.

    PubMed

    Sharma, Sunny; Langhendries, Jean-Louis; Watzinger, Peter; Kötter, Peter; Entian, Karl-Dieter; Lafontaine, Denis L J

    2015-02-27

    The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson-Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function. PMID:25653167

  2. In Vivo Spectrum of UVC-induced Mutation in Mouse Skin Epidermis May Reflect the Cytosine Deamination Propensity of Cyclobutane Pyrimidine Dimers.

    PubMed

    Ikehata, Hironobu; Mori, Toshio; Yamamoto, Masayuki

    2015-11-01

    Although ultraviolet radiation (UVR) has a genotoxicity for inducing skin cancers, the skin may tolerate UVC component because the epidermal layer prevents this short wavelength range from passing through. Here, UVC genotoxicity for mouse skin was evaluated in terms of DNA damage formation and mutagenicity. UVC induced UVR photolesions and mutations remarkably in the epidermis but poorly in the dermis, confirming the barrier ability of the epidermis against shorter UVR wavelengths. Moreover, the epidermis itself responded to UVC mutagenicity with mutation induction suppression, which suppressed the mutant frequencies to a remarkably low, constant level regardless of UVC dose. The mutation spectrum observed in UVC-exposed epidermis showed a predominance of UV-signature mutation, which occurred frequently in 5'-TCG-3', 5'-TCA-3' and 5'-CCA-3' contexts. Especially, for the former two contexts, the mutations recurred at several sites with more remarkable recurrences at the 5'-TCG-3' sites. Comparison of the UVC mutation spectrum with those observed in longer UVR wavelength ranges led us to a mechanism that explains why the sequence context preference of UV-signature mutation changes according to the wavelength, which is based on the difference in the mCpG preference of cyclobutane pyrimidine dimer (CPD) formation among UVR ranges and the sequence context-dependent cytosine deamination propensity of CPD. PMID:26335024

  3. Stacked and H-Bonded Cytosine Dimers. Analysis of the Intermolecular Interaction Energies by Parallel Quantum Chemistry and Polarizable Molecular Mechanics.

    PubMed

    Gresh, Nohad; Sponer, Judit E; Devereux, Mike; Gkionis, Konstantinos; de Courcy, Benoit; Piquemal, Jean-Philip; Sponer, Jiri

    2015-07-30

    Until now, atomistic simulations of DNA and RNA and their complexes have been executed using well calibrated but conceptually simple pair-additive empirical potentials (force fields). Although such simulations provided many valuable results, it is well established that simple force fields also introduce errors into the description, underlying the need for development of alternative anisotropic, polarizable molecular mechanics (APMM) potentials. One of the most abundant forces in all kinds of nucleic acids topologies is base stacking. Intra- and interstrand stacking is assumed to be the most essential factor affecting local conformational variations of B-DNA. However, stacking also contributes to formation of all kinds of noncanonical nucleic acids structures, such as quadruplexes or folded RNAs. The present study focuses on 14 stacked cytosine (Cyt) dimers and the doubly H-bonded dimer. We evaluate the extent to which an APMM procedure, SIBFA, could account quantitatively for the results of high-level quantum chemistry (QC) on the total interaction energies, and the individual energy contributions and their nonisotropic behaviors. Good agreements are found at both uncorrelated HF and correlated DFT and CCSD(T) levels. Resorting in SIBFA to distributed QC multipoles and to an explicit representation of the lone pairs is essential to respectively account for the anisotropies of the Coulomb and of the exchange-repulsion QC contributions. PMID:26119247

  4. Cytosine arabinoside potentiates the apoptotic effect of bendamustine on several B- and T-cell leukemia/lymphoma cells and cell lines.

    PubMed

    Castegnaro, Silvia; Visco, Carlo; Chieregato, Katia; Bernardi, Martina; Albiero, Elena; Zanon, Cristina; Madeo, Domenico; Rodeghiero, Francesco

    2012-11-01

    Bendamustine and cytosine arabinoside (ara-c) are commonly used cytotoxic agents with unique mechanisms of action. We have previously reported a striking additive cytotoxic effect of the consecutive combination of bendamustine and ara-c in mantle cell lymphoma (MCL) cell lines. In the present study, cell lines of follicular lymphoma (DOHH-2), chronic lymphocytic leukemia/lymphoma (EHEB), diffuse large B-cell lymphoma (SU-DHL-4), T-cell leukemia/lymphoma (JURKAT and KARPAS-299) and MCL (JEKO-1 and GRANTA-519) were exposed to the two single drugs or the drugs combined, given simultaneously and consecutively. Peripheral blood chronic lymphocytic leukemia (CLL) B-cells from five patients were also analyzed. Apoptosis, cell proliferation/metabolic activity and mitochondrial damage were evaluated. The combination index (CI) was used to assess synergy between the drugs. Bendamustine exhibited a relevant cytotoxic effect that was dose- and time-dependent, except for SU-DHL-4 and T-cell lymphoma cells. The addition of ara-c after bendamustine significantly potentiated the single-drug cytotoxic effect of bendamustine on all cell lines, including 17p - CLL B-cells, JURKAT and SU-DHL-4, the latter presenting the highest synergism (CI < 0.01). Bendamustine and ara-c are highly synergistic on T- and B-cell lymphoma cells and cell lines, similar to MCL, overcoming resistance to the single agents. PMID:22530665

  5. Behenoyl cytosine arabinoside, daunorubicin, 6-mercaptopurine, and prednisolone combination therapy for acute myelogenous leukemia in adults and prognostic factors related to remission duration and survival length.

    PubMed

    Ohno, R; Kato, Y; Nagura, E; Murase, T; Okumura, M; Yamada, H; Ogura, M; Minami, S; Suzuki, H; Morishima, Y

    1986-12-01

    Fifty-one consecutive previously untreated adult patients with acute myelogenous leukemia (AML) were treated with BHAC-DMP (N4-behenoyl-I-beta-D-arabinofuranosyl-cytosine, daunorubicin, 6-mercaptopurine, and prednisolone) therapy. Forty-two patients (82.4%) achieved complete remission (CR). The Kaplan-Meier analysis revealed a probability for remaining in remission of 14% and for survival of 23% at 6 years. Pretreatment factors related to the achievement of CR, such as age, French-American-British (FAB) classification and WBC at the start of treatment, were not identified. Factors related to the CR duration and survival time of the patients who had achieved CR were first analyzed by a univariate analysis with the generalized Wilcoxon test. WBC count at the start of treatment, percent of blasts in the marrow at 1 and 2 weeks after the initiation of therapy, days required until CR, number of courses of induction therapy required until CR, and days required for the disappearance of circulating blasts were identified as statistically significant prognostic factors. When these characteristics were further analyzed by the Cox multivariate regression model, the percent of blasts in the bone marrow at 2 weeks was the most important prognostic factor with a statistical significance, and WBC count at the start of treatment and days required until CR (or number of courses required to achieve CR) were also important factors, with borderline significance. PMID:3465875

  6. Absence of a gene encoding cytosine deaminase in the genome of the agaricomycete Coprinopsis cinerea enables simple marker recycling through 5-fluorocytosine counterselection.

    PubMed

    Nakazawa, Takehito; Honda, Yoichi

    2015-08-01

    Coprinopsis cinerea is a model species for molecular genetics studies of sexual development in agaricomycetes or homobasidiomycetes. Recently, efficient gene targeting was established in this fungus by generating Cc.ku70 or Cc.lig4 disruptants. To determine the molecular mechanisms underlying sexual development, which involves many genes, generating multiple gene disruptants is required. However, the number of transformation markers available for C. cinerea is limited. This problem would be solved by establishing marker recycling. In this study, we found that C. cinerea lacks a gene encoding a homolog of Saccharomyces cerevisiae cytosine deaminase (Fcy1p) in its genome, which is present in many other fungi. We also observed that C. cinerea is resistant to 5-fluorocytosine. Based on these findings, we established a simple marker recycling method in this fungus using 5-fluorocytosine counterselection after heterologous expression of FCY1 derived from Pleurotus ostreatus, together with the hygromycin resistance gene. This study proposes a simple genetic manipulation system that can be performed using wild-type strains of several fungi that lack a gene homologous to S. cerevisiae FCY1 in their genomes. PMID:26223587

  7. Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

    PubMed Central

    Chung, Daehwan; Farkas, Joel; Huddleston, Jennifer R.; Olivar, Estefania; Westpheling, Janet

    2012-01-01

    Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation. PMID:22928042

  8. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

    PubMed Central

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Martinussen, Jan

    2015-01-01

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. PMID:25746996

  9. Epigenetically modified nucleotides in chronic heroin and cocaine treated mice.

    PubMed

    Chao, Mu-Rong; Fragou, Domniki; Zanos, Panos; Hu, Chiung-Wen; Bailey, Alexis; Kouidou, Sofia; Kovatsi, Leda

    2014-09-17

    Epigenetic changes include the addition of a methyl group to the 5' carbon of the cytosine ring, known as DNA methylation, which results in the generation of the fifth DNA base, namely 5-methylcytosine. During active or passive demethylation, an intermediate modified base is formed, 5-hydroxymethylcytosine. We have currently quantified 5-methylcytosine and 5-hydroxymethylcytosine in the liver and brain of mice treated with cocaine or heroin, using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Our results show that global 5-methylcytosine levels are not affected by heroin or cocaine administration, neither in the liver nor in the brain. However, 5-hydroxymethylcytosine levels are reduced in the liver following cocaine administration, while they are not affected by cocaine in the brain or by heroin administration in the liver and the brain. Elucidation of the epigenetic phenomena that takes place with respect to drug abuse and addiction, via quantitative analysis of different modified bases, may enable a better understanding of the underlying mechanisms and may lead to more personalized and effective treatment options. PMID:25064621

  10. HIF-1α in heart: remodeling nucleotide metabolism

    PubMed Central

    Wu, Joe; Bond, Cherie; Chen, Ping; Chen, Minghua; Li, Ying; Shohet, Ralph V.; Wright, Gary

    2015-01-01

    These studies have examined the effect of hypoxia inducible factor 1α (HIF-1α) on nucleotide metabolism in the ischemic heart using a genetic mouse model with heart-specific and regulated expression of a stable form of HIF-1α. We find that AMP deaminase (AMPD), the entry point of the purine nucleotide cycle (PNC), is induced by HIF-1α at the level of mRNA, protein, and activity. AMP that accumulates during ischemia can be metabolized to adenosine by 5′-nucleotidase or to IMP by AMPD. Consistent with the finding of AMPD induction, adenosine accumulation during ischemia was much attenuated in HIF-1α-expressing hearts. Further investigation of nucleotide salvage enzymes found that hypoxanthine phosphoribosyl transferase (HPRT) is also upregulated in HIF-1α-expressing hearts. Treatment of hearts with an inhibitor of the PNC, hadacidin, hastens the fall of the adenylate energy charge during ischemia and the accumulation of AMP. The results provide new insight into the role of the PNC in heart, especially as it relates to ischemia, and indicate that HIF-1α regulates nucleotide metabolism as a compensatory response to hypoxia. PMID:25681585

  11. DNA Nucleotides Detection via capacitance properties of Graphene

    NASA Astrophysics Data System (ADS)

    Khadempar, Nahid; Berahman, Masoud; Yazdanpanah, Arash

    2016-05-01

    In the present paper a new method is suggested to detect the DNA nucleotides on a first-principles calculation of the electronic features of DNA bases which chemisorbed to a graphene sheet placed between two gold electrodes in a contact-channel-contact system. The capacitance properties of graphene in the channel are surveyed using non-equilibrium Green's function coupled with the Density Functional Theory. Thus, the capacitance properties of graphene are theoretically investigated in a biological environment, and, using a novel method, the effect of the chemisorbed DNA nucleotides on electrical charges on the surface of graphene is deciphered. Several parameters in this method are also extracted including Electrostatic energy, Induced density, induced electrostatic potential, Electron difference potential and Electron difference density. The qualitative and quantitative differences among these parameters can be used to identify DNA nucleotides. Some of the advantages of this approach include its ease and high accuracy. What distinguishes the current research is that it is the first experiment to investigate the capacitance properties of gaphene changes in the biological environment and the effect of chemisorbed DNA nucleotides on the surface of graphene on the charge.

  12. XPA: A key scaffold for human nucleotide excision repair.

    PubMed

    Sugitani, Norie; Sivley, Robert M; Perry, Kelly E; Capra, John A; Chazin, Walter J

    2016-08-01

    Nucleotide excision repair (NER) is essential for removing many types of DNA lesions from the genome, yet the mechanisms of NER in humans remain poorly understood. This review summarizes our current understanding of the structure, biochemistry, interaction partners, mechanisms, and disease-associated mutations of one of the critical NER proteins, XPA. PMID:27247238

  13. DNA sequence representation by trianders and determinative degree of nucleotides

    PubMed Central

    Duplij, Diana; Duplij, Steven

    2005-01-01

    A new version of DNA walks, where nucleotides are regarded unequal in their contribution to a walk is introduced, which allows us to study thoroughly the “fine structure” of nucleotide sequences. The approach is based on the assumption that nucleotides have an inner abstract characteristic, the determinative degree, which reflects genetic code phenomenological properties and is adjusted to nucleotides physical properties. We consider each codon position independently, which gives three separate walks characterized by different angles and lengths, and that such an object is called triander which reflects the “strength” of branch. A general method for identifying DNA sequence “by triander” which can be treated as a unique “genogram” (or “gene passport”) is proposed. The two- and three-dimensional trianders are considered. The difference of sequences fine structure in genes and the intergenic space is shown. A clear triplet signal in coding sequences was found which is absent in the intergenic space and is independent from the sequence length. This paper presents the topological classification of trianders which can allow us to provide a detailed working out signatures of functionally different genomic regions. PMID:16052707

  14. Single Nucleotide Polymorphisms Predict Symptom Severity of Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Jiao, Yun; Chen, Rong; Ke, Xiaoyan; Cheng, Lu; Chu, Kangkang; Lu, Zuhong; Herskovits, Edward H.

    2012-01-01

    Autism is widely believed to be a heterogeneous disorder; diagnosis is currently based solely on clinical criteria, although genetic, as well as environmental, influences are thought to be prominent factors in the etiology of most forms of autism. Our goal is to determine whether a predictive model based on single-nucleotide polymorphisms (SNPs)…

  15. The role of nucleotide sugar transporters in development of eukaryotes

    PubMed Central

    Liu, Li; Xu, Yu-Xin; Hirschberg, Carlos B.

    2010-01-01

    The Golgi apparatus membrane of all eukaryotes has nucleotide sugar transporters which play essential roles in the glycosylation of glycoproteins, proteoglycans and glycolipids. Mutations of these transporters have broad developmental phenotypes across many species including diseases in humans and cattle. PMID:20144721

  16. HIF-1α in the heart: remodeling nucleotide metabolism.

    PubMed

    Wu, Joe; Bond, Cherie; Chen, Ping; Chen, Minghua; Li, Ying; Shohet, Ralph V; Wright, Gary

    2015-05-01

    These studies have examined the effect of hypoxia inducible factor 1α (HIF-1α) on nucleotide metabolism in the ischemic heart using a genetic mouse model with heart-specific and regulated expression of a stable form of HIF-1α. We find that AMP deaminase (AMPD), the entry point of the purine nucleotide cycle (PNC), is induced by HIF-1α at the level of mRNA, protein, and activity. AMP that accumulates during ischemia can be metabolized to adenosine by 5'-nucleotidase or to IMP by AMPD. Consistent with the finding of AMPD induction, adenosine accumulation during ischemia was much attenuated in HIF-1α-expressing hearts. Further investigation of nucleotide salvage enzymes found that hypoxanthine phosphoribosyl transferase (HPRT) is also upregulated in HIF-1α-expressing hearts. Treatment of hearts with an inhibitor of the PNC, hadacidin, hastens the fall of the adenylate energy charge during ischemia and the accumulation of AMP. The results provide new insight into the role of the PNC in the heart, especially as it relates to ischemia, and indicate that HIF-1α regulates nucleotide metabolism as a compensatory response to hypoxia. PMID:25681585

  17. A Laboratory Exercise for Genotyping Two Human Single Nucleotide Polymorphisms

    ERIC Educational Resources Information Center

    Fernando, James; Carlson, Bradley; LeBard, Timothy; McCarthy, Michael; Umali, Finianne; Ashton, Bryce; Rose, Ferrill F., Jr.

    2016-01-01

    The dramatic decrease in the cost of sequencing a human genome is leading to an era in which a wide range of students will benefit from having an understanding of human genetic variation. Since over 90% of sequence variation between humans is in the form of single nucleotide polymorphisms (SNPs), a laboratory exercise has been devised in order to…

  18. Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified approximately 13000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel ...

  19. Complete nucleotide sequence of Nootka lupine vein-clearing virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete genome sequence of Nootka lupine vein-clearing virus (NLVCV) was determined to be 4,172 nucleotides in length containing four open reading frames ORFs with a similar genetic organization and conceptual translations of virus species in the genus Carmovirus, family Tombusviridae. The orde...

  20. Guanosine nucleotide precursor for flavinogenesis of Eremothecium Ashbyii.

    PubMed

    Mitsuda, H; Nakajima, K

    1975-01-01

    The purine precursor in the riboflavin biosynthetic pathway in Eremothecium ashbyii was examined using a guanine analogue, 8-azaguanine, with non-growing cell systems. 1. Riboflavin formation in the culture filtrate was determined at 0, 5, 10 and 20 hr after start of the incubation of the non-growing cells in the presence of xanthine or 8-azaguanine (1 mM, respectively). At 20 hr of incubation, the addition of xanthine stimulated riboflavin formation by 36% and the addition of 8-azaguanine inhibited the formation by 57%. 2. Acid soluble nucleotide pools in the cells were followed at 0, 5, 10 and 20 hr of the incubation period in the presence of xanthine or 8-azaguanine by means of anion exchange column chromatography. The result showed that the GTP pool changed markedly despite the fact that the adenosine nucleotide pool was almost constant irrespective of the presence or absence of these purines till 10 hr of incubation. But, the decrease of the former was overcome in part by the addition of flavinogenic xanthine. Furthermore, the total amounts of GTP and guanosine accumulated in cells in the presence of 8-azaguanine reached the maximum already at 5 hr, attaining a level twice as much as the GTP contents of the control. 3. The role of guanosine nucleotide pool in riboflavin formation was further examined using 8-azaguanine. In this experiment the drug was added to the suspension of non-growing cells at 3 hr or 6 hr after the incubation was started and the reaction was continued till the 12th hr. A more clear-cut correlationship between riboflavin formation and guanosine nucleotide pool was oberved by this experiment. The guanosine nucleotide pool (consisting of GMP, GDP and GTP) increased simultaneously with the inhibition of riboflavin formation. Of the guanosine nucleotides pools, the GMP pool increased 2.7 times above normal upon the addition of 8-azaguanine during the incubation for 6 hr and 5.3 fold for 9 hr. While, the GTP pool increased 1.9 fold above

  1. A study into the effects of protein binding on nucleotide conformation.

    PubMed Central

    Moodie, S L; Thornton, J M

    1993-01-01

    In this study, we examine the effects of binding to protein upon nucleotide conformation, by the comparison of X-ray crystal structures of free and protein-bound nucleotides. A dataset of structurally non-homologous protein-nucleotide complexes was derived from the Brookhaven Protein Data Bank by a novel protocol of dual sequential and structural alignments, and a dataset of native nucleotide structures was obtained from the Cambridge Structural Database. The nucleotide torsion angles and sugar puckers, which describe nucleotide conformation, were analysed in both datasets and compared. Differences between them are described and discussed. Overall, the nucleotides were found to bind in low energy conformations, not significantly different from their 'free' conformations except that they adopted an extended conformation in preference to the 'closed' structure predominantly observed by free nucleotide. The archetypal conformation of a protein-bound nucleotide is derived from these observations. PMID:8464727

  2. Conformational changes of P-glycoprotein by nucleotide binding.

    PubMed Central

    Wang, G; Pincheira, R; Zhang, M; Zhang, J T

    1997-01-01

    P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[beta,gamma-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport. PMID:9396736

  3. Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis

    PubMed Central

    Pisithkul, Tippapha; Jacobson, Tyler B.; O'Brien, Thomas J.; Stevenson, David M.

    2015-01-01

    An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. PMID:26070680

  4. Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

    PubMed

    Pisithkul, Tippapha; Jacobson, Tyler B; O'Brien, Thomas J; Stevenson, David M; Amador-Noguez, Daniel

    2015-09-01

    An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-labeled sugars and [(15)N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. PMID:26070680

  5. Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK

    PubMed Central

    Bauer, Daniela; Merz, Dale R.; Pelz, Benjamin; Theisen, Kelly E.; Yacyshyn, Gail; Mokranjac, Dejana; Dima, Ruxandra I.; Rief, Matthias; Žoldák, Gabriel

    2015-01-01

    The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily. PMID:26240360

  6. High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation.

    PubMed

    Villiger, Thomas K; Steinhoff, Robert F; Ivarsson, Marija; Solacroup, Thomas; Stettler, Matthieu; Broly, Hervé; Krismer, Jasmin; Pabst, Martin; Zenobi, Renato; Morbidelli, Massimo; Soos, Miroslav

    2016-07-10

    Recent advances in miniaturized cell culture systems have facilitated the screening of media additives on productivity and protein quality attributes of mammalian cell cultures. However, intracellular components are not routinely measured due to the limited throughput of available analytical techniques. In this work, time profiling of intracellular nucleotides and nucleotide sugars of CHO-S cell fed-batch processes in a micro-scale bioreactor system was carried out using a recently developed high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS). Supplementation of various media additives significantly altered the intracellular nucleotides and nucleotide sugars that are inextricably linked to the process of glycosylation. The results revealed that UDP-Gal synthesis appeared to be particularly limiting whereas the impact of elevated UDP-GlcNAc and GDP-Fuc levels on the final glycosylation patterns was only marginally important. In contrast, manganese and asparagine supplementation altered the glycan profiles without affecting intracellular components. The combination of miniaturized cell cultures and high-throughput analytical techniques serves therefore as a useful tool for future quality driven media optimization studies. PMID:27131894

  7. Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK.

    PubMed

    Bauer, Daniela; Merz, Dale R; Pelz, Benjamin; Theisen, Kelly E; Yacyshyn, Gail; Mokranjac, Dejana; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

    2015-08-18

    The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily. PMID:26240360

  8. Method for the detection of specific nucleic acid sequences by polymerase nucleotide incorporation

    DOEpatents

    Castro, Alonso

    2004-06-01

    A method for rapid and efficient detection of a target DNA or RNA sequence is provided. A primer having a 3'-hydroxyl group at one end and having a sequence of nucleotides sufficiently homologous with an identifying sequence of nucleotides in the target DNA is selected. The primer is hybridized to the identifying sequence of nucleotides on the DNA or RNA sequence and a reporter molecule is synthesized on the target sequence by progressively binding complementary nucleotides to the primer, where the complementary nucleotides include nucleotides labeled with a fluorophore. Fluorescence emitted by fluorophores on single reporter molecules is detected to identify the target DNA or RNA sequence.

  9. Effects of bovine cytochrome P450 single-nucleotide polymorphism, forage type and body condition on production traits in cattle.

    PubMed

    Sales, M A; Larson, M J; Reiter, S T; Brown, A H; Brown, M A; Looper, M L; Coffey, K P; Rosenkrans, C F

    2012-08-01

    Relating single-nucleotide polymorphisms (SNP) to cows with acceptable productivity could benefit cattle breeders in areas where tall fescue is the predominant forage. This study aimed to (i) identify SNPs in bovine cytochrome P450 3A28 (CYP3A28) and (ii) determine the associations between SNP genotype, forage and cow body condition (BC). Genotype (CC, CG or GG) and forage [Kentucky-31 wild-type endophyte-infected tall fescue (KY+) vs. bermudagrass] effects on milk volume and quality were determined in Herd 1 cows (123 cows); in Herd 2 (99 cows), genotype and BC (low vs. moderate) effects on ovarian follicle size, calving date and calving per cent were determined; and in Herd 3 (114 cows), effects of genotype and fescue cultivar [KY+ vs. non-toxic endophyte-infected tall fescue (HiMag4)] were related to calving per cent, calving date and weaning weights of both cow and her calf. A cytosine (C) to guanine (G) transversion at base 994 (C994G) in CYP3A28 was identified. There was a genotype × forage type interaction (p < 0.05) on milk protein in Herd 1 cows; CC cows grazing bermudagrass had greater milk protein percentage in relation to other cows in the herd. In Herd 2, BC and genotype × BC tended (p < 0.10) to influence follicle size and Julian calving date respectively. Diameter of the largest follicle tended to be larger in moderate BC than in low-BC cows; whereas, CC and CG cows in moderate BC and homozygous (CC and GG) cows in low BC tended to calve 14 days earlier in relation to CG cows in low BC. In Herd 3, there was a genotype × forage type interaction (p < 0.05) on calving per cent, Julian calving date and calf weaning weight. In this study, genetic alterations (G allele at C994G) coupled with nutritional factors (low BC and toxic tall fescue) resulted in overall lower productivity in cows. PMID:21668515

  10. Chemotherapy for acute myeloid leukemias with cytosine arabinoside, daunorubicin, etoposide, and mitoxantrone may cause permanent oligoasthenozoospermia or amenorrhea in middle-aged patients.

    PubMed

    Lemez, P; Urbánek, V

    2005-01-01

    The aim was to follow-up gonadal functions in long-term survivors of acute myeloid leukemias (AML) after intensive chemotherapy based on high-doses of cytosine arabinoside (Ara-C) and anthracyclines in the study UHKT-911. Adult patients were treated with at least 3 cycles of chemotherapy including 1-3 courses of Ara-C 10 x 2000 mg/m2/12 h and daunorubicin (DNR) 2 x 45 mg/m2/d. Spermiologic examinations were performed in 7 men by the classic microscopic method and results were evaluated according to the WHOcriteria. Two patients (42- and 47-year-old) after DNR and Ara-C chemotherapy had nearly normal spermiologic findings. The semen of a 49-year-old patient contained normal numbers of spermatozoa with decreased velocity when examined 1 year after chemotherapy but 4 years later exhibited oligoasthenozoospermia. The patient received 4 cycles of Ara-C and DNR plus one cycle with etoposide 350 mg/m2 and mitoxantrone 30 mg/m2. Semen examination of two patients 55- and 59-year-old showed permanent oligoasthenozoospermia with only sporadic progressively motile spermatozoa which might not be compatible with fertilization by sexual intercourse. They received the same chemotherapy including cumulative doses of etoposide 500 mg/m2 and mitoxantrone 36 mg/m2. Semen of two patients after allogeneic bone marrow transplantation exhibited severe oligoasthenozoospermia with no motile spermatozoa. Permanent amenorrhea developed in two women (42- and 46-year-old) during chemotherapy with DNR, Ara-C, etoposide, and mitoxantrone which was not the case in three women (29-40 years old) treated without etoposide and mitoxantrone. Intensive chemotherapy with high-doses of Ara-C and DNR plus one cycle of etoposide and mitoxantrone may cause permanent gonadal dysfunction in middle-aged patients with AML. PMID:16151584

  11. Tomato yellow leaf curl virus resistance by Ty-1 involves increased cytosine methylation of viral genomes and is compromised by cucumber mosaic virus infection

    PubMed Central

    Butterbach, Patrick; Verlaan, Maarten G.; Dullemans, Annette; Lohuis, Dick; Visser, Richard G. F.; Bai, Yuling; Kormelink, Richard

    2014-01-01

    Tomato yellow leaf curl virus (TYLCV) and related begomoviruses are a major threat to tomato production worldwide and, to protect against these viruses, resistance genes from different wild tomato species are introgressed. Recently, the Ty-1 resistance gene was identified, shown to code for an RNA-dependent RNA polymerase and to be allelic with Ty-3. Here we show that upon TYLCV challenging of resistant lines carrying Ty-1 or Ty-3, low virus titers were detected concomitant with the production of relatively high levels of siRNAs whereas, in contrast, susceptible tomato Moneymaker (MM) revealed higher virus titers but lower amounts of siRNAs. Comparative analysis of the spatial genomic siRNA distribution showed a consistent and subtle enrichment for siRNAs derived from the V1 and C3 genes in Ty-1 and Ty-3. In plants containing Ty-2 resistance the virus was hardly detectable, but the siRNA profile resembled the one observed in TYLCV-challenged susceptible tomato (MM). Furthermore, a relative hypermethylation of the TYLCV V1 promoter region was observed in genomic DNA collected from Ty-1 compared with that from (MM). The resistance conferred by Ty-1 was also effective against the bipartite tomato severe rugose begomovirus, where a similar genome hypermethylation of the V1 promoter region was discerned. However, a mixed infection of TYLCV with cucumber mosaic virus compromised the resistance. The results indicate that Ty-1 confers resistance to geminiviruses by increasing cytosine methylation of viral genomes, suggestive of enhanced transcriptional gene silencing. The mechanism of resistance and its durability toward geminiviruses under natural field conditions is discussed. PMID:25136118

  12. Preparation and in vitro antitumor effects of cytosine arabinoside-loaded genipin-poly-l-glutamic acid-modified bacterial magnetosomes

    PubMed Central

    Liu, Yuan-Gang; Dai, Qing-Lei; Wang, Shi-Bin; Deng, Qiong-Jia; Wu, Wen-Guo; Chen, Ai-Zheng

    2015-01-01

    To solve the problem of synthesized magnetic nanoparticles in cancer therapy, a new drug delivery system synthesized from bacteria was used to load cytosine arabinoside (Ara-C). Genipin (GP) and poly-l-glutamic acid (PLGA) were selected as dual cross-linkers. The preparation and characterization of Ara-C-loaded GP-PLGA-modified bacterial magnetosomes (BMs) (ABMs-P), as well as their in vitro antitumor effects, were all investigated. Transmission electron micrographs (TEM) and Fourier transform infrared (FTIR) spectroscopy suggested that Ara-C could be bound to the membrane of BMs modified by GP-PLGA. The diameters of the BMs and ABMs-P were 42.0±8.6 nm and 74.9±8.2 nm, respectively. The zeta potential revealed that the nanoparticles were stable. Moreover, this system exhibited optimal drug-loading properties and long-term release behavior. The optimal encapsulation efficiency and drug-loading were 64.1%±6.6% and 38.9%±2.4%, respectively, and ABMs-P could effectively release 90% Ara-C within 40 days, without the release of an initial burst. In addition, in vitro antitumor experiments elucidated that ABMs-P is cytotoxic to HL-60 cell lines, with an inhibition rate of 95%. The method of coupling drugs on BMs using dual cross-linkers is effective, and our results reveal that this new system has potential applications for drug delivery in the future. PMID:25733831

  13. Effect of nucleobase sequence on the proton-transfer reaction and stability of the guanine-cytosine base pair radical anion.

    PubMed

    Chen, Hsing-Yin; Yeh, Shu-Wen; Hsu, Sodio C N; Kao, Chai-Lin; Dong, Teng-Yuan

    2011-02-21

    The formation of base pair radical anions is closely related to many fascinating research fields in biology and chemistry such as radiation damage to DNA and electron transport in DNA. However, the relevant knowledge so far mainly comes from studies on isolated base pair radical anions, and their behavior in the DNA environment is less understood. In this study, we focus on how the nucleobase sequence affects the properties of the guanine-cytosine (GC) base pair radical anion. The energetic barrier and reaction energy for the proton transfer along the N(1)(G)-H···N(3)(C) hydrogen bond and the stability of GC˙(-) (i.e., electron affinity of GC) embedded in different sequences of base-pair trimer were evaluated using density functional theory. The computational results demonstrated that the presence of neighboring base pairs has an important influence on the behavior of GC˙(-) in the gas phase. The excess electron was found to be localized on the embedded GC and the charge leakage to neighboring base pairs was very minor in all of the investigated sequences. Accordingly, the sequence behavior of the proton-transfer reaction and the stability of GC˙(-) is chiefly governed by electrostatic interactions with adjacent base pairs. However, the effect of base stacking, due to its electrostatic nature, is severely screened upon hydration, and thus, the sequence dependence of the properties of GC˙(-) in aqueous environment becomes relatively weak and less than that observed in the gas phase. The effect of geometry relaxation associated with neighboring base pairs as well as the possibility of proton transfer along the N(2)(G)-H···O(2)(C) channel have also been investigated. The implications of the present findings to the electron transport and radiation damage of DNA are discussed. PMID:21152551

  14. Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene

    PubMed Central

    2011-01-01

    Background Eukaryotic DNA methylation is one of the most studied epigenetic processes, as it results in a direct and heritable covalent modification triggered by external stimuli. In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites. A genome-wide analysis of DNA methylation in Arabidopsis has shown that CNN methylation is preferentially concentrated in transposon genes and non-coding repetitive elements. We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress. Results We describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region. Conclusions These results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in

  15. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas

    PubMed Central

    Chung, Taemoon; Na, Juri; Kim, Young-il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  16. Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression.

    PubMed

    Young, Rosanna E B; Purton, Saul

    2014-12-01

    Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5' UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5' UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes. PMID:25234691

  17. Cytosine derivatized bis(2,2'-bithienyl)methane molecularly imprinted polymer for selective recognition of 6-thioguanine, an antitumor drug.

    PubMed

    Huynh, Tan-Phat; Wojnarowicz, Agnieszka; Sosnowska, Marta; Srebnik, Simcha; Benincori, Tiziana; Sannicolò, Francesco; D'Souza, Francis; Kutner, Wlodzimierz

    2015-08-15

    A molecularly imprinted polymer (MIP) was designed and synthesized to serve as a functional material for selective recognition of 6-thioguanine (6TG), an antitumor drug. For that, the newly synthesized functional monomer, cytosine-bis(2,2'-bithienyl)-(4-carboxyphenyl)methane ester (Cyt-S4), revealed Watson-Crick type nucleobase pairing of 6TG. Formation of the Cyt-S4 and 6TG complex of the 2:1 stoichiometry was postulated based on the DFT calculations at the B3LYP/3-21G((⁎)) level and experimentally confirmed by fluorescence titration. The molecularly imprinted polymer (MIP) film was deposited by potentiodynamic electropolymerization on a Pt disk electrode as well as on an Au-coated glass slide and on an Au-quartz crystal resonator. The statistical model of formation of this film was successfully simulated by molecular dynamics. Completeness of the subsequent 6TG template extraction from MIP was confirmed by the UV-visible spectroscopy. An imprinting factor of 2.9 for the MIP film was determined by piezoelectric microgravimetry using ECQM. The double-layer capacity and alternating current measurements under flow-injection analysis (FIA) conditions were selected to transduce the 6TG recognition signal into the change of the double-layer capacity dependence on the 6TG concentration in solution for different supporting electrolyte concentrations. Detectability of the resulting chemosensor was 10 µM 6TG for the 0.5 M KF carrier solution in FIA. Selectivity of the chemosensor with respect to common interferences was high, e.g., it exceeded 130 to 2-amino-6-methylmercaptopurine, a 6TG metabolite. PMID:25805629

  18. Cytosine-Phosphorothionate-Guanine Oligodeoxynucleotides Exacerbates Hemophagocytosis by Inducing Tumor Necrosis Factor-Alpha Production in Mice after Bone Marrow Transplantation.

    PubMed

    Liu, Jiajia; Guo, Yong-Mei; Onai, Nobuyuki; Ohyagi, Hideaki; Hirokawa, Makoto; Takahashi, Naoto; Tagawa, Hiroyuki; Ubukawa, Kumi; Kobayashi, Isuzu; Tezuka, Hiroyuki; Minamiya, Yoshihiro; Ohteki, Toshiaki; Sawada, Kenichi

    2016-04-01

    Hemophagocytic syndrome (HPS) is frequently associated with hematopoietic stem cell transplantation and is treated with some benefit derived from TNF-α inhibitors. However, the mechanisms of how HPS occurs and how a TNF-α inhibitor exerts some benefit to HPS management have remained unclear. We evaluated the effect of toll-like receptor (TLR) ligands, especially focusing on cytosine-phosphorothionate-guanine oligodeoxynucleotide (CpG), a TLR9 ligand, on HPS in mice that underwent transplantation with syngeneic or allogeneic bone marrow (BM) cells (Syn-BMT, Allo-BMT), or with allogeneic BM cells plus splenocytes to promote graft-versus-host disease (GVHD mice). Hemophagocytosis was a common feature early after all BMT, but it subsided in Syn-BMT and Allo-BMT mice. In GVHD mice, however, hemophagocytosis persisted and was accompanied by upregulated production of IFN-γ but not TNF-α, and it was suppressed by blockade of IFN-γ but not TNF-α. A single injection of the TLR9 ligand CpG promoted HPS in all BMT mice and was lethal in GVHD mice, accompanied by greatly upregulated production of TNF-α, IL-6, and IFN-γ. Blocking of TNF-α, but not IL-6 or IFN-γ, suppressed CpG-induced HPS in all BMT mice and rescued GVHD mice from CpG-induced mortality. Thus, TLR9 signaling mediates TNF-α-driven HPS in BMT mice and is effectively treated through TNF-α inhibition. PMID:26740374

  19. Studies on the antitumor activity and biochemical actions of cyclopentenyl cytosine against human colon carcinoma HT-29 in vitro and in vivo.

    PubMed

    Gharehbaghi, K; Zhen, W; Fritzer-Szekeres, M; Szekeres, T; Jayaram, H N

    1999-01-01

    Cyclopentenyl cytosine (CPEC) is cytotoxic to several tumor cell lines. CPEC inhibits CTP synthesis resulting in depletion of cytidylate pools. The aim of this study was to examine CPEC's cytotoxic and antitumor activity in vitro and in vivo against human colon carcinoma HT-29, and to relate its action on CTP synthesis. CPEC exhibits potent cytotoxicity in vitro to HT-29 cells with an LC50 (concentration that is lethal to the survival of 50% cell colonies) of 2.4 microM and 0.46 microM following 2 h and 24 h exposure, respectively. Incubation of cells with CPEC for 2 h resulted in a dose-dependent decrease in cytidylate pools. The in vivo antitumor activity of CPEC in athymic mice transplanted subcutaneously (s.c.) with 3 million HT-29 cells was examined. Antitumor activity of CPEC was elucidated in early-staged tumor, wherein CPEC (1.5 mg/kg, QD x 9 or 3 mg/kg, QOD x 9) was administered intraperitoneally (i.p.) 24 h after tumor implantation and it resulted in a significant reduction in tumor weight to 48% of control. The effect of CPEC on established solid tumors in vivo was examined in athymic mice transplanted s.c. 14 days earlier with HT-29 cells and treated i.p. with 1.5 mg/kg CPEC, QD x 5 for 4 courses, with a 10 day-interval between courses. This treatment resulted in a significant reduction in tumor weight (72%) in the treated group. HPLC analysis of HT-29 tumor obtained from mice after treatment with CPEC showed a depletion of the CTP concentration reaching a nadir at 8 h. In conclusion, the present studies demonstrate potent antitumor activity of CPEC against freshly transplanted and established human colon carcinoma which can be corroborated with the drug's biochemical actions. PMID:10069488

  20. Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene.

    PubMed

    Wang, Frederick; Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Chun, Changho; Kwon, Young Jik; Berg, Kristian; Madsen, Steen J; Hirschberg, Henry

    2014-05-01

    Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80% of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect. PMID:24610460

  1. A single nucleotide insertion in the canine interleukin-2 receptor gamma chain results in X-linked severe combined immunodeficiency disease.

    PubMed

    Somberg, R L; Pullen, R P; Casal, M L; Patterson, D F; Felsburg, P J; Henthorn, P S

    1995-08-01

    The immunologic and genetic analysis of a 14-week-old-male cardigan Welsh corgi puppy that presented with failure to thrive, diarrhea, and intermittent vomiting are described. The lack of palpable lymph nodes, the premature death of a male sibling, and similar clinical signs in a male cousin suggested that a primary immunodeficiency disease might be responsible for his poor clinical condition. Quantitation of serum immunoglobulins revealed low concentrations of IgG and undetectable IgA, yet normal concentrations of IgM. A complete blood cell count showed a slight anemia and lymphopenia. Although the peripheral blood contained a normal percentage of T cells, with an increased CD4:CD8 ratio, they were unable to proliferate in response to phytohemagglutinin (PHA) and/or interleukin 2 (IL-2). Furthermore, following PHA activation, the peripheral blood lymphocytes (PBL) demonstrated a nearly complete lack of IL-2 binding. All of these laboratory findings were identical with our previous findings from dogs with X-linked severe combined immunodeficiency (XSCID) that is due to a mutation in their IL-2 receptor gamma (IL-2R gamma) chain. Examination of the corgi's IL-2R gamma cDNA revealed an insertion of a cytosine following nucleotide 582, resulting in a premature stop codon prior to the transmembrane domain. The insertion also created an EcoO109 restriction enzyme site that enabled us to detect the mutation in the patient's genomic DNA. This new mutation in the IL-2R gamma chain discovered in a cardigan Welsh corgi puppy results in XSCID with similar immunologic abnormalities as observed in dogs with the same disease resulting from a different IL-2R gamma chain mutation. PMID:8571541

  2. Nucleotide sequence and genome organization of tomato leaf curl geminivirus.

    PubMed

    Dry, I B; Rigden, J E; Krake, L R; Mullineaux, P M; Rezaian, M A

    1993-01-01

    The genome of tomato leaf curl virus (TLCV) from Australia was cloned and its complete nucleotide sequence determined. It is a single circular ssDNA of 2766 nucleotides containing the consensus nonanucleotide sequence present in all geminiviruses. It has six open reading frames with an organization resembling that of certain other dicotyledonous plant-infecting monopartite geminiviruses, i.e. tomato yellow leaf curl and beet curly top viruses. The regulatory sequences present indicate a bidirectional mode of transcription. A dimeric TLCV DNA clone was constructed in a binary vector and used to agroinoculate three different host species. Typical virus infections were produced, confirming that the single DNA component is sufficient for infectivity. PMID:8423446

  3. The primary nucleotide sequence of U4 RNA.

    PubMed

    Reddy, R; Henning, D; Busch, H

    1981-04-10

    U4 RNA is one of the "capped" nuclear snRNAs recently found to be precipitable by anti-Sm antibodies as ribonucleoprotein particles. U4 RNA, along with other snRNAs, has been implicated in hnRNA processing, mRNA transport, or both (Lerner, M. R., Boyle, J., Mount, S., Wolin, S., and Steitz, J. A. (1980) Nature 283, 220-224). Since the proteins bound to different snRNAs appear to be the same, the functions of different snRNPs might be dependent on the RNA components. To help understand the function of U4 RNP, the nucleotide sequence of U4 RNA was determined. The sequence is (formula see text) In addition to the modified nucleotides in the "cap," U4 RNA contains Am at position 63 and m6A at position 98. It also exhibited A-C microheterogeneity at position 97. PMID:6162848

  4. Roles for RNA in Telomerase Nucleotide and Repeat Addition Processivity

    PubMed Central

    Lai, Cary K.; Miller, Michael C.; Collins, Kathleen

    2010-01-01

    Summary Telomerase is a ribonucleoprotein reverse transcriptase with two subunits critical for catalytic activity, the protein telomerase reverse transcriptase (TERT) and telomerase RNA. In this study, we establish additional roles of the telomerase RNA subunit by demonstrating that RNA motifs stimulate the processivity of nucleotide and repeat addition. These functions are both functionally and physically separable from the roles of other RNA motifs in establishing a properly defined template. Binding of Tetrahymena telomerase RNA stem IV to TERT enhances nucleotide addition processivity, while a cooperation of the RNA pseudoknot and stem IV promotes repeat addition processivity. The low processivity of DNA synthesis by telomerase ribonucleoproteins lacking the pseudoknot and/or stem IV can be rescued by addition of the deleted region in trans. These findings demonstrate RNA elements with roles in telomerase elongation processivity that are distinct from RNA elements that specify the internal template. PMID:12820978

  5. The nucleotide exchange factors of Hsp70 molecular chaperones

    PubMed Central

    Bracher, Andreas; Verghese, Jacob

    2015-01-01

    Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell. PMID:26913285

  6. Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy

    PubMed Central

    Schmid, Andreas K.; Davis, Ronald W.

    2016-01-01

    DNA sequencing by imaging in an electron microscope is an approach that holds promise to deliver long reads with low error rates and without the need for amplification. Earlier work using transmission electron microscopes, which use high electron energies on the order of 100 keV, has shown that low contrast and radiation damage necessitates the use of heavy atom labeling of individual nucleotides, which increases the read error rates. Other prior work using scattering electrons with much lower energy has shown to suppress beam damage on DNA. Here we explore possibilities to increase contrast by employing two methods, X-ray photoelectron and Auger electron spectroscopy. Using bulk DNA samples with monomers of each base, both methods are shown to provide contrast mechanisms that can distinguish individual nucleotides without labels. Both spectroscopic techniques can be readily implemented in a low energy electron microscope, which may enable label-free DNA sequencing by direct imaging. PMID:27149617

  7. Single nucleotide polymorphism analysis using different colored dye dimer probes

    NASA Astrophysics Data System (ADS)

    Marmé, Nicole; Friedrich, Achim; Denapaite, Dalia; Hakenbeck, Regine; Knemeyer, Jens-Peter

    2006-09-01

    Fluorescence quenching by dye dimer formation has been utilized to develop hairpin-structured DNA probes for the detection of a single nucleotide polymorphism (SNP) in the penicillin target gene pbp2x, which is implicated in the penicillin resistance of Streptococcus pneumoniae. We designed two specific DNA probes for the identification of the pbp2x genes from a penicillin susceptible strain R6 and a resistant strain Streptococcus mitis 661 using green-fluorescent tetramethylrhodamine (TMR) and red-fluorescent DY-636, respectively. Hybridization of each of the probes to its respective target DNA sequence opened the DNA hairpin probes, consequently breaking the nonfluorescent dye dimers into fluorescent species. This hybridization of the target with the hairpin probe achieved single nucleotide specific detection at nanomolar concentrations via increased fluorescence.

  8. The nucleotide sequence of cowpea mosaic virus B RNA

    PubMed Central

    Lomonossoff, G.P.; Shanks, M.

    1983-01-01

    The complete sequence of the bottom component RNA (B RNA) of cowpea mosaic virus (CPMV) has been determined. Restriction enzyme fragments of double-stranded cDNA were cloned in M13 and the sequence of the inserts was determined by a combination of enzymatic and chemical sequencing techniques. Additional sequence information was obtained by primed synthesis on first strand cDNA. The complete sequence deduced is 5889 nucleotides long excluding the 3' poly(A), and contains an open reading frame sufficient to code for a polypeptide of mol. wt. 207 760. The coding region is flanked by a 5' leader sequence of 206 nucleotides and a 3' non-coding region of 82 residues which does not contain a polyadenylation signal. PMID:16453487

  9. Rapid purification of iodinated ligands for cyclic nucleotide radioimmunoassays

    SciTech Connect

    Wilson, S.P.

    1988-01-01

    The tyrosine methyl esters of succinyl cyclic AMP and succinyl cyclic GMP were iodinated by the chloramine T method and individually applied to C18 cartridges. A solution of 1-propanol/0.1 M sodium acetate pH 4.75 (17.5:82.5) was then pumped onto each cartridge and the eluate collected. A large peak of radioactivity, containing primarily the monoiodo and diiodo derivatives, was eluted. Radioactivity in peak fractions was greater than or equal to 95% the monoiodo derivative and represented 20 to 25% of the starting radioactivity. Contamination by the native cyclic nucleotide analogs was less than 5%. These peak fractions containing primarily monoiodinated products worked well in cyclic nucleotide radioimmunoassays. This fractionation required less than 30 min.

  10. RIBOSE MODIFIED NUCLEOSIDES AND NUCLEOTIDES AS LIGANDS FOR PURINE RECEPTORS

    PubMed Central

    Ravi, R. G.; Nandanan, E.; Kim, H. S.; Moro, S.; Kim, Y. C.; Lee, K.; Barak, D.; Marquez, V. E.; Ji, X. D.

    2016-01-01

    Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3′,5′-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs. PMID:11563046

  11. Spontaneous formation and base pairing of plausible prebiotic nucleotides in water.

    PubMed

    Cafferty, Brian J; Fialho, David M; Khanam, Jaheda; Krishnamurthy, Ramanarayanan; Hud, Nicholas V

    2016-01-01

    The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life. PMID:27108699

  12. Spontaneous formation and base pairing of plausible prebiotic nucleotides in water

    PubMed Central

    Cafferty, Brian J.; Fialho, David M.; Khanam, Jaheda; Krishnamurthy, Ramanarayanan; Hud, Nicholas V.

    2016-01-01

    The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life. PMID:27108699

  13. Cyclic nucleotides in tissues during long-term hypokinesia

    NASA Technical Reports Server (NTRS)

    Makeyeva, V. F.; Komolova, G. S.; Yegorov, I. A.; Serova, L. V.; Chelnaya, N. A.

    1981-01-01

    Male Wistar rates were kept hypokinetic by placing them in small containers for 22 days. Blood plasma cAMP content was subsequently found increased, and cGMP content decreased, in the experimental animals. Liver and thymus cAMP content was similar in the control and experimental animals. There was a 20 and 38% decrease of cAMP content in the kidneys and spleen, respectively. Hypokinesia's reduction of cyclic nucleotides seems to inhibit RNA and protein synthesis.

  14. Transepithelial pressure pulses induce nucleotide release in polarized MDCK cells.

    PubMed

    Praetorius, H A; Frøkiaer, J; Leipziger, J

    2005-01-01

    The release of nucleotides is involved in mechanosensation in various epithelial cells. Intriguingly, kidney epithelial cells are absolutely dependent on the primary cilium to sense changes in apical laminar flow. During fluid passage, the renal epithelial cells are subjected to various mechanical stimuli in addition to changes in the laminar flow rate. In the distal part of the collecting duct, the epithelial cells are exposed to pressure changes and possibly distension during papillary contractions. The aim of the present study was to determine whether nucleotide release contributes to mechanosensation in kidney epithelial cells, thereby establishing whether pressure changes are sufficient to produce nucleotide-mediated responses. Madin-Darby canine kidney (MDCK) cells grown on permeable supports were mounted in a closed double perfusion chamber on an inverted microscope. The intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored with the Ca(2+)-sensitive fluorescence probe fluo 4. Transepithelial pressure pulses of 30-80 mm Hg produced a transient increase in [Ca(2+)](i) of MDCK cells. This response is independent of the primary cilium, since it is readily observed in immature cells that do not yet express primary cilia. The amplitudes of the pressure-induced Ca(2+) transients varied with the applied chamber pressure in a quantity-dependent manner. The ATPase apyrase and the P2Y antagonist suramin significantly reduced the pressure-induced Ca(2+) transients. Applying apyrase or suramin to both sides of the preparation simultaneously nearly abolished the pressure-induced Ca(2+) response. In conclusion, these observations suggest that rapid pressure changes induce both apical and basolateral nucleotide release that contribute to mechanosensation in kidney epithelial cells. PMID:15367389

  15. Monovar: single-nucleotide variant detection in single cells.

    PubMed

    Zafar, Hamim; Wang, Yong; Nakhleh, Luay; Navin, Nicholas; Chen, Ken

    2016-06-01

    Current variant callers are not suitable for single-cell DNA sequencing, as they do not account for allelic dropout, false-positive errors and coverage nonuniformity. We developed Monovar (https://bitbucket.org/hamimzafar/monovar), a statistical method for detecting and genotyping single-nucleotide variants in single-cell data. Monovar exhibited superior performance over standard algorithms on benchmarks and in identifying driver mutations and delineating clonal substructure in three different human tumor data sets. PMID:27088313

  16. Nucleotide sequence of SHV-2 beta-lactamase gene

    SciTech Connect

    Garbarg-Chenon, A.; Godard, V.; Labia, R.; Nicolas, J.C. )

    1990-07-01

    The nucleotide sequence of plasmid-mediated beta-lactamase SHV-2 from Salmonella typhimurium (SHV-2pHT1) was determined. The gene was very similar to chromosomally encoded beta-lactamase LEN-1 of Klebsiella pneumoniae. Compared with the sequence of the Escherichia coli SHV-2 enzyme (SHV-2E.coli) obtained by protein sequencing, the deduced amino acid sequence of SHV-2pHT1 differed by three amino acid substitutions.

  17. Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase.

    PubMed

    Salaemae, Wanisa; Yap, Min Y; Wegener, Kate L; Booker, Grant W; Wilce, Matthew C J; Polyak, Steven W

    2015-05-01

    Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue. PMID:25801336

  18. Alteration of nucleotide metabolism: a new mechanism for mitochondrial disorders.

    PubMed

    Martí, Ramon; Nishigaki, Yutaka; Vilá, Maya R; Hirano, Michio

    2003-07-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). TP deficiency alters the metabolism of the nucleosides thymidine and deoxyuridine, which, in turn, produces abnormalities of mitochondrial DNA (mtDNA) including depletion, deletions, and point mutations. MNGIE is the best characterized of the expanding number of mitochondrial disorders caused by alterations in the metabolism of nucleosides/nucleotides. Because mitochondria contain their own machinery for nucleoside and nucleotide metabolism and have physically separate nucleotide pools, it is not surprising that disorders of these pathways cause human diseases. Other diseases in this group include mtDNA depletion syndromes caused by mutations on the nuclear genes encoding the mitochondrial thymidine kinase and deoxyguanosine kinase; autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA due to mutations in the genes encoding the muscle-isoform of mitochondrial ADP/ATP translocator; and mitochondrial DNA depletion due to toxicities of nucleoside analogues. Mutations in the deoxynucleotide carrier, a transporter of deoxynucleoside diphosphates, have been identified as a cause of congenital microcephaly. However, alterations of mtDNA have not yet been established in this disorder. Future studies are likely to reveal additional diseases and provide further insight into this new subject. PMID:12940507

  19. Nucleotide Sequencing and Identification of Some Wild Mushrooms

    PubMed Central

    Das, Sudip Kumar; Mandal, Aninda; Datta, Animesh K.; Gupta, Sudha; Paul, Rita; Saha, Aditi; Sengupta, Sonali; Dubey, Priyanka Kumari

    2013-01-01

    The rDNA-ITS (Ribosomal DNA Internal Transcribed Spacers) fragment of the genomic DNA of 8 wild edible mushrooms (collected from Eastern Chota Nagpur Plateau of West Bengal, India) was amplified using ITS1 (Internal Transcribed Spacers 1) and ITS2 primers and subjected to nucleotide sequence determination for identification of mushrooms as mentioned. The sequences were aligned using ClustalW software program. The aligned sequences revealed identity (homology percentage from GenBank data base) of Amanita hemibapha [CN (Chota Nagpur) 1, % identity 99 (JX844716.1)], Amanita sp. [CN 2, % identity 98 (JX844763.1)], Astraeus hygrometricus [CN 3, % identity 87 (FJ536664.1)], Termitomyces sp. [CN 4, % identity 90 (JF746992.1)], Termitomyces sp. [CN 5, % identity 99 (GU001667.1)], T. microcarpus [CN 6, % identity 82 (EF421077.1)], Termitomyces sp. [CN 7, % identity 76 (JF746993.1)], and Volvariella volvacea [CN 8, % identity 100 (JN086680.1)]. Although out of 8 mushrooms 4 could be identified up to species level, the nucleotide sequences of the rest may be relevant to further characterization. A phylogenetic tree is constructed using Neighbor-Joining method showing interrelationship between/among the mushrooms. The determined nucleotide sequences of the mushrooms may provide additional information enriching GenBank database aiding to molecular taxonomy and facilitating its domestication and characterization for human benefits. PMID:24489501

  20. Nucleotide sequencing and identification of some wild mushrooms.

    PubMed

    Das, Sudip Kumar; Mandal, Aninda; Datta, Animesh K; Gupta, Sudha; Paul, Rita; Saha, Aditi; Sengupta, Sonali; Dubey, Priyanka Kumari

    2013-01-01

    The rDNA-ITS (Ribosomal DNA Internal Transcribed Spacers) fragment of the genomic DNA of 8 wild edible mushrooms (collected from Eastern Chota Nagpur Plateau of West Bengal, India) was amplified using ITS1 (Internal Transcribed Spacers 1) and ITS2 primers and subjected to nucleotide sequence determination for identification of mushrooms as mentioned. The sequences were aligned using ClustalW software program. The aligned sequences revealed identity (homology percentage from GenBank data base) of Amanita hemibapha [CN (Chota Nagpur) 1, % identity 99 (JX844716.1)], Amanita sp. [CN 2, % identity 98 (JX844763.1)], Astraeus hygrometricus [CN 3, % identity 87 (FJ536664.1)], Termitomyces sp. [CN 4, % identity 90 (JF746992.1)], Termitomyces sp. [CN 5, % identity 99 (GU001667.1)], T. microcarpus [CN 6, % identity 82 (EF421077.1)], Termitomyces sp. [CN 7, % identity 76 (JF746993.1)], and Volvariella volvacea [CN 8, % identity 100 (JN086680.1)]. Although out of 8 mushrooms 4 could be identified up to species level, the nucleotide sequences of the rest may be relevant to further characterization. A phylogenetic tree is constructed using Neighbor-Joining method showing interrelationship between/among the mushrooms. The determined nucleotide sequences of the mushrooms may provide additional information enriching GenBank database aiding to molecular taxonomy and facilitating its domestication and characterization for human benefits. PMID:24489501

  1. Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

    PubMed

    Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

    2015-11-01

    The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. PMID:27491034

  2. Genome-Wide Patterns of Nucleotide Polymorphism in Domesticated Rice

    PubMed Central

    Hernandez, Ryan D; Boyko, Adam; Fledel-Alon, Adi; York, Thomas L; Polato, Nicholas R; Olsen, Kenneth M; Nielsen, Rasmus; McCouch, Susan R; Bustamante, Carlos D; Purugganan, Michael D

    2007-01-01

    Domesticated Asian rice (Oryza sativa) is one of the oldest domesticated crop species in the world, having fed more people than any other plant in human history. We report the patterns of DNA sequence variation in rice and its wild ancestor, O. rufipogon, across 111 randomly chosen gene fragments, and use these to infer the evolutionary dynamics that led to the origins of rice. There is a genome-wide excess of high-frequency derived single nucleotide polymorphisms (SNPs) in O. sativa varieties, a pattern that has not been reported for other crop species. We developed several alternative models to explain contemporary patterns of polymorphisms in rice, including a (i) selectively neutral population bottleneck model, (ii) bottleneck plus migration model, (iii) multiple selective sweeps model, and (iv) bottleneck plus selective sweeps model. We find that a simple bottleneck model, which has been the dominant demographic model for domesticated species, cannot explain the derived nucleotide polymorphism site frequency spectrum in rice. Instead, a bottleneck model that incorporates selective sweeps, or a more complex demographic model that includes subdivision and gene flow, are more plausible explanations for patterns of variation in domesticated rice varieties. If selective sweeps are indeed the explanation for the observed nucleotide data of domesticated rice, it suggests that strong selection can leave its imprint on genome-wide polymorphism patterns, contrary to expectations that selection results only in a local signature of variation. PMID:17907810

  3. Bijective transformation circular codes and nucleotide exchanging RNA transcription.

    PubMed

    Michel, Christian J; Seligmann, Hervé

    2014-04-01

    The C(3) self-complementary circular code X identified in genes of prokaryotes and eukaryotes is a set of 20 trinucleotides enabling reading frame retrieval and maintenance, i.e. a framing code (Arquès and Michel, 1996; Michel, 2012, 2013). Some mitochondrial RNAs correspond to DNA sequences when RNA transcription systematically exchanges between nucleotides (Seligmann, 2013a,b). We study here the 23 bijective transformation codes ΠX of X which may code nucleotide exchanging RNA transcription as suggested by this mitochondrial observation. The 23 bijective transformation codes ΠX are C(3) trinucleotide circular codes, seven of them are also self-complementary. Furthermore, several correlations are observed between the Reading Frame Retrieval (RFR) probability of bijective transformation codes ΠX and the different biological properties of ΠX related to their numbers of RNAs in GenBank's EST database, their polymerization rate, their number of amino acids and the chirality of amino acids they code. Results suggest that the circular code X with the functions of reading frame retrieval and maintenance in regular RNA transcription, may also have, through its bijective transformation codes ΠX, the same functions in nucleotide exchanging RNA transcription. Associations with properties such as amino acid chirality suggest that the RFR of X and its bijective transformations molded the origins of the genetic code's machinery. PMID:24565870

  4. Cyclic nucleotide regulation of cardiac sympatho-vagal responsiveness.

    PubMed

    Li, Dan; Paterson, David J

    2016-07-15

    Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are now recognized as important intracellular signalling molecules that modulate cardiac sympatho-vagal balance in the progression of heart disease. Recent studies have identified that a significant component of autonomic dysfunction associated with several cardiovascular pathologies resides at the end organ, and is coupled to impairment of cyclic nucleotide targeted pathways linked to abnormal intracellular calcium handling and cardiac neurotransmission. Emerging evidence also suggests that cyclic nucleotide coupled phosphodiesterases (PDEs) play a key role limiting the hydrolysis of cAMP and cGMP in disease, and as a consequence this influences the action of the nucleotide on its downstream biological target. In this review, we illustrate the action of nitric oxide-CAPON signalling and brain natriuretic peptide on cGMP and cAMP regulation of cardiac sympatho-vagal transmission in hypertension and ischaemic heart disease. Moreover, we address how PDE2A is now emerging as a major target that affects the efficacy of soluble/particulate guanylate cyclase coupling to cGMP in cardiac dysautonomia. PMID:26915722

  5. Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

    PubMed Central

    Gantt, Richard W.; Peltier-Pain, Pauline; Singh, Shanteri; Zhou, Maoquan; Thorson, Jon S.

    2013-01-01

    We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in kcat/Km of >400-fold and >15-fold for formation of NDP–glucoses and UDP–sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP–sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides. PMID:23610417

  6. Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

    PubMed Central

    Galibert, F; Alexandraki, D; Baur, A; Boles, E; Chalwatzis, N; Chuat, J C; Coster, F; Cziepluch, C; De Haan, M; Domdey, H; Durand, P; Entian, K D; Gatius, M; Goffeau, A; Grivell, L A; Hennemann, A; Herbert, C J; Heumann, K; Hilger, F; Hollenberg, C P; Huang, M E; Jacq, C; Jauniaux, J C; Katsoulou, C; Karpfinger-Hartl, L

    1996-01-01

    The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non-canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome. Images PMID:8641269

  7. KATP channels process nucleotide signals in muscle thermogenic response

    PubMed Central

    Reyes, Santiago; Park, Sungjo; Terzic, Andre; Alekseev, Alexey E.

    2014-01-01

    Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K+ (KATP) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, KATP channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how KATP channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the KATP channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with KATP channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal KATP channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of KATP channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of KATP channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders. PMID:20925594

  8. Scambio, a novel guanine nucleotide exchange factor for Rho

    PubMed Central

    Curtis, Christina; Hemmeryckx, Bianca; Haataja, Leena; Senadheera, Dinithi; Groffen, John; Heisterkamp, Nora

    2004-01-01

    Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho. PMID:15107133

  9. Cyclic Nucleotide Phosphodiesterases: important signaling modulators and therapeutic targets

    PubMed Central

    Ahmad, Faiyaz; Murata, Taku; Simizu, Kasumi; Degerman, Eva; Maurice, Donald; Manganiello, Vincent

    2014-01-01

    By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases are critical regulators of their intracellular concentrations and their biological effects. Since these intracellular second messengers control many cellular homeostatic processes, dysregulation of their signals and signaling pathways initiate or modulate pathophysiological pathways related to various disease states, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication, chronic obstructive pulmonary disease, and psoriasis. Alterations in expression of PDEs and PDE-gene mutations (especially mutations in PDE6, PDE8B, PDE11A and PDE4) have been implicated in various diseases and cancer pathologies. PDEs also play important role in formation and function of multi-molecular signaling/regulatory complexes called signalosomes. At specific intracellular locations, individual PDEs, together with pathway-specific signaling molecules, regulators, and effectors, are incorporated into specific signalosomes, where they facilitate and regulate compartmentalization of cyclic nucleotide signaling pathways and specific cellular functions. Currently, only a limited number of PDE inhibitors (PDE3, PDE4, PDE5 inhibitors) are used in clinical practice. Future paths to novel drug discovery include the crystal structure-based design approach, which has resulted in generation of more effective family-selective inhibitors, as well as burgeoning development of strategies to alter compartmentalized cyclic nucleotide signaling pathways by selectively targeting individual PDEs and their signalosome partners. PMID:25056711

  10. Nucleotide sequences specific to Brucella and methods for the detection of Brucella

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.

    2009-02-24

    Nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  11. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2007-02-06

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  12. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2009-02-24

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  13. Nucleotide sequences specific to Yersinia pestis and methods for the detection of Yersinia pestis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Motin, Vladinir L.

    2009-02-24

    Nucleotide sequences specific to Yersinia pestis that serve as markers or signatures for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  14. n-Nucleotide circular codes in graph theory.

    PubMed

    Fimmel, Elena; Michel, Christian J; Strüngmann, Lutz

    2016-03-13

    The circular code theory proposes that genes are constituted of two trinucleotide codes: the classical genetic code with 61 trinucleotides for coding the 20 amino acids (except the three stop codons {TAA,TAG,TGA}) and a circular code based on 20 trinucleotides for retrieving, maintaining and synchronizing the reading frame. It relies on two main results: the identification of a maximal C(3) self-complementary trinucleotide circular code X in genes of bacteria, eukaryotes, plasmids and viruses (Michel 2015 J. Theor. Biol. 380, 156-177. (doi:10.1016/j.jtbi.2015.04.009); Arquès & Michel 1996 J. Theor. Biol. 182, 45-58. (doi:10.1006/jtbi.1996.0142)) and the finding of X circular code motifs in tRNAs and rRNAs, in particular in the ribosome decoding centre (Michel 2012 Comput. Biol. Chem. 37, 24-37. (doi:10.1016/j.compbiolchem.2011.10.002); El Soufi & Michel 2014 Comput. Biol. Chem. 52, 9-17. (doi:10.1016/j.compbiolchem.2014.08.001)). The univerally conserved nucleotides A1492 and A1493 and the conserved nucleotide G530 are included in X circular code motifs. Recently, dinucleotide circular codes were also investigated (Michel & Pirillo 2013 ISRN Biomath. 2013, 538631. (doi:10.1155/2013/538631); Fimmel et al. 2015 J. Theor. Biol. 386, 159-165. (doi:10.1016/j.jtbi.2015.08.034)). As the genetic motifs of different lengths are ubiquitous in genes and genomes, we introduce a new approach based on graph theory to study in full generality n-nucleotide circular codes X, i.e. of length 2 (dinucleotide), 3 (trinucleotide), 4 (tetranucleotide), etc. Indeed, we prove that an n-nucleotide code X is circular if and only if the corresponding graph [Formula: see text] is acyclic. Moreover, the maximal length of a path in [Formula: see text] corresponds to the window of nucleotides in a sequence for detecting the correct reading frame. Finally, the graph theory of tournaments is applied to the study of dinucleotide circular codes. It has full equivalence between the combinatorics

  15. The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

    PubMed Central

    Root, D. D.; Reisler, E.

    1992-01-01

    Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304380

  16. Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Aleksandrov, L; Mengos, A; Chang, X; Aleksandrov, A; Riordan, J R

    2001-04-20

    After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing. PMID:11279083

  17. Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110.

    PubMed Central

    Matsumura, M; Katakura, Y; Imanaka, T; Aiba, S

    1984-01-01

    The product of a kanamycin resistance gene encoded by plasmid pTB913 isolated from a thermophilic bacillus was identified as a kanamycin nucleotidyltransferase which is similar to that encoded by plasmid pUB110 from a mesophile, Staphylococcus aureus. The enzyme encoded by pTB913 was more thermostable than that encoded by pUB110. In view of a close resemblance of restriction endonuclease cleavage maps around the BglII site in the structural genes of both enzymes, ca. 1,200 base pairs were sequenced, followed by amino-terminal amino acid sequencing of the enzyme. The two nucleotide sequences were found to be identical to each other except for only one base in the midst of the structural gene. Each structural gene, initiating from a GUG codon as methionine, was composed of 759 base pairs and 253 amino acid residues (molecular weight, ca. 29,000). The sole difference was transversion from a cytosine (pUB110) to an adenine (pTB913) at a position + 389, counting the first base of the initiation codon as + 1. That is, a threonine at position 130 for the pUB110-coded kanamycin nucleotidyltransferase was replaced by a lysine for the pTB913-coded enzyme. The difference in thermostability between the two enzymes caused by a single amino acid replacement is discussed in light of electrostatic effects. Images PMID:6090428

  18. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    PubMed

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  19. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    PubMed Central

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  20. Aberrant Promoter Methylation at CpG Cytosines Induce the Upregulation of the E2F5 Gene in Breast Cancer

    PubMed Central

    Ali, Arshad; Ullah, Farman; Ali, Irum Sabir; Faraz, Ahmad; Khan, Mumtaz; Shah, Syed Tahir Ali; Ali, Nawab

    2016-01-01

    Purpose The promoter methylation status of cell cycle regulatory genes plays a crucial role in the regulation of the eukaryotic cell cycle. CpG cytosines are actively subjected to methylation during tumorigenesis, resulting in gain/loss of function. E2F5 gene has growth repressive activities; various studies suggest its involvement in tumorigenesis. This study aims to investigate the epigenetic regulation of E2F5 in breast cancer to better understand tumor biology. Methods The promoter methylation status of 50 breast tumor tissues and adjacent normal control tissues was analyzed. mRNA expression was determined using SYBR® green quantitative polymerase chain reaction (PCR), and methylation-specific PCR was performed for bisulfite-modified genomic DNA using E2F5-specific primers to assess promoter methylation. Data was statistically analyzed. Results Significant (p<0.001) upregulation was observed in E2F5 expression among tumor tissues, relative to the control group. These samples were hypo-methylated at the E2F5 promoter region in the tumor tissues, compared to the control. Change in the methylation status (Δmeth) was significantly lower (p=0.022) in the tumor samples, indicating possible involvement in tumorigenesis. Patients at the postmenopausal stage showed higher methylation (75%) than those at the premenopausal stage (23.1%). Interestingly, methylation levels gradually increased from the early to the advanced stages of the disease (p<0.001), which suggests a putative role of E2F5 methylation in disease progression that can significantly modulate tumor biology at more advanced stage and at postmenopausal age (Pearson's r=0.99 and 0.86, respectively). Among tissues with different histological status, methylation frequency was higher in invasive lobular carcinoma (80.0%), followed by invasive ductal carcinoma (46.7%) and ductal carcinoma in situ (20.0%). Conclusion Methylation is an important epigenetic factor that might be involved in the upregulation of E2F5

  1. Pharmacokinetic Profile and Acute Toxicological Properties of a Novel Radiosensitizer Cytosine-Phosphate-Guanosine Oligodeoxynucleotide 107 in Mice Following Intravenous and Orthotopic Administration.

    PubMed

    Cen, Yanyan; Li, Xiaoli; Yin, Zhiwei; Yan, Zifei; Liu, Dan; Peng, Wei; Pan, Feng; Zhou, Hong

    2015-10-01

    The synthetic cytosine-phosphate-guanosine oligodeoxynucleotide 107 (CpG ODN107) is a novel radiosensitizer for glioma treatment. However, the information related to its pharmacokinetics and toxicity remains unclear. Therefore, the plasma pharmacokinetics, distribution, elimination, and acute toxicity of CpG ODN107 in mice were investigated in the present experiments. The results from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that the plasma elimination half-life (t1/2β) of CpG ODN107 in BALB/c mice varied slightly with the dose, and it was 0.65, 0.49, and 0.50 h at the intravenous doses of 2.5, 5, and 10 mg/kg, respectively. CpG ODN107 rapidly and widely distributed in organs/tissues, except the brain and testes. The highest concentrations were found in the liver (28.6% of the administered dose after 0.5 h) and the kidneys (5.7% of the administered dose after 1 h). CpG ODN107 (0.3, 3, and 30 μg/mL) could highly bind to human and mouse plasma proteins in vitro. CpG ODN107 in the forms of prototype was excreted in urine (1.79%) and feces (0.91%), and its shortened metabolites were excreted in urine (2.1%) and feces (2.2%) within the first 24 h. The mice in vivo optical image showed CpG ODN107 labeled with Alexa Fluor 680 fluorochrome (AF680) accumulated in the brain after orthotopic injection, eliminated very slowly, and excreted in urine compared with poly T labeled with AF680. The median lethal dose (LD50) of CpG ODN107 was 75.7 mg/kg for mice; this dose only could produce apparent spleen and liver damage, in line with the distribution features of CpG ODN. In conclusion, our present pharmacokinetic and toxicity investigation will provide helpful information to further pharmacodynamic and pharmacokinetic research of CpG ODN107 and other oligodeoxynucleotide drugs in the future. PMID:26213852

  2. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

    PubMed

    Xiao, Yongsheng; Wang, Yinsheng

    2016-09-01

    Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016. PMID:25376990

  3. Cyclic nucleotide phosphodiesterases in heart and vessels: A therapeutic perspective.

    PubMed

    Bobin, Pierre; Belacel-Ouari, Milia; Bedioune, Ibrahim; Zhang, Liang; Leroy, Jérôme; Leblais, Véronique; Fischmeister, Rodolphe; Vandecasteele, Grégoire

    2016-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac and vascular muscle functions. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families that are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP, controlling specific cell functions in response to various neurohormonal stimuli. In the myocardium and vascular smooth muscle, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling and smooth muscle contractile tone. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important for the degradation of cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. There is experimental evidence that these PDEs, as well as other PDE families, including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure, as well as several vascular diseases. After a brief presentation of the cyclic nucleotide pathways in cardiac and vascular cells, and the major characteristics of the PDE superfamily, this review will focus on the current use of PDE inhibitors in cardiovascular diseases, and the recent research developments that could lead to better exploitation of the therapeutic potential of these enzymes in the future. PMID:27184830

  4. Nucleotide metabolic mismatches in mammalian hearts: implications for transplantation

    PubMed Central

    Yacoub, MH; Smolenski, RT

    2013-01-01

    Introduction Human donor organ shortages have led surgeons and scientists to explore the use of animals as alternative organ sources. Acute thrombovascular rejection (AVR) is the main hurdle in xenotransplantation. Disparities in nucleotide metabolism in the vessels of different species may contribute significantly to the microvascular component of AVR. Methods We evaluated the extent of nucleotide metabolism mismatch in selected organs and endothelial cells of different mammals with particular focus on the changes in activity of ecto-5’-nucleotidase (E5’N) elicited by exposure of porcine hearts or endothelial cells to human blood (ex vivo) or human plasma (in vitro). Results E5’N activity in the rat heart was significantly higher than in other species. We noted a significant difference (p<0.001) in E5’N activity between human and pig endothelial cell lines. Initial pig aortic endothelial E5’N activity decreased in vitro after a three-hour exposure to human and porcine plasma while remaining constant in controls. Ex vivo perfusion with fresh human blood for four hours resulted in a significant decrease of E5’N activity in both wild type and transgenic pig hearts overexpressing human decay accelerating factor (p<0.001). Conclusions This study provides evidence that mismatches in basal mammalian metabolic pathways and humoral immunity interact in a xenogeneic environment. Understanding the role of nucleotide metabolism and signalling in xenotransplantation may identify new targets for genetic modifications and may lead to the development of new therapies extending graft survival. PMID:23317713

  5. Y-Single Nucleotide Polymorphisms Diversity in Chinese Indigenous Horse

    PubMed Central

    Han, Haoyuan; Zhang, Qin; Gao, Kexin; Yue, Xiangpeng; Zhang, Tao; Dang, Ruihua; Lan, Xianyong; Chen, Hong; Lei, Chuzhao

    2015-01-01

    In contrast to high genetic diversity of mitochondrial DNA (mtDNA), equine Y chromosome shows extremely low variability, implying limited patrilines in the domesticated horse. In this study, we applied direct sequencing and restriction fragment length polymorphism (RFLP) methods to investigate the polymorphisms of 33 Y chromosome specific loci in 304 Chinese indigenous horses from 13 breeds. Consequently, two Y-single nucleotide polymorphisms (SNPs) (Y-45701/997 and Y-50869) and one Y-indel (Y-45288) were identified. Of those, the Y-50869 (T>A) revealed the highest variation frequency (24.67%), whereas it was only 3.29% and 1.97% in Y-45288 (T/-) and Y-45701/997 (G>T) locus, respectively. These three mutations accounted for 27.96% of the total samples and identified five Y-SNP haplotypes, demonstrating genetic diversity of Y chromosome in Chinese horses. In addition, all the five Y-SNP haplotypes were shared by different breeds. Among 13 horse breeds analyzed, Balikun horse displayed the highest nucleotide diversity (π = 5.6×10−4) and haplotype diversity (h = 0.527), while Ningqiang horse showed the lowest nucleotide diversity (π = 0.00000) and haplotype diversity (h = 0.000). The results also revealed that Chinese horses had a different polymorphic pattern of Y chromosome from European and American horses. In conclusion, Chinese horses revealed genetic diversity of Y chromosome, however more efforts should be made to better understand the domestication and paternal origin of Chinese indigenous horses. PMID:26104513

  6. Study of sperm cell phosphorylating systems using nucleotide photoaffinity probes

    SciTech Connect

    Khatoon, S.

    1983-01-01

    The major thrust of the research presented in this thesis was to identify specific nucleotide binding proteins and phosphoproteins of rat caput and cauda sperm. Also, the differences in these proteins between caput and cauda sperm were investigated as well as determination of the membrane sidedness of the proteins and their location in either the head or tail/mid-piece region. In addition, the effects of small molecular weight modifers such as cGMP, cAMP and Ca/sup 2 +/ on the detection of binding proteins and phosphorylated proteins was studied. The technique used to identify and locate nucleotide binding proteins was photoaffinity labeling using the proven 8-azidopurine nucleotide analogs of cAMP, ATP and GTP in radioactive form. The first study presented involved the use of (/sup 32/P)8-N /sub 3/cAMP which showed that both caput and cauda sperm contained both type I and type II regulatory subunits (R/sub I/ and R/sub II/, respectively) of the cAMP dependent kinases and that the great majority of the regulatory subunits were located in the tail/mid-piece section and not in the sperm head. The second phase of this study involved the use of (..gamma../sup 32/P)8-azidoadensosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/ATP) and (..gamma../sup 32/P)8-azidoguanosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/GTP) to photolable specific ATP and GTP binding proteins and to phosphorylate specific phosphoproteins. Again, this was done on caput versus cauda sperm and the location of the majority of the photolabeled or phosphorylated proteins was shown to be in the tail/mid-piece fraction. In addition, considerable differences were found in both the phosphorylated and photolabeled proteins of caput versus cauda sperm.

  7. Bioinformatics comparison of sulfate-reducing metabolism nucleotide sequences

    NASA Astrophysics Data System (ADS)

    Tremberger, G.; Dehipawala, Sunil; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

    2015-09-01

    The sulfate-reducing bacteria can be traced back to 3.5 billion years ago. The thermodynamics details of the sulfur cycle have been well documented. A recent sulfate-reducing bacteria report (Robator, Jungbluth, et al , 2015 Jan, Front. Microbiol) with Genbank nucleotide data has been analyzed in terms of the sulfite reductase (dsrAB) via fractal dimension and entropy values. Comparison to oil field sulfate-reducing sequences was included. The AUCG translational mass fractal dimension versus ATCG transcriptional mass fractal dimension for the low temperature dsrB and dsrA sequences reported in Reference Thirteen shows correlation R-sq ~ 0.79 , with a probably of about 3% in simulation. A recent report of using Cystathionine gamma-lyase sequence to produce CdS quantum dot in a biological method, where the sulfur is reduced just like in the H2S production process, was included for comparison. The AUCG mass fractal dimension versus ATCG mass fractal dimension for the Cystathionine gamma-lyase sequences was found to have R-sq of 0.72, similar to the low temperature dissimilatory sulfite reductase dsr group with 3% probability, in contrary to the oil field group having R-sq ~ 0.94, a high probable outcome in the simulation. The other two simulation histograms, namely, fractal dimension versus entropy R-sq outcome values, and di-nucleotide entropy versus mono-nucleotide entropy R-sq outcome values are also discussed in the data analysis focusing on low probability outcomes.

  8. Labeling of mitochondrial adenine nucleotides of bovine sperm

    SciTech Connect

    Cheetham, J.; Lardy, H.A.

    1986-05-01

    Incorporation of /sup 32/P/sub i/ into the adenine nucleotide pool of intact bovine spermatozoa utilizing endogenous substrates results in a specific activity (S.A.) ratio ATP/ADP of 0.3 to 0.5, suggesting compartmentation of nucleotide pools or a pathway for phosphorylation of AMP in addition to the myokinase reaction. Incubation of filipin-permeabilized cells with pyruvate, acetylcarnitine, or ..cap alpha..-ketoglutarate (..cap alpha..KG) resulted in ATP-ADP S.A. ratios of 0.5, 0.8, and 1.6, respectively, for mitochondrial nucleotides. However, when malate was included with pyruvate or acetylcarnitine, the ATP/ADP S.A. ratio increased by 400% to 2.0 for pyruvate/malate and by 290% to 2.8 for acetylcarnitine/malate, while the ATP/ADP ratio increased by less than 100% in both cases. These results may indicate that under conditions of limited flux through the citric acid cycle a pathway for phosphorylation of AMP from a precursor other than ATP exists or that ATP is compartmented within the mitochondrion. In the presence of uncoupler and oligomycin with ..cap alpha..KG, pyruvate/malate, or acetylcarnitine/malate, /sup 32/P/sub i/ is incorporated primarily into ATP, resulting in an ATP/ADP S.A. ratio of 4.0 for ..cap alpha..KG, 2.7 for pyruvate/malate, and 2.8 for acetylcarnitine/malate. These data are consistent with phosphorylation of ADP during substrate level phosphorylation in the citric acid cycle.

  9. Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study.

    PubMed

    Cervoni, L; Lascu, I; Xu, Y; Gonin, P; Morr, M; Merouani, M; Janin, J; Giartosio, A

    2001-04-17

    The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications. PMID:11294625

  10. P2Y nucleotide receptors: Promise of therapeutic applications

    PubMed Central

    Jacobson, Kenneth A.; Boeynaems, Jean-Marie

    2010-01-01

    Extracellular nucleotides, such as ATP and UTP, have distinct signaling roles through a class of G protein-coupled receptors, termed P2Y. However, the receptor ligands are typically charged molecules of low bioavailability and stability in vivo. Recent progress in the development of selective agonists and antagonists for P2Y receptors and study of knockout mice have led to new drug concepts based on these receptors. The rapidly accelerating progress in this field has already resulted in drug candidates for cystic fibrosis, dry eye disease, and thrombosis. On the horizon are novel treatments of cardiovascular diseases, inflammatory diseases, and neurodegeneration. PMID:20594935

  11. Isolation and characterization of phosmidosine. A new antifungal nucleotide antibiotic.

    PubMed

    Uramoto, M; Kim, C J; Shin-Ya, K; Kusakabe, H; Isono, K; Phillips, D R; McCloskey, J A

    1991-04-01

    A new nucleotide antibiotic, phosmidosine was isolated from a culture filtrate of a newly isolated streptomycete identified as Streptomyces sp. RK-16. HRFAB-MS and elemental analysis established the molecular formula of C16H24N7O8P. 1H, 13C and 31P NMR indicated the presence of a methyl phosphate group and UV spectra were similar to those of 8-hydroxyadenosine. The antibiotic inhibited spore formation of Botrytis cinerea at the concentration of 0.25 micrograms/ml. PMID:2032945

  12. The complete nucleotide sequence of pelargonium leaf curl virus.

    PubMed

    McGavin, Wendy J; MacFarlane, Stuart A

    2016-05-01

    Investigation of a tombusvirus isolated from tulip plants in Scotland revealed that it was pelargonium leaf curl virus (PLCV) rather than the originally suggested tomato bushy stunt virus. The complete sequence of the PLCV genome was determined for the first time, revealing it to be 4789 nucleotides in size and to have an organization similar to that of the other, previously described tombusviruses. Primers derived from the sequence were used to construct a full-length infectious clone of PLCV that recapitulates the disease symptoms of leaf curling in systemically infected pelargonium plants. PMID:26906694

  13. ATCG nucleotide fluctuation of Deinococcus radiodurans radiation genes

    NASA Astrophysics Data System (ADS)

    Holden, Todd; Subramaniam, R.; Sullivan, R.; Cheung, E.; Schneider, C.; Tremberger, G., Jr.; Flamholz, A.; Lieberman, D. H.; Cheung, T. D.

    2007-09-01

    The radiation resistance-repair genes in Deinococcus radiodurans (DR) and E-coli were analyzed in terms of the A, T, C, G nucleotide fluctuations. The studied genes were Rec-A, Rec-Q, and the unique DR PprA gene. In an ATCG sequence, each base was assigned a number equal to its atomic number. The resulting numerical sequence was the basis of the statistical analysis. Fractal analysis using the Higuchi method gave a fractal dimension increase of the Deinococcus radiodurans genes as compared to E-coli, which is comparable to the enhancement observed in the human HAR1 region (HAR1F gene) over that of the chimpanzee. Near neighbor fluctuation was also studied via the Black-Scholes model where the increment sequence was treated as a random walk series. The Deinococcus radiodurans radiation gene standard deviations were consistently higher than that of the E-coli deviations, and agree with the fractal analysis results. The sequence stacking interaction was studied using the published nucleotide-pair melting free energy values and Deinococcus radiodurans radiation genes were shown to possess larger negative free energies. The high sensitivity of the fractal dimension as a biomarker was tested with correlation analysis of the gamma ray dose versus fractal dimension, and the R square values were found to be above 0.9 (N=5). When compared with other nucleotide sequences such as the rRNA sequences, HAR1 and its chimpanzee counterpart, the higher fluctuation (correlated randomness) and larger negative free energy of a DR radiation gene suggested that a radiation resistance-repair sequence exhibited higher complexity. As the HAR1 nucleotide sequence complexity and its transcription activity of co-expressing cortex protein reelin supported a positive selection event in humans, a similar inference of positive selection of coding genes could be drawn for Deinococcus radiodurans when compared to E-coli. The origin of such a positive selection would be consistent with that of a

  14. Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms.

    PubMed

    Fakhrai-Rad, Hossein; Pourmand, Nader; Ronaghi, Mostafa

    2002-05-01

    Pyrosequencing, a non-electrophoretic method for DNA sequencing, is emerging as a popular platform for analysis of single nucleotide polymorphisms (SNPs). This technology has the advantage of accuracy, ease-of-use, and high flexibility for different applications. Here, we review the methodology and the use of this technique for SNP genotyping, SNP discovery, haplotyping, and allelic frequency studies. In addition, we describe new schemes for template preparation and multiplexing as an effort for cost reduction in large-scale studies. PMID:11968080

  15. May Cyclic Nucleotides Be a Source for Abiotic RNA Synthesis?

    NASA Astrophysics Data System (ADS)

    Costanzo, Giovanna; Pino, Samanta; Botta, Giorgia; Saladino, Raffaele; di Mauro, Ernesto

    2011-12-01

    Nucleic bases are obtained by heating formamide in the presence of various catalysts. Formamide chemistry also allows the formation of acyclonucleosides and the phosphorylation of nucleosides in every possible position, also affording 2',3' and 3',5' cyclic forms. We have reported that 3',5' cyclic GMP and 3',5' cyclic AMP polymerize in abiotic conditions yielding short oligonucleotides. The characterization of this reaction is being pursued, several of its parameters have been determined and experimental caveats are reported. The yield of non-enzymatic polymerization of cyclic purine nucleotides is very low. Polymerization is strongly enhanced by the presence of base-complementary RNA sequences.

  16. Regulation of adenylyl cyclase from Blastocladiella emersonii by guanine nucleotides.

    PubMed

    Terenzi, H; Maia, J C

    1993-11-01

    GTP gamma S stimulates adenylyl cyclase in particulate fractions of Blastocladiella emersonii zoospores. Cholera toxin catalyses the ADP-ribosylation of a membrane protein of a molecular weight (46,000) similar to that of the alpha subunit of Gs found in vertebrate cells. A membrane protein of 46 kDa can also be recognized in Western blots by an antipeptide antiserum (RM/1) raised against the C-terminus of G alpha 2-subunits. These results suggest that a G-protein mediates the regulation of Blastocladiella adenylyl cyclase by guanine nucleotides. PMID:8224237

  17. Use of phosphorus oxychloride in synthesizing nucleotides and oligonucleotides

    PubMed Central

    Mungall, W.S.; Greene, G.L.; Miller, P.S.; Letsinger, R.L.

    1974-01-01

    Procedures are described for phosphorylating protected nucleotides, oligonucleotides and phosphoramidate oligonucleotide derivatives at the 3′-hydroxyl group. The conditions (phosphorylation with phosphorus oxychloride and pyridine in dioxane followed by hydrolysis with aqueous pyridine) are sufficiently mild that base labile (trifluoroacetylamino; β-cyanoethyl phosphotriester) and acid labile (O-monomethoxytrityl; phosphoramidate) functions are retained intact. Application of the technique is illustrated by the synthesis of dpT, dTp, d(CF3CONH)Tp, dTpNTp, and dTpNTpNTp. In addition, the utilization of phosphorus oxychloride in joining thymidine derivatives and dinucleoside phosphotriester blocks via phosphodiester links is described. PMID:10793743

  18. Electron capture induced dissociation of nucleotide anions in water nanodroplets.

    PubMed

    Liu, B; Haag, N; Johansson, H; Schmidt, H T; Cederquist, H; Brondsted Nielsen, S; Zettergren, H; Hvelplund, P; Manil, B; Huber, B A

    2008-02-21

    We have studied the outcome of collisions between the hydrated nucleotide anion adenosine 5'-monophosphate (AMP) and sodium. Electron capture leads to hydrogen loss as well as water evaporation regardless of the initial number m of water molecules attached to the parent ion (m< or =16). The yield of dianions with microsecond lifetimes increases strongly with m, which is explained from dielectric screening of the two charges by the water nanodroplet. For comparison, collision induced dissociation results in water losses with no or very little damage of the AMP molecule itself. PMID:18298174

  19. Heavy atom labeled nucleotides for measurement of kinetic isotope effects.

    PubMed

    Weissman, Benjamin P; Li, Nan-Sheng; York, Darrin; Harris, Michael; Piccirilli, Joseph A

    2015-11-01

    Experimental analysis of kinetic isotope effects represents an extremely powerful approach for gaining information about the transition state structure of complex reactions not available through other methodologies. The implementation of this approach to the study of nucleic acid chemistry requires the synthesis of nucleobases and nucleotides enriched for heavy isotopes at specific positions. In this review, we highlight current approaches to the synthesis of nucleic acids enriched site specifically for heavy oxygen and nitrogen and their application in heavy atom isotope effect studies. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment. PMID:25828952

  20. Comparing compressed sequences for faster nucleotide BLAST searches.

    PubMed

    Cameron, Michael; Williams, Hugh E

    2007-01-01

    Molecular biologists, geneticists, and other life scientists use the BLAST homology search package as their first step for discovery of information about unknown or poorly annotated genomic sequences. There are two main variants of BLAST: BLASTP for searching protein collections and BLASTN for nucleotide collections. Surprisingly, BLASTN has had very little attention; for example, the algorithms it uses do not follow those described in the 1997 BLAST paper and no exact description has been published. It is important that BLASTN is state-of-the-art: Nucleotide collections such as GenBank dwarf the protein collections in size, they double in size almost yearly, and they take many minutes to search on modern general purpose workstations. This paper proposes significant improvements to the BLASTN algorithms. Each of our schemes is based on compressed bytepacked formats that allow queries and collection sequences to be compared four bases at a time, permitting very fast query evaluation using lookup tables and numeric comparisons. Our most significant innovations are two new, fast gapped alignment schemes that allow accurate sequence alignment without decompression of the collection sequences. Overall, our innovations more than double the speed of BLASTN with no effect on accuracy and have been integrated into our new version of BLAST that is freely available for download from http://www.fsa-blast.org/. PMID:17666756

  1. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    SciTech Connect

    Stoll, V.S.; Blanchard, J.S.

    1988-02-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between (/sup 14/C)NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.

  2. Endoplasmic reticulum: Where nucleotide sugar transport meets cytokinin control mechanisms

    PubMed Central

    Niemann, Michael CE; Werner, Tomáš

    2015-01-01

    The endoplasmic reticulum (ER) is a multifunctional eukaryotic organelle where the vast majority of secretory proteins are folded and assembled to achieve their correct tertiary structures. The lumen of the ER and Golgi apparatus also provides an environment for numerous glycosylation reactions essential for modifications of proteins and lipids, and for cell wall biosynthesis. These glycosylation reactions require a constant supply of cytosolically synthesized substrate precursors, nucleotide sugars, which are transported by a group of dedicated nucleotide sugar transporters (NST). Recently, we have reported on the identification of a novel ER-localized NST protein, ROCK1, which mediates the transport of UDP-linked acetylated hexosamines across the ER membrane in Arabidopsis. Interestingly, it has been demonstrated that the activity of ROCK1 is important for the regulation of cytokinin-degrading enzymes, cytokinin oxidases/dehydrogenases (CKX), in the ER and, thus, for cytokinin responses. In this addendum we will address the biochemical and cellular activity of the ROCK1 transporter and its phylogenetic relation to other NST proteins. PMID:26418963

  3. Premature aging and cancer in nucleotide excision repair-disorders

    PubMed Central

    Diderich, K.; Alanazi, M.; Hoeijmakers, J.H.J.

    2014-01-01

    During past decades the major impact of DNA damage on cancer as ‘disease of the genes’ has become abundantly apparent. In addition to cancer recent years have also uncovered a very strong association of DNA damage with many features of (premature) aging. The notion that DNA repair systems not only protect against cancer but equally against too fast aging has become evident from a systematic, integral analysis of a variety of mouse mutants carrying defects in e.g. transcription-coupled repair with or without an additional impairment of global genome nucleotide excision repair and the corresponding segmental premature aging syndromes in man. A striking correlation between the degree of the DNA repair deficiency and the acceleration of specific progeroid symptoms has been discovered for those repair systems that primarily protect from the cytotoxic and cytostatic effects of DNA damage. These observations are explained from the perspective of nucleotide excision repair mouse mutant and human syndromes. However, similar principles likely apply to other DNA repair pathways including interstrand crosslink repair and double strand break repair and genome maintenance systems in general, supporting the notion that DNA damage constitutes an important intermediate in the process of aging. PMID:21680258

  4. Gene transfer in the evolution of parasite nucleotide biosynthesis.

    PubMed

    Striepen, Boris; Pruijssers, Andrea J P; Huang, Jinling; Li, Catherine; Gubbels, Marc-Jan; Umejiego, Nwakaso N; Hedstrom, Lizbeth; Kissinger, Jessica C

    2004-03-01

    Nucleotide metabolic pathways provide numerous successful targets for antiparasitic chemotherapy, but the human pathogen Cryptosporidium parvum thus far has proved extraordinarily refractory to classical treatments. Given the importance of this protist as an opportunistic pathogen afflicting immunosuppressed individuals, effective treatments are urgently needed. The genome sequence of C. parvum is approaching completion, and we have used this resource to critically assess nucleotide biosynthesis as a target in C. parvum. Genomic analysis indicates that this parasite is entirely dependent on salvage from the host for its purines and pyrimidines. Metabolic pathway reconstruction and experimental validation in the laboratory further suggest that the loss of pyrimidine de novo synthesis is compensated for by possession of three salvage enzymes. Two of these, uridine kinase-uracil phosphoribosyltransferase and thymidine kinase, are unique to C. parvum within the phylum Apicomplexa. Phylogenetic analysis suggests horizontal gene transfer of thymidine kinase from a proteobacterium. We further show that the purine metabolism in C. parvum follows a highly streamlined pathway. Salvage of adenosine provides C. parvum's sole source of purines. This renders the parasite susceptible to inhibition of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the multistep conversion of AMP to GMP. The inosine 5' monophosphate dehydrogenase inhibitors ribavirin and mycophenolic acid, which are already in clinical use, show pronounced anticryptosporidial activity. Taken together, these data help to explain why widely used drugs fail in the treatment of cryptosporidiosis and suggest more promising targets. PMID:14973196

  5. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  6. Analysis of enzyme-catalyzed nucleotide modification by aldose reductase

    SciTech Connect

    Grimshaw, C.E.

    1987-05-01

    Homogeneous bovine kidney aldose reductase catalyzes two reactions in addition to the normal aldehyde-dependent oxidation of NADPH. First, adduct formation between the oxidized nucleotide and the oxidized substrate is observed during turnover due to initial formation of a reversible E:NADP/sup +/:R-CHO ternary complex, which subsequently reacts to give the covalent complex (E:NADP/sup +/-R-CHO). The reaction is enzyme-catalyzed with substantial enhancement of both the pseudo-first order rate constant and the overall K/sub eq/ relative to the reaction with free NADP/sup +/ in aqueous buffer. Analysis of the concentration dependence and time-course for reversible dead-end and covalent complex formation are described for several aldehyde and nucleotide substrates. Non-linear time courses for aldehyde reduction and substrate inhibition by the aldehyde substrate in initial velocity studies are completely accounted for by this mechanism, thereby eliminating a simple Dalziel-type explanation for the substrate activation by aldehyde which is also observed. Second, enzyme-catalyzed oxidation of NADPH occurs in the absence of aldehyde substrate with a rate equal to .03% of V/sub max/ for the normal reduction of glyceraldehyde. By 500 MHz /sup 1/H-NMR, the enzyme-catalyzed oxidation of (4-/sup 2/H)NADPH appears to be greater than 95% stereospecific. Spectroscopic evidence for a similar oxidation reaction is observed for the covalent E:NADP/sup +/-R-CHO adduct with glyceraldehyde, but not with glycolaldehyde.

  7. Identification of single nucleotides in MoS2 nanopores.

    PubMed

    Feng, Jiandong; Liu, Ke; Bulushev, Roman D; Khlybov, Sergey; Dumcenco, Dumitru; Kis, Andras; Radenovic, Aleksandra

    2015-12-01

    The size of the sensing region in solid-state nanopores is determined by the size of the pore and the thickness of the pore membrane, so ultrathin membranes such as graphene and single-layer molybdenum disulphide could potentially offer the necessary spatial resolution for nanopore DNA sequencing. However, the fast translocation speeds (3,000-50,000 nt ms(-1)) of DNA molecules moving across such membranes limit their usability. Here, we show that a viscosity gradient system based on room-temperature ionic liquids can be used to control the dynamics of DNA translocation through MoS2 nanopores. The approach can be used to statistically detect all four types of nucleotide, which are identified according to current signatures recorded during their transient residence in the narrow orifice of the atomically thin MoS2 nanopore. Our technique, which exploits the high viscosity of room-temperature ionic liquids, provides optimal single nucleotide translocation speeds for DNA sequencing, while maintaining a signal-to-noise ratio higher than 10. PMID:26389660

  8. Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.

    PubMed Central

    Hofer, F; Fields, S; Schneider, C; Martin, G S

    1994-01-01

    The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The interaction of the RalGDS C-terminal segment with Ras is specific, dependent on activation of Ras by GTP, and blocked by a mutation that affects Ras effector function. These characteristics are similar to those previously demonstrated for the interaction between Ras and its putative effector, Raf, suggesting that the RalGDS may also be a Ras effector. Consistent with this idea, the RalGDS was found to inhibit the binding of Raf to Ras. Images PMID:7972015

  9. Nucleotide Substitution and Recombination at Orthologous Loci in Staphylococcus aureus†

    PubMed Central

    Hughes, Austin L.; Friedman, Robert

    2005-01-01

    The pattern of nucleotide substitution was examined at 2,129 orthologous loci among five genomes of Staphylococcus aureus, which included two sister pairs of closely related genomes (MW2/MSSA476 and Mu50/N315) and the more distantly related MRSA252. A total of 108 loci were unusual in lacking any synonymous differences among the five genomes; most of these were short genes encoding proteins highly conserved at the amino acid sequence level (including many ribosomal proteins) or unknown predicted genes. In contrast, 45 genes were identified that showed anomalously high divergence at synonymous sites. The latter genes were evidently introduced by homologous recombination from distantly related genomes, and in many cases, the pattern of nucleotide substitution made it possible to reconstruct the most probable recombination event involved. These recombination events introduced genes encoding proteins that differed in amino acid sequence and thus potentially in function. Several of the proteins are known or likely to be involved in pathogenesis (e.g., staphylocoagulase, exotoxin, Ser-Asp fibrinogen-binding bone sialoprotein-binding protein, fibrinogen and keratin-10 binding surface-anchored protein, fibrinogen-binding protein ClfA, and enterotoxin P). Therefore, the results support the hypothesis that exchange of homologous genes among S. aureus genomes can play a role in the evolution of pathogenesis in this species. PMID:15805516

  10. Nucleotide sequence and expression of a Drosophila metallothionein.

    PubMed

    Lastowski-Perry, D; Otto, E; Maroni, G

    1985-02-10

    A Drosophila melanogaster cDNA clone was isolated based on its more intense hybridization to RNA sequences from copper-fed larvae than from control larval RNA. This clone showed strong hybridization to mouse metallothionein I cDNA at reduced stringency. Its nucleotide sequence includes an open reading segment which codes for a 40-amino acid protein; this protein is identified as metallothionein based on its similarity to the amino-terminal portion of mammalian and crab metalloproteins. The 10 cysteine residues present occur in five pairs of near vicinal cysteines (Cys-X-Cys). This cDNA sequence hybridized to a 400-nucleotide polyadenylated RNA whose presence in the cells of the alimentary canal of larvae was stimulated by ingestion of cadmium or copper; in other tissues this RNA was present at much lower levels. Mercury, silver, and zinc induced metallothionein to a lesser extent. The level of metallothionein RNA increased very soon after the initiation of metal treatment and reached a maximum after approximately 36 h. PMID:2578462

  11. Gene transfer in the evolution of parasite nucleotide biosynthesis

    PubMed Central

    Striepen, Boris; Pruijssers, Andrea J. P.; Huang, Jinling; Li, Catherine; Gubbels, Marc-Jan; Umejiego, Nwakaso N.; Hedstrom, Lizbeth; Kissinger, Jessica C.

    2004-01-01

    Nucleotide metabolic pathways provide numerous successful targets for antiparasitic chemotherapy, but the human pathogen Cryptosporidium parvum thus far has proved extraordinarily refractory to classical treatments. Given the importance of this protist as an opportunistic pathogen afflicting immunosuppressed individuals, effective treatments are urgently needed. The genome sequence of C. parvum is approaching completion, and we have used this resource to critically assess nucleotide biosynthesis as a target in C. parvum. Genomic analysis indicates that this parasite is entirely dependent on salvage from the host for its purines and pyrimidines. Metabolic pathway reconstruction and experimental validation in the laboratory further suggest that the loss of pyrimidine de novo synthesis is compensated for by possession of three salvage enzymes. Two of these, uridine kinase-uracil phosphoribosyltransferase and thymidine kinase, are unique to C. parvum within the phylum Apicomplexa. Phylogenetic analysis suggests horizontal gene transfer of thymidine kinase from a proteobacterium. We further show that the purine metabolism in C. parvum follows a highly streamlined pathway. Salvage of adenosine provides C. parvum's sole source of purines. This renders the parasite susceptible to inhibition of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the multistep conversion of AMP to GMP. The inosine 5′ monophosphate dehydrogenase inhibitors ribavirin and mycophenolic acid, which are already in clinical use, show pronounced anticryptosporidial activity. Taken together, these data help to explain why widely used drugs fail in the treatment of cryptosporidiosis and suggest more promising targets. PMID:14973196

  12. Extreme accumulation of nucleotides in simulated hydrothermal pore systems

    PubMed Central

    Baaske, Philipp; Weinert, Franz M.; Duhr, Stefan; Lemke, Kono H.; Russell, Michael J.; Braun, Dieter

    2007-01-01

    We simulate molecular transport in elongated hydrothermal pore systems influenced by a thermal gradient. We find extreme accumulation of molecules in a wide variety of plugged pores. The mechanism is able to provide highly concentrated single nucleotides, suitable for operations of an RNA world at the origin of life. It is driven solely by the thermal gradient across a pore. On the one hand, the fluid is shuttled by thermal convection along the pore, whereas on the other hand, the molecules drift across the pore, driven by thermodiffusion. As a result, millimeter-sized pores accumulate even single nucleotides more than 108-fold into micrometer-sized regions. The enhanced concentration of molecules is found in the bulk water near the closed bottom end of the pore. Because the accumulation depends exponentially on the pore length and temperature difference, it is considerably robust with respect to changes in the cleft geometry and the molecular dimensions. Whereas thin pores can concentrate only long polynucleotides, thicker pores accumulate short and long polynucleotides equally well and allow various molecular compositions. This setting also provides a temperature oscillation, shown previously to exponentially replicate DNA in the protein-assisted PCR. Our results indicate that, for life to evolve, complicated active membrane transport is not required for the initial steps. We find that interlinked mineral pores in a thermal gradient provide a compelling high-concentration starting point for the molecular evolution of life. PMID:17494767

  13. Nucleotide Docking: Prediction of Reactant State Complexes for Ribonuclease Enzymes

    SciTech Connect

    Elsasser, Brigitta M.; Fels, Gregor

    2010-12-01

    Ribonuclease enzymes (RNases) play key roles in the maturation and metabolism of all RNA molecules. Computational simulations of the processes involved can help to elucidate the underlying enzymatic mechanism and is often employed in a synergistic approach together with biochemical experiments. Theoretical calculations require atomistic details regarding the starting geometries of the molecules involved, which, in the absence of crystallographic data, can only be achieved from computational docking studies. Fortunately, docking algorithms have improved tremendously in recent years, so that reliable structures of enzyme-ligand complexes can now be successfully obtained from computation. However, most docking programs are not particularly optimized for nucleotide docking. In order to assist our studies on the cleavage of RNA by the two most important ribonuclease enzymes, RNase A and RNase H, we evaluated four docking tools - MOE2009, Glide 5.5, QXP-Flo+0802, and Autodock 4.0 - for their ability to simulate complexes between these enzymes and RNA oligomers. To validate our results, we analyzed the docking results with respect to the known key interactions between the protein and the nucleotide. In addition, we compared the predicted complexes with X-ray structures of the mutated enzyme as well as with structures obtained from previous calculations. In this manner, we were able to prepare the desired reaction state complex so that it could be used as the starting structure for further DFT/B3LYP QM/MM reaction mechanism studies.

  14. Single Nucleotide Polymorphism Analysis of European Archaeological M. leprae DNA

    PubMed Central

    Watson, Claire L.; Lockwood, Diana N. J.

    2009-01-01

    Background Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae ancient DNA (aDNA) from medieval bones and single nucleotide polymorphism (SNP) typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution. Methods and Findings Skeletons have been exhumed from 3 European countries (the United Kingdom, Denmark and Croatia) and are dated around the medieval period (476 to 1350 A.D.). we tested for the presence of 3 previously identified single nucleotide polymorphisms (SNPs) in 10 aDNA extractions. M. leprae aDNA was extracted from 6 of the 10 bone samples. SNP analysis of these 6 extractions were compared to previously analysed European SNP data using the same PCR assays and were found to be the same. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3). Conclusions These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide. PMID:19847306

  15. Nucleotide variation in the Toxoplasma gondii micronemal protein 8 gene.

    PubMed

    Li, Z Y; Song, H Q; Wang, C R; Zhu, X Q

    2016-01-01

    Toxoplasma gondii is a successful opportunistic protozoan distributed worldwide, which can infect all vertebrates, leading to serious infection, blindness, and abortion. Micronemal (MIC) proteins are critically important for T. gondii infection, as they participate in various stages of the Toxoplasma life cycle, including invasion and attachment to host cells. MIC8 secretion relies on the concentration of intracellular calcium, and can mediate the invasion of T. gondii by interacting with soluble MIC3. To investigate genetic diversity of the MIC8 gene, 16 T. gondii strains from different hosts and geographical locations, and two reference isolates (ToxoDB: TGME49_245490 and TGVEG_245490) were examined in this study. The results showed that all the examined MIC8 genes are 2055 bp, with an A+T content ranging from 50.2 to 50.6%. Conversely, lower levels of variation were detected within their nucleotide and amino acid sequences. Phylogenetic analyses indicated that three classical genotypes of T. gondii and the ToxoDB#9 genotype did not group exclusively via Bayesian inference, maximum parsimony, neighbor joining, and/or maximum likelihood assays based on the nucleotide and amino acid sequences of the MIC8 gene. In summary, the T. gondii MIC8 gene is not a suitable marker for population genetic studies of this parasite. PMID:27173337

  16. Nucleotide sequence corresponding to five chemotaxis genes in Escherichia coli.

    PubMed Central

    Mutoh, N; Simon, M I

    1986-01-01

    The nucleotide sequence of DNA which contains five chemotaxis-related genes of Escherichia coli, cheW, cheR, cheB, cheY, and cheZ, and part of the cheA gene was determined. Molecular weights of the polypeptides encoded by these genes were calculated from translated amino acid sequences, and they were 18,100 for cheW, 32,700 for cheR, 37,500 for cheB, 14,100 for cheY, and 24,000 for cheZ. Nucleotide sequences which could act as ribosome-binding sites were found in the upstream region of each gene. After the termination codon of the cheW gene, a typical rho-independent transcription termination signal was observed. There are no other open reading frames long enough to encode polypeptides in this region except those which code for the two previously reported genes tar and tap. PMID:3510184

  17. Evaluation of published single nucleotide polymorphisms associated with acute GVHD.

    PubMed

    Chien, Jason W; Zhang, Xinyi Cindy; Fan, Wenhong; Wang, Hongwei; Zhao, Lue Ping; Martin, Paul J; Storer, Barry E; Boeckh, Michael; Warren, Edus H; Hansen, John A

    2012-05-31

    Candidate genetic associations with acute GVHD (aGVHD) were evaluated with the use of genotyped and imputed single-nucleotide polymorphism data from genome-wide scans of 1298 allogeneic hematopoietic cell transplantation (HCT) donors and recipients. Of 40 previously reported candidate SNPs, 6 were successfully genotyped, and 10 were imputed and passed criteria for analysis. Patient and donor genotypes were assessed for association with grades IIb-IV and III-IV aGVHD, stratified by donor type, in univariate and multivariate allelic, recessive and dominant models. Use of imputed genotypes to replicate previous IL10 associations was validated. Similar to previous publications, the IL6 donor genotype for rs1800795 was associated with a 20%-50% increased risk for grade IIb-IV aGVHD after unrelated HCT in the allelic (adjusted P = .011) and recessive (adjusted P = .0013) models. The donor genotype was associated with a 60% increase in risk for grade III-IV aGVHD after related HCT (adjusted P = .028). Other associations were found for IL2, CTLA4, HPSE, and MTHFR but were inconsistent with original publications. These results illustrate the advantages of using imputed single-nucleotide polymorphism data in genetic analyses and demonstrate the importance of validation in genetic association studies. PMID:22282500

  18. Mutants affecting nucleotide recognition by T7 DNA polymerase.

    PubMed

    Donlin, M J; Johnson, K A

    1994-12-13

    Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in

  19. Neighboring group catalysis in the design of nucleotide prodrugs.

    PubMed

    Khamnei, S; Torrence, P F

    1996-09-27

    An approach is described for potential application to the delivery of polar nucleosides and nucleotides across lipophilic membranes, namely, nucleotide prodrugs based on salicyl phosphate. 3'-Azido-3'-deoxythymidine (AZT) and 3'-deoxythymidine (ddT) were chosen as models. For the synthesis of prototype compounds 1 and 2, the approach was first to react either methyl salicylate (for 1) or phenyl salicylate (for 2) with phosphorus oxychloride in dry methylene chloride at 0 degree C with the addition of triethylamine as acid scavenger. The resulting intermediate phosphorodichloridate was reacted immediately with excess nucleoside under the same conditions. The control model compound 3 was prepared by reaction of phenyl phosphorodichloridate and excess nucleoside in pyridine/methylene chloride at 0 degree C to give 3 in 82% yield. The synthesis of triester 7 involved reaction of alpha-(chloroacetyl)salicyl chloride with 2,3,4,6-tetra-O-benzyl-D-glucopyranose to give [[(2,3,4,6-tetra-O-benzyl-D-glucopyranosyl)-oxy]carbonyl]-2- (1-chloroacetoxy)benzene (4) which was dechloroacetylated to 5,2,3,4,6-tetra-O-benzyl-D-glucopyranosyl salicylate. Phosphorylation of 5 with phosphorus oxychloride provided the phosphorodichloridate which was directly converted to 6 by reaction with dideoxythymidine. Removal of benzyl groups by catalytic hydrogenation gave compound 7, bis(2',3'-dideoxythymidin-5'-yl) D-glucopyranosyl phosphate. The AZT prodrug triesters, 1 and 2, underwent much more rapid hydrolysis than the triester 3, most probably due to the formation of an acyl phosphate complex from the attack on phosphorus of the salicylate carboxylate. The hydrolysis of the less lipophilic 7 was significantly slower than that of 1 or 2. Both pig liver esterase and rat brain cytosol were able to effect the cleavage to dinucleotide or mononucleotide of prodrug forms 2 and 7, much more rapidly than either 3 or 1, suggesting that the esterase-like enzymatic activity of rat brain was similar to

  20. Enzymatic polymerisation involving 2'-amino-LNA nucleotides.

    PubMed

    Johannsen, Marie W; Veedu, Rakesh N; Madsen, Andreas Stahl; Wengel, Jesper

    2012-05-15

    The triphosphate of the thymine derivative of 2'-amino-LNA (2'-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion® HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2'-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2'-Amino-LNA-T in the template and 2'-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2'-amino-LNA-T nucleotides. PMID:22503454

  1. Empirical Bayes Estimation of Coalescence Times from Nucleotide Sequence Data.

    PubMed

    King, Leandra; Wakeley, John

    2016-09-01

    We demonstrate the advantages of using information at many unlinked loci to better calibrate estimates of the time to the most recent common ancestor (TMRCA) at a given locus. To this end, we apply a simple empirical Bayes method to estimate the TMRCA. This method is both asymptotically optimal, in the sense that the estimator converges to the true value when the number of unlinked loci for which we have information is large, and has the advantage of not making any assumptions about demographic history. The algorithm works as follows: we first split the sample at each locus into inferred left and right clades to obtain many estimates of the TMRCA, which we can average to obtain an initial estimate of the TMRCA. We then use nucleotide sequence data from other unlinked loci to form an empirical distribution that we can use to improve this initial estimate. PMID:27440864

  2. A single natural nucleotide mutation alters bacterial pathogen host tropism.

    PubMed

    Viana, David; Comos, María; McAdam, Paul R; Ward, Melissa J; Selva, Laura; Guinane, Caitriona M; González-Muñoz, Beatriz M; Tristan, Anne; Foster, Simon J; Fitzgerald, J Ross; Penadés, José R

    2015-04-01

    The capacity of microbial pathogens to alter their host tropism leading to epidemics in distinct host species populations is a global public and veterinary health concern. To investigate the molecular basis of a bacterial host-switching event in a tractable host species, we traced the evolutionary trajectory of the common rabbit clone of Staphylococcus aureus. We report that it evolved through a likely human-to-rabbit host jump over 40 years ago and that only a single naturally occurring nucleotide mutation was required and sufficient to convert a human-specific S. aureus strain into one that could infect rabbits. Related mutations were identified at the same locus in other rabbit strains of distinct clonal origin, consistent with convergent evolution. This first report of a single mutation that was sufficient to alter the host tropism of a microorganism during its evolution highlights the capacity of some pathogens to readily expand into new host species populations. PMID:25685890

  3. Single nucleotide variations: biological impact and theoretical interpretation.

    PubMed

    Katsonis, Panagiotis; Koire, Amanda; Wilson, Stephen Joseph; Hsu, Teng-Kuei; Lua, Rhonald C; Wilkins, Angela Dawn; Lichtarge, Olivier

    2014-12-01

    Genome-wide association studies (GWAS) and whole-exome sequencing (WES) generate massive amounts of genomic variant information, and a major challenge is to identify which variations drive disease or contribute to phenotypic traits. Because the majority of known disease-causing mutations are exonic non-synonymous single nucleotide variations (nsSNVs), most studies focus on whether these nsSNVs affect protein function. Computational studies show that the impact of nsSNVs on protein function reflects sequence homology and structural information and predict the impact through statistical methods, machine learning techniques, or models of protein evolution. Here, we review impact prediction methods and discuss their underlying principles, their advantages and limitations, and how they compare to and complement one another. Finally, we present current applications and future directions for these methods in biological research and medical genetics. PMID:25234433

  4. Insights into how nucleotide-binding domains power ABC transport.

    PubMed

    Newstead, Simon; Fowler, Philip W; Bilton, Paul; Carpenter, Elisabeth P; Sadler, Peter J; Campopiano, Dominic J; Sansom, Mark S P; Iwata, So

    2009-09-01

    The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered. PMID:19748342

  5. [Application of single nucleotide polymorphism in crop genetics and improvement].

    PubMed

    Du, Chun-Fang; Liu, Hui-Min; Li, Run-Zhi; Li, Peng-Bo; Ren, Zhi-Qiang

    2003-11-01

    Single nucleotide polymorphism(SNP) is the most common type of sequence difference between alleles, which can be used as a kind of high-throughput genetic marker. Several different routes have been developed to discover and identify SNP. These include the direct sequencing of PCR amplicons, electronic SNP(eSNP) and so on. SNP assays have been made in many crop species such as maize and soybean. The elite germplasm of some crops have been narrowed in genetic diversity, increasing the amount of linkage disequilibrium (LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes. SNP analysis has been broadly used in the field of plant gene mapping, integration of genetic and physical maps, DNA marker-assisted breeding and functional genomics. PMID:15639972

  6. Syndrome-based discrimination of single nucleotide polymorphism.

    PubMed

    May, E E; Dolan, P; Crozier, P; Brozik, S

    2006-01-01

    The ability to discriminate nucleic acid sequences is necessary for a wide variety of applications: high throughput screening, distinguishing genetically modified organisms (GMOs), molecular computing, differentiating biological markers, fingerprinting a specific sensor response for complex systems, etc. Hybridization-based target recognition and discrimination is central to the operation of nucleic acid sensor systems. Therefore developing a quantitative correlation between mishybridization events and sensor out put is critical to the accurate interpretation of results. In this work, using experimental data produced by introducing single mutations (single nucleotide polymorphisms, SNPs) in the probe sequence of computational catalytic molecular beacons (deoxyribozyme gates) [1], we investigate coding theory algorithms for uniquely categorizing SNPs based on the calculation of syndromes. PMID:17947098

  7. A nucleotide-level coarse-grained model of RNA

    SciTech Connect

    Šulc, Petr; Ouldridge, Thomas E.; Louis, Ard A.; Romano, Flavio; Doye, Jonathan P. K.

    2014-06-21

    We present a new, nucleotide-level model for RNA, oxRNA, based on the coarse-graining methodology recently developed for the oxDNA model of DNA. The model is designed to reproduce structural, mechanical, and thermodynamic properties of RNA, and the coarse-graining level aims to retain the relevant physics for RNA hybridization and the structure of single- and double-stranded RNA. In order to explore its strengths and weaknesses, we test the model in a range of nanotechnological and biological settings. Applications explored include the folding thermodynamics of a pseudoknot, the formation of a kissing loop complex, the structure of a hexagonal RNA nanoring, and the unzipping of a hairpin motif. We argue that the model can be used for efficient simulations of the structure of systems with thousands of base pairs, and for the assembly of systems of up to hundreds of base pairs. The source code implementing the model is released for public use.

  8. The nucleotide sequence of the bacteriophage T5 ltf gene.

    PubMed

    Kaliman, A V; Kulshin, V E; Shlyapnikov, M G; Ksenzenko, V N; Kryukov, V M

    1995-06-01

    The nucleotide sequence of the bacteriophage T5 Bg/II-BamHI fragment (4,835 bp in length) known to carry a gene encoding the LTF protein which forms the phage L-shaped tail fibers was determined. It was shown to contain an open reading frame for 1,396 amino acid residues that corresponds to a protein of 147.8 kDa. The coding region of ltf gene is preceded by a typical Shine-Dalgarno sequence. Downstream from the ltf gene there is a strong transcription terminator. Data bank analysis of the LTF protein sequence reveals 55.1% identity to the hypothetical protein ORF 401 of bacteriophage lambda in a segment of 118 amino acids overlap. PMID:7789514

  9. A fluorescence polarization assay for cyclic nucleotide phosphodiesterases.

    PubMed

    Huang, Wei; Zhang, Yan; Sportsman, J Richard

    2002-06-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3'-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented. PMID:12097184

  10. Fluorogenic Labeling of 5-Formylpyrimidine Nucleotides in DNA and RNA.

    PubMed

    Samanta, Biswajit; Seikowski, Jan; Höbartner, Claudia

    2016-01-26

    5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA. PMID:26679556

  11. Nucleotide sequence of Bacillus phage Nf terminal protein gene.

    PubMed Central

    Leavitt, M C; Ito, J

    1987-01-01

    The nucleotide sequence of Bacillus phage Nf gene E has been determined. Gene E codes for phage terminal protein which is the primer necessary for the initiation of DNA replication. The deduced amino acid sequence of Nf terminal protein is approximately 66% homologous with the terminal proteins of Bacillus phages PZA and luminal diameter 29, and shows similar hydropathy and secondary structure predictions. A serine which has been identified as the residue which covalently links the protein to the 5' end of the genome in luminal diameter 29, is conserved in all three phages. The hydropathic and secondary structural environment of this serine is similar in these phage terminal proteins and also similar to the linking serine of adenovirus terminal protein. PMID:3601672

  12. Nucleotide excision repair deficient mouse models and neurological disease.

    PubMed

    Niedernhofer, Laura J

    2008-07-01

    Nucleotide excision repair (NER) is a highly conserved mechanism to remove helix-distorting DNA base damage. A major substrate for NER is DNA damage caused by environmental genotoxins, most notably ultraviolet radiation. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three human diseases caused by inherited defects in NER. The symptoms and severity of these diseases vary dramatically, ranging from profound developmental delay to cancer predisposition and accelerated aging. All three syndromes include neurological disease, indicating an important role for NER in protecting against spontaneous DNA damage as well. To study the pathophysiology caused by DNA damage, numerous mouse models of NER-deficiency were generated by knocking-out genes required for NER or knocking-in disease-causing human mutations. This review explores the utility of these mouse models to study neurological disease caused by NER-deficiency. PMID:18272436

  13. Current research status, databases and application of single nucleotide polymorphism.

    PubMed

    Javed, R; Mukesh

    2010-07-01

    Single Nucleotide Polymorphisms (SNPs) are the most frequent form of DNA variation in the genome. SNPs are genetic markers which are bi-allelic in nature and grow at a very fast rate. Current genomic databases contain information on several million SNPs. More than 6 million SNPs have been identified and the information is publicly available through the efforts of the SNP Consortium and others data bases. The NCBI plays a major role in facillating the identification and cataloging of SNPs through creation and maintenance of the public SNP database (dbSNP) by the biomedical community worldwide and stimulate many areas of biological research including the identification of the genetic components of disease. In this review article, we are compiling the existing SNP databases, research status and their application. PMID:21717869

  14. Nucleotide sequences of five anti-lysozyme monoclonal antibodies.

    PubMed Central

    Darsley, M J; Rees, A R

    1985-01-01

    The nucleotide sequences of the heavy and light chain immunoglobulin mRNAs derived from five hybridomas (Gloop 1-5) secreting IgGs specific for the loop region of hen egg lysozyme were determined. These monoclonal antibodies recognise three distinct but overlapping epitopes within the loop region. The sequences of two pairs of antibodies with indistinguishable fine specificities were similar in both chains whereas the sequences of antibodies of non-identical specificities were very different. It is proposed that the D-segments expressed in two of the antibodies (Gloop3 and Gloop4) are the products of one, or perhaps two, previously unidentified germ line D-genes. Gloop1 and Gloop2 use a D-segment previously identified in antibodies specific for the hapten 2-phenyloxazolone; however it is recombined in a different reading frame in the anti-lysozyme antibodies, producing a different amino acid sequence. PMID:2410256

  15. Osmotic surveillance mediates rapid wound closure through nucleotide release

    PubMed Central

    Gault, William J.; Enyedi, Balázs

    2014-01-01

    Osmotic cues from the environment mediate rapid detection of epithelial breaches by leukocytes in larval zebrafish tail fins. Using intravital luminescence and fluorescence microscopy, we now show that osmolarity differences between the interstitial fluid and the external environment trigger ATP release at tail fin wounds to initiate rapid wound closure through long-range activation of basal epithelial cell motility. Extracellular nucleotide breakdown, at least in part mediated by ecto-nucleoside triphosphate diphosphohydrolase 3 (Entpd3), restricts the range and duration of osmotically induced cell migration after injury. Thus, in zebrafish larvae, wound repair is driven by an autoregulatory circuit that generates pro-migratory tissue signals as a function of environmental exposure of the inside of the tissue. PMID:25533845

  16. The adsorption and reaction of adenine nucleotides on montmorillonite

    NASA Technical Reports Server (NTRS)

    Ferris, James P.; Hagan, William J., Jr.

    1986-01-01

    The binding of AMP to Zn(2+)-montmorillonite is investigated in the presence of salts and Good's zwitterion buffers, PIPES and MES. The initial concentrations of nucleotide and the percent adsorbtion are used to calculate the adsorption isotherms, and the Langmuir adsorption equation is used for the analysis of data. The adsorption coefficient was found to be three times greater in the presence of 0.2 M PIPES than in its absence. In addition, basal spacings measured by X-ray diffraction were increased by the buffer. These results are interpreted in terms of a model in which the adsorption of AMP is mediated by a Zn(2+) complex of PIPES in different orientations in the interlamellar region of the montmorillonite. Mixed ligand complexes of this type are reminiscent of the complexes observed between metal ions and biological molecules in living systems.

  17. A nucleotide-level coarse-grained model of RNA

    NASA Astrophysics Data System (ADS)

    Šulc, Petr; Romano, Flavio; Ouldridge, Thomas E.; Doye, Jonathan P. K.; Louis, Ard A.

    2014-06-01

    We present a new, nucleotide-level model for RNA, oxRNA, based on the coarse-graining methodology recently developed for the oxDNA model of DNA. The model is designed to reproduce structural, mechanical, and thermodynamic properties of RNA, and the coarse-graining level aims to retain the relevant physics for RNA hybridization and the structure of single- and double-stranded RNA. In order to explore its strengths and weaknesses, we test the model in a range of nanotechnological and biological settings. Applications explored include the folding thermodynamics of a pseudoknot, the formation of a kissing loop complex, the structure of a hexagonal RNA nanoring, and the unzipping of a hairpin motif. We argue that the model can be used for efficient simulations of the structure of systems with thousands of base pairs, and for the assembly of systems of up to hundreds of base pairs. The source code implementing the model is released for public use.

  18. Countermeasure for space flight effects on immune system: nutritional nucleotides

    NASA Technical Reports Server (NTRS)

    Kulkarni, A. D.; Yamauchi, K.; Sundaresan, A.; Ramesh, G. T.; Pellis, N. R.

    2005-01-01

    Microgravity and its environment have adverse effects on the immune system. Abnormal immune responses observed in microgravity may pose serious consequences, especially for the recent directions of NASA for long-term space missions to Moon, Mars and deep Space exploration. The study of space flight immunology is limited due to relative inaccessibility, difficulty of performing experiments in space, and inadequate provisions in this area in the United States and Russian space programs (Taylor 1993). Microgravity and stress experienced during space flights results in immune system aberration (Taylor 1993). In ground-based mouse models for some of the microgravity effects on the human body, hindlimb unloading (HU) has been reported to cause abnormal cell proliferation and cytokine production (Armstrong et al., 1993, Chapes et al. 1993). In this report, we document that a nutritional nucleotide supplementation as studied in ground-based microgravity analogs, has potential to serve as a countermeasure for the immune dysfunction observed in space travel.

  19. Single Nucleotide Polymorphism Mapping Using Genome-Wide Unique Sequences

    PubMed Central

    Chen, Leslie Y.Y.; Lu, Szu-Hsien; Shih, Edward S.C.; Hwang, Ming-Jing

    2002-01-01

    As more and more genomic DNAs are sequenced to characterize human genetic variations, the demand for a very fast and accurate method to genomically position these DNA sequences is high. We have developed a new mapping method that does not require sequence alignment. In this method, we first identified DNA fragments of 15 bp in length that are unique in the human genome and then used them to position single nucleotide polymorphism (SNP) sequences. By use of four desktop personal computers with AMD K7 (1 GHz) processors, our new method mapped more than 1.6 million SNP sequences in 20 hr and achieved a very good agreement with mapping results from alignment-based methods. PMID:12097348

  20. Computational learning on specificity-determining residue-nucleotide interactions

    PubMed Central

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Moses, Alan M.; Zhang, Zhaolei

    2015-01-01

    The protein–DNA interactions between transcription factors and transcription factor binding sites are essential activities in gene regulation. To decipher the binding codes, it is a long-standing challenge to understand the binding mechanism across different transcription factor DNA binding families. Past computational learning studies usually focus on learning and predicting the DNA binding residues on protein side. Taking into account both sides (protein and DNA), we propose and describe a computational study for learning the specificity-determining residue-nucleotide interactions of different known DNA-binding domain families. The proposed learning models are compared to state-of-the-art models comprehensively, demonstrating its competitive learning performance. In addition, we describe and propose two applications which demonstrate how the learnt models can provide meaningful insights into protein–DNA interactions across different DNA binding families. PMID:26527718

  1. Polymorphisms of nucleotide excision repair genes predict melanoma survival.

    PubMed

    Li, Chunying; Yin, Ming; Wang, Li-E; Amos, Christopher I; Zhu, Dakai; Lee, Jeffrey E; Gershenwald, Jeffrey E; Grimm, Elizabeth A; Wei, Qingyi

    2013-07-01

    Melanoma is the most highly malignant skin cancer, and nucleotide excision repair (NER) is involved in melanoma susceptibility. In this analysis of 1,042 melanoma patients, we evaluated whether genetic variants of NER genes may predict survival outcome of melanoma patients. We used genotyping data of 74 tagging single-nucleotide polymorphisms (tagSNPs) in eight core NER genes from our genome-wide association study (including two in XPA, 14 in XPC, three in XPE, four in ERCC1, 10 in ERCC2, eight in ERCC3, 14 in ERCC4, and 19 in ERCC5) and evaluated their associations with prognosis of melanoma patients. Using the Cox proportional hazards model and Kaplan-Meier analysis, we found a predictive role of XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871 SNPs in the prognosis of melanoma patients (rs28720291: AG vs. GG, adjusted hazard ratio (adjHR)=11.2, 95% confidence interval (CI) 3.04-40.9, P=0.0003; rs4150314: AG vs. GG, adjHR=4.76, 95% CI 1.09-20.8, P=0.038; rs2470458: AA vs. AG/GG, adjHR=2.11, 95% CI 1.03-4.33, P=0.040; and rs50871: AA vs. AC/CC adjHR=2.27, 95% CI 1.18-4.35, P=0.015). Patients with an increasing number of unfavorable genotypes had markedly increased death risk. Genetic variants of NER genes, particularly XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871, may independently or jointly modulate survival outcome of melanoma patients. Because our results were based on a median follow-up of 3 years without multiple test corrections, additional large prospective studies are needed to confirm our findings. PMID:23407396

  2. Structural basis for a six nucleotide genetic alphabet.

    PubMed

    Georgiadis, Millie M; Singh, Isha; Kellett, Whitney F; Hoshika, Shuichi; Benner, Steven A; Richards, Nigel G J

    2015-06-01

    Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by standard duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a standard "edge on" Watson-Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystallized with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystallized without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology. PMID:25961938

  3. Evolution of Plant Nucleotide-Sugar Interconversion Enzymes

    PubMed Central

    Yin, Yanbin; Huang, Jinling; Gu, Xiaogang; Bar-Peled, Maor; Xu, Ying

    2011-01-01

    Nucleotide-diphospho-sugars (NDP-sugars) are the building blocks of diverse polysaccharides and glycoconjugates in all organisms. In plants, 11 families of NDP-sugar interconversion enzymes (NSEs) have been identified, each of which interconverts one NDP-sugar to another. While the functions of these enzyme families have been characterized in various plants, very little is known about their evolution and origin. Our phylogenetic analyses indicate that all the 11 plant NSE families are distantly related and most of them originated from different progenitor genes, which have already diverged in ancient prokaryotes. For instance, all NSE families are found in the lower land plant mosses and most of them are also found in aquatic algae, implicating that they have already evolved to be capable of synthesizing all the 11 different NDP-sugars. Particularly interesting is that the evolution of RHM (UDP-L-rhamnose synthase) manifests the fusion of genes of three enzymatic activities in early eukaryotes in a rather intriguing manner. The plant NRS/ER (nucleotide-rhamnose synthase/epimerase-reductase), on the other hand, evolved much later from the ancient plant RHMs through losing the N-terminal domain. Based on these findings, an evolutionary model is proposed to explain the origin and evolution of different NSE families. For instance, the UGlcAE (UDP-D-glucuronic acid 4-epimerase) family is suggested to have evolved from some chlamydial bacteria. Our data also show considerably higher sequence diversity among NSE-like genes in modern prokaryotes, consistent with the higher sugar diversity found in prokaryotes. All the NSE families are widely found in plants and algae containing carbohydrate-rich cell walls, while sporadically found in animals, fungi and other eukaryotes, which do not have or have cell walls with distinct compositions. Results of this study were shown to be highly useful for identifying unknown genes for further experimental characterization to determine

  4. Cyclic nucleotide-activated channels in carp olfactory receptor cells.

    PubMed

    Kolesnikov, S S; Kosolapov, A V

    1993-07-25

    When applied from the cytoplasmic side, cyclic 3',5'-adenosine and guanosine monophosphates reversibly increased the ion permeability of inside-out patches of carp olfactory neuron plasma membrane. The cAMP (cGMP)-induced permeability via cAMP (cGMP) concentration was fitted by Hill's equation with the exponents of 1.07 +/- 0.15 (1.12 +/- 0.05) and EC50 = 1.3 +/- 0.6 microM (0.9 +/- 0.3 microM). Substitution of NaCl in the bathing solution by chlorides of other alkali metals resulted in a slight shift of reversal potential of the cyclic nucleotide-dependent (CN) current, which indicates a weak selectivity of the channels. Permeability coefficients calculated by Goldman-Hodgkin-Katz's equation corresponded to the following relation: PNa/PK/PLi/PRb/PCs = 1:0.98:0.94:0.70:0.61. Ca2+ and Mg2+ in physiological concentrations blocked the channels activated by cyclic nucleotides (CN-channels). In the absence of divalent cations the conductance of single CN-channels was equal to 51 +/- 9 pS in 100 mM NaCl solution. Channel density did not exceed 1 micron-2. The maximal open state probability of the channel (Po) tended towards 1.0 at a high concentration of cAMP or cGMP. Dichlorobenzamil decreased Po without changing the single CN-channel' conductance. CN-channels exhibited burst activity. Mean open and closed times as well as the burst duration depended on agonist concentration. A kinetic model with four states (an inactivated, a closed and two open ones) is suggested to explain the regularities of CN-channel gating and dose-response relations. PMID:8334139

  5. Complete nucleotide sequence of Nootka lupine vein-clearing virus.

    PubMed

    Robertson, Nancy L; Côté, Fabien; Paré, Christine; Leblanc, Eric; Bergeron, Michel G; Leclerc, Denis

    2007-12-01

    The complete genome sequence of Nootka lupine vein-clearing virus (NLVCV) was determined to be 4,172 nucleotides in length containing four open reading frames (ORFs) with a similar genetic organization of virus species in the genus Carmovirus, family Tombusviridae. The order and gene product size, starting from the 5'-proximal ORF consisted of: (1) polymerase/replicase gene, ORF1 (p27) and ORF1RT (readthrough) (p87), (2) movement proteins ORF2 (p7) and ORF3 (p9), and, (3) the 3'-proximal coat protein ORF4, (p37). The genomic 5'- and 3'-proximal termini contained a short (59 nt) and a relatively longer 405 nt untranslated region, respectively. The longer replicase gene product contained the GDD motif common to RNA-dependent RNA polymerases. Phylogenetically, NLVCV formed a subgroup with the following four carmoviruses when separately comparing the amino acids of the coat protein or replicase protein: Angelonia flower break virus (AnFBV), Carnation mottle virus (CarMV), Pelargonium flower break virus (PFBV), and Saguaro cactus virus (SgCV). Whole genome nucleotide analysis (percent identities) among the carmoviruses with NLVCV suggested a similar pattern. The species demarcation criteria in the genus Carmovirus for the amino acid sequence identity of the polymerase (<52%) and coat (<41%) protein genes restricted NLVCV as a distinct species, and instead, placed it as a tentative strain of CarMV, PFBV, or SgCV when both the polymerase and CP were used as the determining factors. In contrast, the species criteria that included different host ranges with no overlap and lack of serology relatedness between NLVCV and the carmoviruses, suggested that NLVCV was a distinct species. The relatively low cutoff percentages allowed for the polymerase and CP genes to dictate the inclusion/exclusion of a distinct carmovirus species should be reevaluated. Therefore, at this time we have concluded that NLVCV should be classified as a tentative new species in the genus Carmovirus

  6. ENGINES: exploring single nucleotide variation in entire human genomes

    PubMed Central

    2011-01-01

    Background Next generation ultra-sequencing technologies are starting to produce extensive quantities of data from entire human genome or exome sequences, and therefore new software is needed to present and analyse this vast amount of information. The 1000 Genomes project has recently released raw data for 629 complete genomes representing several human populations through their Phase I interim analysis and, although there are certain public tools available that allow exploration of these genomes, to date there is no tool that permits comprehensive population analysis of the variation catalogued by such data. Description We have developed a genetic variant site explorer able to retrieve data for Single Nucleotide Variation (SNVs), population by population, from entire genomes without compromising future scalability and agility. ENGINES (ENtire Genome INterface for Exploring SNVs) uses data from the 1000 Genomes Phase I to demonstrate its capacity to handle large amounts of genetic variation (>7.3 billion genotypes and 28 million SNVs), as well as deriving summary statistics of interest for medical and population genetics applications. The whole dataset is pre-processed and summarized into a data mart accessible through a web interface. The query system allows the combination and comparison of each available population sample, while searching by rs-number list, chromosome region, or genes of interest. Frequency and FST filters are available to further refine queries, while results can be visually compared with other large-scale Single Nucleotide Polymorphism (SNP) repositories such as HapMap or Perlegen. Conclusions ENGINES is capable of accessing large-scale variation data repositories in a fast and comprehensive manner. It allows quick browsing of whole genome variation, while providing statistical information for each variant site such as allele frequency, heterozygosity or FST values for genetic differentiation. Access to the data mart generating scripts and to

  7. The nucleotide sequence of the uvrD gene of E. coli.

    PubMed Central

    Finch, P W; Emmerson, P T

    1984-01-01

    The nucleotide sequence of a cloned section of the E. coli chromosome containing the uvrD gene has been determined. The coding region for the UvrD protein consists of 2,160 nucleotides which would direct the synthesis of a polypeptide 720 amino acids long with a calculated molecular weight of 82 kd. The predicted amino acid sequence of the UvrD protein has been compared with the amino acid sequences of other known adenine nucleotide binding proteins and a common sequence has been identified, thought to contribute towards adenine nucleotide binding. PMID:6379604

  8. A theoretical study of Ru(II) polypyridyl DNA intercalators structure and electronic absorption spectroscopy of [Ru(phen)2(dppz)]2+ and [Ru(tap)2(dppz)]2+ complexes intercalated in guanine-cytosine base pairs.

    PubMed

    Ambrosek, David; Loos, Pierre-François; Assfeld, Xavier; Daniel, Chantal

    2010-09-01

    The structural and spectroscopic properties of [Ru(phen)(2)(dppz)](2+) and [Ru(tap)(2)(dppz)](2+) (phen=1,10-phenanthroline; tap=1,4,5,8-tetraazaphenanthrene; dppz=dipyridophenazine ) have been investigated by means of density functional theory (DFT), time-dependent DFT (TD-DFT) within the polarized continuum model (IEF-PCM) and quantum mechanics/molecular mechanics (QM/MM) calculations. The model of the Delta and Lambda enantiomers of Ru(II) intercalated in DNA in the minor and major grooves is limited to the metal complexes intercalated in two guanine-cytosine base pairs. The main experimental spectral features of these complexes reported in DNA or synthetic polynucleotides are better reproduced by the theoretical absorption spectra of the Delta enantiomers regardless of intercalation mode (major or minor groove). This is especially true for [Ru(phen)(2)(dppz)](2+). The visible absorption of [Ru(tap)(2)(dppz)](2+) is governed by the MLCT(tap) transitions regardless of the environment (water, acetonitrile or bases pair), the visible absorption of [Ru(phen)(2)(dppz)](2+) is characterized by transitions to metal-to-ligand-charge-transfer MLCT(dppz) in water and acetonitrile and to MLCT(phen) when intercalated in DNA. The response of the IL(dppz) state to the environment is very sensitive. In vacuum, water and acetonitrile these transitions are characterized by significant oscillator strengths and their positions depend significantly on the medium with blue shifts of about 80 nm when going from vacuum to solvent. When the complex is intercalated in the guanine-cytosine base pairs the (1)IL(dppz) transition contributes mainly to the band at 370 nm observed in the spectrum of [Ru(phen)(2)(dppz)](2+) and to the band at 362 nm observed in the spectrum of [Ru(tap)(2)(dppz)](2+). PMID:20554006

  9. Energy efficiency trade-offs drive nucleotide usage in transcribed regions.

    PubMed

    Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J

    2016-01-01

    Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of 'A' versus 'T' and 'G' versus 'C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides 'U' and 'C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides 'A' and 'G' at non-synonymous coding sites. PMID:27098217

  10. The complete nucleotide sequence and genome organization of Red clover vein mosaic virus (genus Carlavirus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover vein mosaic virus (RCVMV) is a serious pathogen of legume crops including pea, chickpea and lentil. The complete nucleotide sequence was generated from an isolate obtained from chickpea in Washington State. The complete genome of RCVMV consists of 8605 nucleotides excluding the poly(A) ...

  11. A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms.

    PubMed

    Blondal, Thorarinn; Waage, Benedikt G; Smarason, Sigurdur V; Jonsson, Frosti; Fjalldal, Sigridur B; Stefansson, Kari; Gulcher, Jeffery; Smith, Albert V

    2003-12-15

    A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis. PMID:14654708

  12. Complete nucleotide sequence of a maize chlorotic mottle virus isolate from Nebraska

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete genome of a maize chlorotic mottle virus isolate from Nebraska (MCMV-NE) was cloned and sequenced. The MCMV-NE genome consists of 4,436 nucleotides and shares 99.5% nucleotide sequence identity with an MCMV isolate from Kansas (MCMV-KS). Of 22 polymorphic sites, most resulted from t...

  13. Energy efficiency trade-offs drive nucleotide usage in transcribed regions

    PubMed Central

    Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J.

    2016-01-01

    Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of ‘A' versus ‘T' and ‘G' versus ‘C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides ‘U' and ‘C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides ‘A' and ‘G' at non-synonymous coding sites. PMID:27098217

  14. The application and performance of single nucleotide polymorphism markers for population genetic analyses of Lepidoptera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNPs) are nucleotide substitution mutations that tend to be at high densities within eukaryotic genomes. The development of assays that detect allelic variation at SNP loci is attractive for genome mapping, population genetics, and phylogeographic applications. A p...

  15. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of WIPO... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid...

  16. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of WIPO... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid...

  17. Dietary nucleotides protect against alcoholic liver injury by attenuating inflammation and regulating gut microbiota in rats.

    PubMed

    Cai, Xiaxia; Bao, Lei; Wang, Nan; Ren, Jinwei; Chen, Qihe; Xu, Meihong; Li, Di; Mao, Ruixue; Li, Yong

    2016-06-15

    Nucleotides have been reported to be effective in attenuating liver damage and regulating gut microbiota. However, the protective effect of nucleotides against alcoholic liver injury remains unknown. The present study aims to investigate whether nucleotides ameliorate alcoholic liver injury and explores the possible mechanism. Male Wistar rats were given alcohol, equivalent distilled water or an isocaloric amount of dextrose intragastrically twice daily for up to 6 weeks respectively. Two subgroups of alcohol-treated rats were fed with a nucleotide-supplemented AIN-93G rodent diet. Serum enzymes, inflammatory cytokines and microbiota composition of the caecum content were evaluated. We found that nucleotides could significantly decrease serum alanine aminotransferase and aspartate aminotransferase, plasma lipopolysaccharide and inflammatory cytokine levels. Sequencing of 16S rRNA genes revealed that nucleotide-treated rats showed a higher abundance of Firmicutes and a lower abundance of Bacteroidetes than alcohol-treated rats. Moreover, nucleotide treatment inhibited the protein expression of toll-like receptor 4, CD14 and repressed the phosphorylation of inhibitor kappa Bα and nuclear factor-κB p65 in the liver. These results suggested that nucleotides suppressed the inflammatory response and regulated gut microbiota in alcoholic liver injury. The partial inhibition of lipopolysaccharide - toll-like receptor 4-nuclear factor-κB p65 signaling in the liver may be attributed to this mechanism. PMID:27247978

  18. Nucleotide Metabolism in Salt-Stressed Zea mays L. Root Tips

    PubMed Central

    Peterson, Todd A.; Nieman, Richard H.; Clark, Robert A.

    1987-01-01

    Corn plants (Zea mays L. cv Pioneer 3906) were grown in a glass house on control and saline nutrient solutions, in winter and summer. There were two saline treatments, both with osmotic potential = −0.4 megapascal but with different Ca2+/Na+ ratios: 0.03 and 0.73. Root tips and shoot meristems (culm tissue) of 26 day-old plants were analyzed for nucleotides to ascertain if there were correlations between nucleotide pool size and the reduced growth on saline cultures. Several other cell components also were determined. Plants grown in winter were only half as large as those grown in summer mainly because of the lower light intensity and lower temperature. But the relative yield reduction on salt treatment compared to the control was similar in winter and summer. The two different salt treatments caused similar yield reductions. Neither salt treatment affected nucleotide pools in culm tissue, with the possible exception of UDPG in winter. In the case of root tips, salt treatment had little or no effect on nucleotide pool sizes in winter when many already seemed near a critical minimum, but in summer it reduced several pools including ATP, total adenine nucleotide, UTP, total uridine nucleotide, and UDP-glucose. The reductions were greatest on the salt treatment with low Ca2+/Na+. There was no simple correlation between the effects of salt stress on growth and on nucleotide pool size. The nucleotide pools of culm tissue indicated that in some respects this tissue was effectively insulated from the salt stress. Roots that were in direct contact with the saline solution indicated significant reductions in nucleotide pools only in the summer whereas growth was reduced both summer and winter. It is possible that the nucleotide concentrations of root cells in winter were already near a critical minimum so that nucleotide synthesis and growth were tightly linked. Significant reductions in nucleotide pools that would be expected to affect growth were more evident in summer

  19. Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases.

    PubMed

    Welter, Moritz; Verga, Daniela; Marx, Andreas

    2016-08-16

    DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution. PMID:27392211

  20. A/T Run Geometry of B-form DNA Is Independent of Bound Methyl-CpG Binding Domain, Cytosine Methylation and Flanking Sequence

    PubMed Central

    Chia, Jyh Yea; Tan, Wen Siang; Ng, Chyan Leong; Hu, Nien-Jen; Foo, Hooi Ling; Ho, Kok Lian

    2016-01-01

    DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed. PMID:27502833