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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. 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

  9. 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

  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 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

  17. 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

  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. 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

  10. 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.

  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. 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.

  15. 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

  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. 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

  20. 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

  1. 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

  2. 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.

  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. 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

  4. 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

  5. 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

  6. 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

  7. 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

  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

    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 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

  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 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

  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 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

  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. 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

  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. 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

  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. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  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. 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).

  13. 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

  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 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

  19. 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

  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 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

  4. 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

  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. 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

  15. 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

  16. 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

  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. 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.

  3. 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.

  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. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. [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

  7. 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

  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. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  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. 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

  6. 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.

  7. 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

  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. 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.

  10. 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

  11. 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.

  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. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  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. 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

  7. 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

  8. 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

  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. 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

  11. 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

  12. 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

  13. 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

  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. 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

  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 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

  19. 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

  20. 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

  1. 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

  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 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

  4. 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

  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. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. (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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. [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

  7. 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.

  8. 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

  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