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1

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

PubMed Central

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

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

2008-01-01

2

Stabilization of a transition-state analogue at the active site of yeast cytosine deaminase: importance of proton transfers.  

PubMed

It is believed that the binding of pyrimidin-2-one to cytosine deaminase (CD) leads to the formation of 4-[R]-hydroxyl-3,4-dihydropyrimidine (DHP). Here the formation of transition-state analogue (TSA) at the active site of yeast cytosine deaminase (yCD) is investigated by quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations. It is shown that DHP may in fact be unstable in the active site and a proton transfer from the Zn hydroxide group to Glu-64 may occur during the nucleophilic attack, leading to an alkoxide-like TSA complex instead. The free energy simulations for the nucleophilic attack process show that the proton transfer from the Zn hydroxide to Glu-64 may play an important role in stabilizing the TSA complex. PMID:17506543

Xu, Qin; Guo, Haobo; Gorin, Andrey; Guo, Hong

2007-06-14

3

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

PubMed Central

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 intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both binding and chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil. PMID:22208667

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

2012-01-01

4

Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase  

SciTech Connect

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.

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

2011-12-31

5

Targeted endostatin-cytosine deaminase fusion gene therapy plus 5-fluorocytosine suppresses ovarian tumor growth.  

PubMed

There are currently no effective therapies for cancer patients with advanced ovarian cancer, therefore developing an efficient and safe strategy is urgent. To ensure cancer-specific targeting, efficient delivery, and efficacy, we developed an ovarian cancer-specific construct (Survivin-VISA-hEndoyCD) composed of the cancer specific promoter survivin in a transgene amplification vector (VISA; VP16-GAL4-WPRE integrated systemic amplifier) to express a secreted human endostatin-yeast cytosine deaminase fusion protein (hEndoyCD) for advanced ovarian cancer treatment. hEndoyCD contains an endostatin domain that has tumor-targeting ability for anti-angiogenesis and a cytosine deaminase domain that converts the prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic drug, 5-fluorouracil. Survivin-VISA-hEndoyCD was found to be highly specific, selectively express secreted hEndoyCD from ovarian cancer cells, and induce cancer-cell killing in vitro and in vivo in the presence of 5-FC without affecting normal cells. In addition, Survivin-VISA-hEndoyCD plus 5-FC showed strong synergistic effects in combination with cisplatin in ovarian cancer cell lines. Intraperitoneal (i.p.) treatment with Survivin-VISA-hEndoyCD coupled with liposome attenuated tumor growth and prolonged survival in mice bearing advanced ovarian tumors. Importantly, there was virtually no severe toxicity when hEndoyCD is expressed by Survivin-VISA plus 5-FC compared with CMV plus 5-FC. Thus, the current study demonstrates an effective cancer-targeted gene therapy that is worthy of development in clinical trials for treating advanced ovarian cancer. PMID:22562248

Sher, Y-P; Chang, C-M; Juo, C-G; Chen, C-T; Hsu, J L; Lin, C-Y; Han, Z; Shiah, S-G; Hung, M-C

2013-02-28

6

Direct and Bystander Killing of Sarcomas by Novel Cytosine Deaminase Fusion Gene1  

Microsoft Academic Search

Soft tissue and bone sarcomas of the extremities can be difficult to eradicate, and standard treatment may require limb amputation. New therapies to decrease tumor size could improve the effectiveness of treat- ment and decrease the frequency of limb amputation. Cytosine deaminase (CD)-based gene therapy has been shown to be effective in decreasing growth of solid tumors when animals with

Margaret Ramnaraine; Weihong Pan; Michael Goblirsch; Christine Lynch; Victor Lewis; Paul Orchard; Patrick Mantyh; Denis R. Clohisy

2003-01-01

7

AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation  

PubMed Central

AID/APOBEC family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, an important process for transcriptional activation. While oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (mC) or 5-hydroxymethylcytosine (hmC) have emerged as alternatives. Here, we have addressed the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on mC relative to cytosine, their canonical substrate, and no detectable deamination of hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of hmC was undetectable in genomic DNA, while oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation. PMID:22772155

Nabel, Christopher S.; Jia, Huijue; Ye, Yu; Shen, Li; Goldschmidt, Hana L.; Stivers, James T.; Zhang, Yi; Kohli, Rahul M.

2012-01-01

8

Tumors Expressing the Cytosine Deaminase Suicide Gene Can Be Eliminated \\/\\/\\/ Vivo with 5-Fluorocytosine and Induce Protective Immunity to Wild Type Tumor  

Microsoft Academic Search

Successful expression of the cytosine deaminase (CD) suicide gene in vivo is demonstrated in three weakly immunogenic murine tumor models: the 102 and 205 fibrosarcomas and the 38 adenocarcinoma. Normal mam malian cells do not contain cytosine deaminase, but tumor cells transduced with retroviral vectors containing the CD gene metabolize the relatively nontoxic prodrug 5-fluorocytosine to the highly toxic 5-fluorouracil.

Craig A. Mullen; Melissa M. Coale; Robert Lowe; R. Michael Blaese

9

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

PubMed

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

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

10

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

PubMed Central

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

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

2013-01-01

11

Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer specific cytotoxicity and tumor growth delay  

PubMed Central

Purpose Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment and novel therapies are therefore in high demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug Experimental design The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter Insulinoma-associated 1 (INSM1). Therapeutic effect was evaluated in vitro in SCLC cell lines and in vivo in SCLC xenografted nude mice using the non-viral nanoparticle, DOTAP:Cholesterol for transgene delivery. Results INSM1-YCD/5-FC and INSM1-YCD-YUPRT/5-FC therapy induced high cytotoxicity in a range of SCLC cell lines. The highest therapeutic effect was obtained from the YCD-YUPRT fusion gene strategy. No cytotoxicity was induced after treatment of cell lines of other origin than SCLC. In addition the INSM1-YCD-YUPRT/5-FC therapy was superior to an established suicide gene system consisting of the Herpes Simplex Virus Thymidine Kinase (HSVTK) gene and prodrug Ganciclovir (GCV). The superior effect was in part due to massive bystander cytotoxicity of YCD-YUPRT-produced toxins. Finally, INSM1-YCD-YUPRT/5-FC therapy induced significant tumor growth delay in SCLC xenografts compared to control treated xenografts. Conclusions The current study is the first to test cytosine deaminase-based suicide gene therapy for SCLC and the first to demonstrate an anti-tumor effect from the delivery of suicide gene therapeutics for SCLC in vivo. PMID:20371678

Christensen, Camilla L.; Gjetting, Torben; Poulsen, Thomas T.; Cramer, Frederik; Roth, Jack A.; Poulsen, Hans S.

2012-01-01

12

A safe, effective in vivo gene therapy for melanoma using tyrosinase promoter-driven cytosine deaminase gene.  

PubMed

This study was designed to develop a safe, effective gene therapy for disseminated melanoma. We constructed retroviral vectors containing a tyrosinase promoter-cytosine deaminase expression cassette (Tyr/CD), and demonstrated that the tyrosinase promoter conferred a selective expression of cytosine deaminase (CD) gene in B16 melanoma cells, especially when the Tyr/CD cassette inserted in 3'LTR region of a retroviral vector. In vivo gene therapy for the intraperitoneally disseminated melanoma using Tyr/CD retrovirus-producing cells and 5-fluorocytosine (5-FC) showed that retroviruses produced in situ were capable of infecting tumor xenografts and bone marrow cells in animal model, and survival rates were prolonged significantly as compared with those treated with CD2 retrovirus-producing cells and 5-FC. Importantly, the treatment-related bone marrow suppression was not observed in the former treatment, while profound bone marrow suppression was observed in the latter treatment. In vivo gene therapy using retrovirus-producing cells containing suicide gene under the control of a tissue-specific promoter and 5-FC administration is safer and more effective for the treatment of disseminated melanoma, as compared with retrovirus-producing cells containing the gene under the control of a universal promoter and 5-FC. PMID:10363176

Cao, G; Zhang, X; He, X; Chen, Q; Qi, Z

1999-01-01

13

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

SciTech Connect

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.

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. (Pitt); (UMASS, MED); (SLUHSC); (UCSF); (UMM)

2012-04-04

14

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

PubMed

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

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

15

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

SciTech Connect

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.

Zhang, Jufeng [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China); Wang, Zhanli [Technology Center, NeoTrident Technology Ltd., Beijing 100080 (China); Wei, Fang [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China); Qiu, Wei [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China); Zhang, Liangren [School of Pharmaceutical Science, Peking University, Beijing 100083 (China); Huang, Qian [Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080 (China)]. E-mail: qhuang@sjtu.edu.cn

2007-08-17

16

Analysis of the human carcinoembryonic antigen promoter core region in colorectal carcinoma-selective cytosine deaminase gene therapy.  

PubMed

We isolated a 204-base pair carcinoembryonic antigen (CEA) promoter core region from a CEA-producing human colorectal carcinoma (CRC) and constructed retrovirus vectors carrying the expression cassette consisting of the CEA promoter core region and the cytosine deaminase (CD) gene. pCD2 retrovirus carrying the CD gene directed by the retrovirus long terminal repeat promoter served as a control vector. An in vitro study showed that the CEA promoter conferred CEA-producing cell-selective CD expression, specifically when the CD expression cassette was inserted into the 3' long terminal repeat of the retrovirus vector. CD-modified CRC xenografts in nude mice were sensitive to 5-fluorocytosine and caused a profound bystander effect on the unmodified CRC. When nude mice harboring intraperitoneally disseminated CRCs were injected intraperitoneally with the CD expression cassette-carrying retrovirus-producing cells, CD transduction into the disseminated CRCs and bone marrow (BM) was observed. CD expression was, however, restricted to CRCs, and it was observed in both CRCs and BM of mice injected with pCD2 retrovirus-producing cells, resulting in better therapeutic outcomes without BM suppression. These results indicate that effective and safe in vivo gene therapy for CRC may be feasible by transferring the CD gene controlled by the CEA promoter core region. PMID:10608354

Cao, G; Kuriyama, S; Cui, L; Nagao, S; Pan, X; Toyokawa, Y; Zhang, X; Nishiwaki, I; Qi, Z

1999-01-01

17

Mutator Effects and Mutation Signatures of Editing Deaminases Produced in Bacteria and Yeast  

PubMed Central

Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This can not be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation. PMID:21568845

Lada, A. G.; Krick, C. Frahm; Kozmin, S. G.; Mayorov, V. I.; Karpova, T. S.; Rogozin, I. B.

2014-01-01

18

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

PubMed Central

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

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

2009-01-01

19

Non-invasive molecular and functional imaging of cytosine deaminase and uracil phosphoribosyltransferase fused with red fluorescence protein  

PubMed Central

Introduction Increased expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) may improve the antitumoral effect of 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC), and thereby enhance the potential of gene-directed enzyme prodrug therapy. For the applicability of gene-directed enzyme prodrug therapy in a clinical setting, it is essential to be able to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using magnetic resonance spectroscopy and optical imaging to measure non-invasively CD and UPRT expression and function. Materials and methods Expression vectors of CD or CD/UPRT fused to monomeric DsRed (mDsRed) were constructed and rat prostate carcinoma (R3327-AT) cell lines stably expressing either CD/mDsRed or CD/UPRT/mDsRed were generated. The expression of the fusion proteins was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. The function of the fusion protein was confirmed in vitro by assessing 5-FC and 5-FU cytotoxicity. In vivo fluorine-19 magnetic resonance spectroscopy (19F MRS) was used to monitor the conversion of 5-FC to 5-FU in mice bearing the R3327-CD/mDsRed and R3327-CD/UPRT/mDsRed tumor xenografts. Results Sensitivity to 5-FC and 5-FU was higher in cells stably expressing the CD/UPRT/mDsRed fusion gene than in cells stably expressing CD/mDsRed alone or wild-type cells. Whole tumor 19F MRS measurements showed rapid conversion of 5-FC to 5-FU within 20 min after 5-FC was administered intravenously in both CD/mDsRed and CD/UPRT/mDsRed tumors with subsequent anabolism to cytotoxic fluoronucleotides (FNucs). CD/UPRT/mDsRed tumor was more efficient in these processes. Conclusion This study demonstrates the utility of these tumor models stably expressing CD or CD/UPRT to non-invasively evaluate the efficacy of the transgene expression/activity by monitoring drug metabolism in vivo using MRS, with potential applications in preclinical and clinical settings. PMID:18661431

XING, LIGANG; DENG, XUELONG; KOTEDIA, KHUSHALI; ACKERSTAFF, ELLEN; PONOMAREV, VLADIMIR; LING, C. CLIFTON; KOUTCHER, JASON A.; LI, GLORIA C.

2014-01-01

20

Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase  

PubMed Central

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis. PMID:24039593

Lada, Artem G.; Stepchenkova, Elena I.; Waisertreiger, Irina S. R.; Noskov, Vladimir N.; Dhar, Alok; Eudy, James D.; Boissy, Robert J.; Hirano, Masayuki; Rogozin, Igor B.; Pavlov, Youri I.

2013-01-01

21

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

SciTech Connect

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

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2008-02-01

22

Cytosine DNA Methylation Is Found in Drosophila melanogaster but Absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Other Yeast Species  

PubMed Central

The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC–MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast. PMID:24640988

2014-01-01

23

Targeted enzyme prodrug therapy for metastatic prostate cancer – a comparative study of L-methioninase, purine nucleoside phosphorylase, and cytosine deaminase  

PubMed Central

Background Enzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3. Results All three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p?

2014-01-01

24

Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene.  

PubMed

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80% of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect. PMID:24610460

Wang, Frederick; Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Chun, Changho; Kwon, Young Jik; Berg, Kristian; Madsen, Steen J; Hirschberg, Henry

2014-05-01

25

Imaging expression of cytosine deaminase-herpes virus thymidine kinase fusion gene (CD/TK) expression with [124I]FIAU and PET.  

PubMed

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)-herpes simplex virus type 1 thymidine kinase (HSV1-tk) fusion gene (CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials. PMID:12920859

Hackman, Trevor; Doubrovin, Michail; Balatoni, Julius; Beresten, Tatiana; Ponomarev, Vladimir; Beattie, Bradly; Finn, Ronald; Bornmann, William; Blasberg, Ronald; Tjuvajev, Juri Gelovani

2002-01-01

26

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes.  

PubMed

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. PMID:25237741

Taylor, Benjamin J M; Wu, Yee Ling; Rada, Cristina

2014-01-01

27

The role of cytidine deaminase and GATA1 mutations in the increased cytosine arabinoside sensitivity of Down syndrome myeloblasts and leukemia cell lines.  

PubMed

Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1-beta-D-arabinofuranosylcytosine (ara-C) and generate higher 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semiquantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine-beta-synthase and deoxycytidine kinase were a median 12.5- and 2.6-fold higher in DS compared with non-DS myeloblasts. The ratio of deoxycytidine kinase/CDA transcripts significantly correlated with ara-C sensitivities and ara-CTP generation. In clinically relevant AML cell line models, high cystathionine-beta-synthase transcripts in DS CMK cells were accompanied by 10-fold greater ara-C sensitivity and 2.4-fold higher levels of ara-CTP compared with non-DS CMS cells. Overexpression of CDA in non-DS THP-1 cells was associated with a 100-fold decreased ara-C sensitivity and 40-fold decreased ara-CTP generation. THP-1 cells secreted CDA into the incubation media and converted extracellular ara-C completely to 1-beta-D-arabinofuranosyluracil within 30 min. Rapid amplification of 5'-cDNA ends (5'-RACE) and reverse transcription-PCR assays identified short- (sf) and long-form (lf) CDA transcripts in THP-1 cells with different 5' untranslated regions and translational start sites; however, only the latter resulted in the active CDA. Although 5' flanking sequences for both CDA transcripts exhibited promoter activity in reporter gene assays, activity for the CDAlf was low. The presence of several GATA1 binding sites in the CDAsf promoter and the uniform detection of GATA1 mutations in DS megakaryocytic leukemia suggested the potential role of GATA1 in regulating CDA transcription and the CDAsf promoter acting as an enhancer. Transfection of GATA1 into Drosophila Mel-2 cells stimulated the CDAlf promoter in a dose-dependent fashion. Additional identification of the mechanisms of differential expression of genes encoding enzymes involved in ara-C metabolism between DS and non-DS myeloblasts may lead to improvements in AML therapy. PMID:14744791

Ge, Yubin; Jensen, Tanya L; Stout, Mark L; Flatley, Robin M; Grohar, Patrick J; Ravindranath, Yaddanapudi; Matherly, Larry H; Taub, Jeffrey W

2004-01-15

28

Cytosine accumulation as a measure of the proton electrochemical gradient acting on the overexpressed cytosine permease of Saccharomyces cerevisiae.  

PubMed

The magnitude of the proton gradient (delta mu H+) driving solute accumulation in Saccharomyces cerevisiae has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (delta psi). In the present work, the size of the cytosine gradient (delta mu cyt) that the yeast generated was used as a measure of the driving gradient (delta mu H+). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+/cytosine system. delta mu cyt, assayed in washed cell suspensions fermenting glucose and containing 0.5 or 50 mM KCl, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, -delta mu H+ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered delta mu cyt to a value roughly equal to the magnitude of the pH gradient (delta pH). A mathematical model was used to correct the first estimates of delta mu H+ for the effect of cytosine leakage outside the symport. In such a system, delta mu cyt cannot exceed the equivalent ratio Vmax/KmL, where Vmax and Km are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying delta mu H+ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, -delta mu H+ during fermentation was estimated to be up to 25 mV larger than delta mu cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of delta pH, delta psi was in turn deduced and compared with the maximum methylamine gradient (delta mu M) the yeast formed. The results supported the claim in the literature that, at acid pH, delta mu M is a measure of delta psi. PMID:8868419

Eddy, A A; Hopkins, P

1996-03-01

29

Rescue of the Orphan Enzyme Isoguanine Deaminase  

SciTech Connect

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k{sub cat} = 49 s{sup -1}, K{sub m} = 72 {micro}M, and k{sub cat}/K{sub m} = 6.7 x 10{sup 5} M{sup -1} s{sup -1}. The kinetic constants for the deamination of cytosine are as follows: k{sub cat} = 45 s{sup -1}, K{sub m} = 302 {micro}M, and k{sub cat}/K{sub m} = 1.5 x 10{sup 5} M{sup -1} s{sup -1}. Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

D Hitchcock; A Fedorov; E Fedorov; L Dangott; S Almo; F Raushel

2011-12-31

30

Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp  

Microsoft Academic Search

This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium. This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene\\u000a in higher plants. Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase\\u000a enzyme assay. Five of them tested positive for ACC deaminase. Induction of

Wenbo Ma; Stepanka B. Sebestianova; Jiri Sebestian; Genrich I. Burd; Frédérique C. Guinel; Bernard R. Glick

2003-01-01

31

Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp.  

PubMed

This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium. This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene in higher plants. Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase enzyme assay. Five of them tested positive for ACC deaminase. Induction of the expression of ACC deaminase was examined in one of the positively tested strains, Rhizobium leguminosarum bv. viciae 128C53K. This rhizobial ACC deaminase had a trace basal level of expression without ACC, but could be induced by a concentration of ACC as low as 1 microM. The more ACC added to this Rhizobium the higher the expression level of the ACC deaminase. PMID:12776924

Ma, Wenbo; Sebestianova, Stepanka B; Sebestian, Jiri; Burd, Genrich I; Guinel, Frédérique C; Glick, Bernard R

2003-01-01

32

Myoadenylate deaminase deficiency  

Microsoft Academic Search

Summary Myoadenylate deaminase (MAD) is the rate-limiting enzyme in the purine nucleotide cycle which is biochemically linked to glycolysis and the citric cycle and thereby providing energy during intense muscular activity. In muscle fibers, myoadenylate deaminase operates at considerably higher activity levels than in other organs. First detected using enzyme-histochemical methods, it now appears that deficiency of myoadenylate deaminase is

H. H. Goebel; A. Bardosi

1987-01-01

33

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase  

PubMed Central

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung+ host proficient in uracil excision repair. PMID:24205358

Stier, Ildiko; Kiss, Antal

2013-01-01

34

Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution  

PubMed Central

Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. In vitro, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an in vivo transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated. PMID:25025377

Morgan, Hugh; Incorvaia, Elisabetta; Rangam, Gopinath; Dean, Wendy; Santos, Fatima; Reik, Wolf; Petersen-Mahrt, Svend K.

2014-01-01

35

Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions.  

PubMed

Mutations are typically perceived as random, independent events. We describe here nonrandom clustered mutations in yeast and in human cancers. Genome sequencing of yeast grown under chronic alkylation damage identified mutation clusters that extend up to 200 kb. A predominance of "strand-coordinated" changes of either cytosines or guanines in the same strand, mutation patterns, and genetic controls indicated that simultaneous mutations were generated by base alkylation in abnormally long single-strand DNA (ssDNA) formed at double-strand breaks (DSBs) and replication forks. Significantly, we found mutation clusters with analogous features in sequenced human cancers. Strand-coordinated clusters of mutated cytosines or guanines often resided near chromosome rearrangement breakpoints and were highly enriched with a motif targeted by APOBEC family cytosine-deaminases, which strongly prefer ssDNA. These data indicate that hypermutation via multiple simultaneous changes in randomly formed ssDNA is a general phenomenon that may be an important mechanism producing rapid genetic variation. PMID:22607975

Roberts, Steven A; Sterling, Joan; Thompson, Cole; Harris, Shawn; Mav, Deepak; Shah, Ruchir; Klimczak, Leszek J; Kryukov, Gregory V; Malc, Ewa; Mieczkowski, Piotr A; Resnick, Michael A; Gordenin, Dmitry A

2012-05-25

36

Reward versus risk: DNA cytidine deaminases triggering immunity and disease.  

PubMed

The enzymatic deamination of cytosine to uracil, using the free base C, its nucleosides, and nucleotides as substrates, is an essential feature of nucleotide metabolism. However, the deamination of C and, especially, 5 methyl C on DNA is typically detrimental, causing mutations leading to serious human disease. Recently, a family of enzymes has been discovered that catalyzes the conversion of C to U on DNA and RNA, generating favorable mutations that are essential for human survival. Members of the Apobec family of nucleic acid-dependent cytidine deaminases include activation-induced cytidine deaminase (AID) and Apobec3G. AID is required for B cells to undergo somatic hypermutation (SHM) and class switch recombination (CSR), two processes that are needed to produce high-affinity antibodies of all isotypes. Apobec3G is responsible for protection against HIV infection. Recent advances in the biochemical and structural analyses of nucleic acid cytidine deaminases will be discussed in relation to their programmed roles in ensuring antibody diversification and in imposing innate resistance against retroviral infection. The serious negative consequences of expressing Apobec deaminases in the wrong place at the wrong time to catalyze aberrant deamination in "at risk" sequences will be discussed in terms of causing genomic instability and disease. PMID:15723516

Pham, Phuong; Bransteitter, Ronda; Goodman, Myron F

2005-03-01

37

Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at GC pairs in immunoglobulin genes  

Microsoft Academic Search

BACKGROUND: Antibody genes are diversified by somatic hypermutation (SHM), gene conversion and class-switch recombination. All three processes are initiated by the activation-induced deaminase (AID). According to a DNA deamination model of SHM, AID converts cytosine to uracil in DNA sequences. The initial deamination of cytosine leads to mutation and recombination in pathways involving replication, DNA mismatch repair and possibly base

Vladimir I Mayorov; Igor B Rogozin; Linda R Adkison; Christin Frahm; Thomas A Kunkel; Youri I Pavlov

2005-01-01

38

Theoretical study of cytosine-Al, cytosine-Cu and cytosine-Ag (neutral, anionic and cationic).  

PubMed

The binding of cytosine to Al, Cu and Ag has been analyzed using the hybrid B3LYP density functional theory method. The three metals all have open shell electronic configuration, with only one unpaired valence electron. Thus it is possible to study the influence of electronic configuration on the stability of these systems. Neutral, cationic and anionic systems were analyzed, in order to assess the influence of atomic charge on bond formation. We argue that in the case of anions, nonconventional hydrogen bonds are formed. It is generally accepted that the hydrogen bond A-H...B is formed by the union of a proton donor group A-H and a proton acceptor B, which contains lone-pair electrons. In this study, we found that in the case of (Cu-cytosine)(-1) and (Ag-cytosine)(-1), N-H...Cu and N-H...Ag bonds are geometrically described as nonconventional hydrogen bonds. Their binding energies fall within the range of -20.0 to -55.4 kcal/mol (depending on the scheme of the reaction) and thus they are classified as examples of strong (>10 kcal/mol) hydrogen bonds. PMID:18193849

Vazquez, Marco-Vinicio; Martínez, Ana

2008-02-01

39

High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase  

PubMed Central

Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James T.; Radhakrishnan, Ravi; Kohli, Rahul M.

2014-01-01

40

Cytidine deaminases: AIDing DNA demethylation?  

PubMed

The presence of 5-methylcytosine (5-mC) in DNA is a vital epigenetic mark in vertebrates. While the enzymes responsible for methylating DNA in vertebrates have been identified, the means by which this mark can be removed are still unclear. Recently, it has been shown that activation-induced cytidine deaminase (AID) contributes to the demethylation of DNA in certain systems. This enzyme has been intensely studied in its role as a key driver of antibody diversification in B cells, but recent observations from early development in zebrafish and mice as well as heterokaryons point to a role beyond immunology. This review takes stock of the reports linking AID and related deaminases to DNA demethylation, and describes the many important questions left to be answered in this field. PMID:20889711

Fritz, Eric L; Papavasiliou, F Nina

2010-10-01

41

Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase  

PubMed Central

Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID’s functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID’s selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2?-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID’s reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2?-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID’s closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2?-fluoro-RNA substrates, AID’s deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID’s DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA. PMID:23942124

Nabel, Christopher S.; Lee, Jae W.; Wang, Laura C.; Kohli, Rahul M.

2013-01-01

42

Arxula adeninivorans recombinant guanine deaminase and its application in the production of food with low purine content.  

PubMed

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods. PMID:24481069

Trautwein-Schult, Anke; Jankowska, Dagmara; Cordes, Arno; Hoferichter, Petra; Klein, Christina; Matros, Andrea; Mock, Hans-Peter; Baronian, Keith; Bode, Rüdiger; Kunze, Gotthard

2014-01-01

43

Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains  

SciTech Connect

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb; Lu, Gang; Bigler, Rebecca; Jezyk, Mark R.; Li, Chunhua; Tanaka Hall, Traci M.; Wang, Zefeng (NIH); (Beijing U); (UNC)

2011-10-28

44

Discovery of Deaminase Activities in COG1816  

E-print Network

between proteins. Proteins previously annotated as adenosine deaminases: Pa0148 (Pseudomonas aeruginosa PAO1), AAur_1117 (Arthrobacter aurescens TC1), Sgx9403e and Sgx9403g, were purified and their substrate profiles revealed that adenine...

Goble, Alissa M

2013-04-24

45

Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant  

PubMed Central

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1+ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ?30-fold and decreases dTTP ?4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1? mutant, fission yeast dcd1? cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1? cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the ?H2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1? cells. We propose that replication forks stall and collapse in dcd1? cells, burdening DNA damage and checkpoint responses to maintain genome integrity. PMID:22927644

Sanchez, Arancha; Sharma, Sushma; Rozenzhak, Sophie; Roguev, Assen; Krogan, Nevan J.; Chabes, Andrei

2012-01-01

46

Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase  

SciTech Connect

Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 {angstrom} resolution (Protein Data Bank entry 2PAJ). This protein folds as a distorted ({beta}/{alpha}){sub 8} barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s{sup -1}, 8.0 {micro}M, and 1.3 x 10{sup 5} M{sup -1} s{sup -1} (k{sub cat}, K{sub m}, and k{sub cat}/K{sub m}, respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin.

Hall, R.S.; Swaminathan, S.; Agarwal, R.; Hitchcock, D.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

2010-05-25

47

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

PubMed Central

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

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

2014-01-01

48

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways  

PubMed Central

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes. PMID:24349193

Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

2013-01-01

49

Arabidopsis MET1 cytosine methyltransferase mutants.  

PubMed Central

We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

Kankel, Mark W; Ramsey, Douglas E; Stokes, Trevor L; Flowers, Susan K; Haag, Jeremy R; Jeddeloh, Jeffrey A; Riddle, Nicole C; Verbsky, Michelle L; Richards, Eric J

2003-01-01

50

Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus.  

PubMed

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision. PMID:23954625

Chowdhury, Sajeda; Kitamura, Kouichi; Simadu, Miyuki; Koura, Miki; Muramatsu, Masamichi

2013-09-17

51

The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1-21.2  

SciTech Connect

The interferon-inducible double-stranded RNA-specific adenosine deaminase is an RNA-modifying enzyme implicated in the generation of biased hypermutations of viral RNAs and the site-selective editing of mammalian mRNAs of neural origin. The gene for the dsRNA-specific adenosine deaminase has been mapped by fluorescence in situ hybridization (FISH) of genomic clones to a single locus on human chromosome 1 bands q21.1-21.2. Simultaneous multicolor FISH including X clones and yeast artificial chromosomes showed a localization of the gene in band 1q21 centromeric of D1S1705. 22 refs., 1 fig.

Weier, H.U.G.; Greulich, K.M. [Lawrence Berkeley National Lab., CA (United States)] [Lawrence Berkeley National Lab., CA (United States); George, C.X.; Samuel, C.E. [Univ. of California, Santa Barbara, CA (United States)] [Univ. of California, Santa Barbara, CA (United States)

1995-11-20

52

Isoenzymatic forms of human cytidine deaminase.  

PubMed

Cytidine deaminase (CDA) purified from human placenta revealed the presence of five isoenzymatic forms that differ only in their isoelectric point. Since human cytidine deaminase exists in two variants (CDA 1 and CDA 2) with a non-conservative amino acid substitution at codon 27, in this work we demonstrate that these two variants may combine together in vitro, giving five CDA isoforms as observed in vivo from human placenta. For this purpose, each of the two forms of CDA was purified close to homogeneity and dissociated into monomers in the presence of a small amount of sodium dodecyl sulfate as a dissociating agent. The monomers were mixed together and subjected to anion-exchange chromatography and to chromatofocusing analysis in order to visualize the formation of the five isoforms. Furthermore, for both CDA 1 and CDA 2 some substrates and inhibitors of CDA were assayed, with the aim of demonstrating different kinetic behavior between the two natural variants. PMID:15713780

Vincenzetti, S; Mariani, P L; Cammertoni, N; Polzonetti, V; Natalini, P; Quadrini, B; Volpini, R; Vita, A

2004-12-01

53

PERSPECTIVE DNA (Cytosine-5) Methyltransferase Inhibitors: A Potential  

E-print Network

occurs at the level of DNA (cytosine-5) methylation. Moreover, the authors present evidence suggestingPERSPECTIVE DNA (Cytosine-5) Methyltransferase Inhibitors: A Potential Therapeutic Agent evidence suggesting that the promot- ers for reelin and GAD67 are coordinately regulated. The reg- ulation

Champagne, Frances A.

54

Crystal Structure of Staphylococcus aureus tRNA Adenosine Deaminase TadA in Complex with RNA  

SciTech Connect

Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.

Losey,H.; Ruthenburg, A.; Verdine, G.

2006-01-01

55

Zinc-binding Domain-dependent, Deaminase-independent Actions of Apolipoprotein B mRNA-editing Enzyme, Catalytic Polypeptide 2 (Apobec2), Mediate Its Effect on Zebrafish Retina Regeneration.  

PubMed

The Apobec/AID family of cytosine deaminases can deaminate cytosine and thereby contribute to adaptive and innate immunity, DNA demethylation, and the modification of cellular mRNAs. Unique among this family is Apobec2, whose enzymatic activity has been questioned and whose function remains poorly explored. We recently reported that zebrafish Apobec2a and Apobec2b (Apobec2a,2b) regulate retina regeneration; however, their mechanism of action remained unknown. Here we show that although Apobec2a,2b lack cytosine deaminase activity, they require a conserved zinc-binding domain to stimulate retina regeneration. Interestingly, we found that human APOBEC2 is able to functionally substitute for Apobec2a,2b during retina regeneration. By identifying Apobec2-interacting proteins, including ubiquitin-conjugating enzyme 9 (Ubc9); topoisomerase I-binding, arginine/serine-rich, E3 ubiquitin protein ligase (Toporsa); and POU class 6 homeobox 2 (Pou6f2), we uncovered that sumoylation regulates Apobec2 subcellular localization and that nuclear Apobec2 controls Pou6f2 binding to DNA. Importantly, mutations in the zinc-binding domain of Apobec2 diminished its ability to stimulate Pou6f2 binding to DNA, and knockdown of Ubc9 or Pou6f2 suppressed retina regeneration. PMID:25190811

Powell, Curtis; Cornblath, Eli; Goldman, Daniel

2014-10-17

56

ORAL PRESENTATION Open Access Double-stranded RNA adenosine deaminase  

E-print Network

ORAL PRESENTATION Open Access Double-stranded RNA adenosine deaminase ADAR1 enhances both T cell), an inhibitor of viral mRNA translation, and the double-stranded RNA adenosine deaminase ADAR1. ADAR1 has the ability to convert adenosine (A) into guanosine (G), thereby introducing mutations in the viral genome

Paris-Sud XI, Université de

57

Dispersed sites of HIV Vif-dependent polyubiquitination in the DNA deaminase APOBEC3F.  

PubMed

APOBEC3F (A3F) and APOBEC3G (A3G) are DNA cytosine deaminases that potently restrict human immunodeficiency virus type 1 replication when the virus is deprived of its accessory protein Vif (virion infectivity factor). Vif counteracts these restriction factors by recruiting A3F and A3G to an E3 ubiquitin (Ub) ligase complex that mediates their polyubiquitination (polyUb) and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream Ub acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in A3G and proposed an antiparallel model of A3G interaction with the Vif-E3 Ub ligase complex wherein Vif binding at one terminus of A3G orients the opposite terminus for polyUb [Iwatani et al. (2009). Proc. Natl. Acad. Sci. USA, 106, 19539-19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in A3F and used a complementary, unbiased proteomic approach to identify Ub acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant Ub acceptor sites in both A3F and A3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyUb of A3F and A3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 Ub ligase complex and diminish enthusiasm for the amenability of APOBEC3 Ub acceptor sites to therapeutic intervention. PMID:23318957

Albin, John S; Anderson, John S; Johnson, Jeffrey R; Harjes, Elena; Matsuo, Hiroshi; Krogan, Nevan J; Harris, Reuben S

2013-04-12

58

tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli  

PubMed Central

We report the characterization of tadA, the first prokaryotic RNA editing enzyme to be identified. Escherichia coli tadA displays sequence similarity to the yeast tRNA deaminase subunit Tad2p. Recom binant tadA protein forms homodimers and is sufficient for site-specific inosine formation at the wobble position (position 34) of tRNAArg2, the only tRNA having this modification in prokaryotes. With the exception of yeast tRNAArg, no other eukaryotic tRNA substrates were found to be modified by tadA. How ever, an artificial yeast tRNAAsp, which carries the anticodon loop of yeast tRNAArg, is bound and modified by tadA. Moreover, a tRNAArg2 minisubstrate containing the anticodon stem and loop is sufficient for specific deamination by tadA. We show that nucleotides at positions 33–36 are sufficient for inosine formation in mutant Arg2 minisubstrates. The anticodon is thus a major determinant for tadA substrate specificity. Finally, we show that tadA is an essential gene in E.coli, underscoring the critical function of inosine at the wobble position in prokaryotes. PMID:12110595

Wolf, Jeannette; Gerber, Andre P.; Keller, Walter

2002-01-01

59

High-throughput sequencing of cytosine methylation in plant DNA  

E-print Network

Abstract 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...

Hardcastle, Thomas J

2013-06-07

60

Long non-coding RNAs as targets for cytosine methylation  

PubMed Central

Post-synthetic modifications of nucleic acids have long been known to affect their functional and structural properties. For instance, numerous different chemical modifications modulate the structural organization, stability or translation efficiency of tRNAs and rRNAs. In contrast, little is known about modifications of poly(A)RNAs. Here, we demonstrate for the first time that the two well-studied regulatory long non-coding RNAs HOTAIR and XIST are targets of site-specific cytosine methylation. In both XIST and HOTAIR, we found methylated cytosines located within or near functionally important regions that are known to mediate interaction with chromatin-associated protein complexes. We show that cytosine methylation in the XIST A structure strongly affects binding to the chromatin-modifying complex PRC2 in vitro. These results suggest that cytosine methylation may serve as a general strategy to regulate the function of long non-coding RNAs. PMID:23595112

Amort, Thomas; Souliere, Marie F.; Wille, Alexandra; Jia, Xi-Yu; Fiegl, Heidi; Worle, Hildegard; Micura, Ronald; Lusser, Alexandra

2013-01-01

61

Cytosine methylation levels in the genome of Stellaria longipes  

Microsoft Academic Search

Environment-induced alteration of DNA methylation levels was investigated inStellaria longipes (Caryophyllaceae). Total cytosine methylation levels were measured using HPLC in 6 genets representing two ecotypes (alpine\\u000a and prairie) grown in short day photoperiod and cold temperature (SDC) and long day photoperiod and warm temperature (LDW)\\u000a conditions. The levels of methylated cytosine were 16.54-22.20% among the three genets from the alpine

Q. Cai; C. C. Chinnappa

1999-01-01

62

Human repair endonuclease incises DNA at cytosine photoproducts  

SciTech Connect

The nature of DNA damage by uvB and uvC irradiation was investigated using a defined sequence of human DNA. A UV-irradiated, 3'-end-labeled, 92 base pair sequence from the human alphoid segment was incubated with a purified human lymphoblast endonuclease that incises DNA at non-dimer photoproducts. Analysis by polyacrylamide gel electrophoresis identified all sites of endonucleolytic incision as cytosines. These were found in regions of the DNA sequence lacking adjacent pyrimidines and therefore are neither cyclobutane pyrimidine dimers nor 6-4'-pyrimidines. Incision at cytosine photoproducts was not detected at loci corresponding to alkali-labile sites in either control or irradiated substrates. This demonstrates that the bands detected after the enzymic reactions were not the result of DNA strand breaks, base loss sites or ring-opened cytosines. The optimal wavelengths for formation of cytosine photoproducts are 270-295 nm, similar to those associated with maximal tumor yields in animal ultraviolet carcinogenesis studies. Irradiation by monochromatic 254 nm light resulted in reduced cytosine photoproduct formation. This human UV endonuclease has an apparently identical substrate specificity to E. coli endonuclease III. Both the human and bacterial enzymes incise cytosine moieties in UV irradiated DNA and modified thymines in oxidized DNA.

Gallagher, P.E.; Weiss, R.B.; Brent, T.P.; Duker, N.J.

1987-05-01

63

Intraspecies diversity of the industrial yeast strains Saccharomyces cerevisiae and Saccharomyces pastorianus based on analysis of the sequences of the internal transcribed spacer (ITS) regions and the D1/D2 region of 26S rDNA.  

PubMed

We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions. PMID:17617725

Kawahata, Miho; Fujii, Tsutomu; Iefuji, Haruyuki

2007-07-01

64

miR-181b negatively regulates activation-induced cytidine deaminase in B cells.  

PubMed

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3' untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation. PMID:18762567

de Yébenes, Virginia G; Belver, Laura; Pisano, David G; González, Susana; Villasante, Aranzazu; Croce, Carlo; He, Lin; Ramiro, Almudena R

2008-09-29

65

Serum adenosine deaminase activity in cutaneous anthrax  

PubMed Central

Background Adenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax. Material/Methods Sixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels. Results Serum ADA activity was significantly higher in patients with cutaneous anthrax than in the controls (p<0.001). A positive correlation was observed between ADA activity and lymphocyte counts (r=0.589, p=0.021) in the patient group. Conclusions This study suggests that serum ADA could be used as a biochemical marker in cutaneous anthrax. PMID:24997584

Sunnetcioglu, Mahmut; Karadas, Sevdegul; Aslan, Mehmet; Ceylan, Mehmet Resat; Demir, Halit; Oncu, Mehmet Resit; Karahocagil, Mustafa Kas?m; Sunnetcioglu, Aysel; Aypak, Cenk

2014-01-01

66

The catalase activity of diiron adenine deaminase.  

PubMed

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. PMID:21998098

Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima; Kumaran, Desigan; Tichy, Shane E; Gheyi, Tarun; Zheng, Xiaojing; Bain, Kevin; Groshong, Chris; Emtage, Spencer; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Lindahl, Paul A; Raushel, Frank M

2011-12-01

67

The catalase activity of diiron adenine deaminase  

PubMed Central

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn2+ before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO4. Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [FeII/FeII]-ADE catalyzed the conversion of H2O2 to O2 and H2O. The values of kcat and kcat/Km for the catalase activity are 200 s?1 and 2.4 × 104 M?1 s?1, respectively. [FeII/FeII]-ADE underwent more than 100 turnovers with H2O2 before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with gave = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H2O2 by [FeII/FeII]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. PMID:21998098

Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima; Kumaran, Desigan; Tichy, Shane E; Gheyi, Tarun; Zheng, Xiaojing; Bain, Kevin; Groshong, Chris; Emtage, Spencer; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Lindahl, Paul A; Raushel, Frank M

2011-01-01

68

ACC Deaminase Containing PGPR for Potential Exploitation in Agriculture  

Microsoft Academic Search

\\u000a The beneficial free-living bacteria present in the plant rhizosphere are usually referred to as plant growth promoting rhizobacteria\\u000a (PGPR). Among the various mechanisms of plant growth promotion, certain PGPR possess the enzyme 1-aminocyclopropane-1-carboxylic\\u000a acid (ACC) deaminase that cleaves plant-produced ACC, the immediate precursor of the stress hormone ethylene. ACC deaminase\\u000a containing PGPR act as a sink for ACC and protects

Venkadasamy Govindasamy; Murugesan Senthilkumar; Pranita Bose; Lakkineni Vithal Kumar; D. Ramadoss; Kannepalli Annapurna

69

Fenpropimorph affects uptake of uracil and cytosine in Saccharomyces cerevisiae.  

PubMed Central

Fenpropimorph was shown to inhibit the accumulation of the pyrimidine bases uracil and cytosine from the growth media in Saccharomyces cerevisiae. Uracil prototrophs of S. cerevisiae were more resistant to the growth-inhibitory effects of fenpropimorph than were uracil auxotrophs. High concentrations of uracil rescued fenpropimorph-treated uracil auxotrophs, and cytosine, which is accumulated by a separate mechanism, could also support growth of treated uracil auxotrophs. Fenpropimorph caused a significant decrease in the uptake of radiolabeled uracil, which was not due to accumulation of ergosta-8,14-dienol (ignosterol) in the treated cultures. Radiolabeled cytosine uptake was unaffected by drug treatment in a wild-type strain but was inhibited in a sterol mutant, in which ergosterol was absent from the cell. The role of fenpropimorph in causing membrane dysfunction through a mechanism other than altered sterol metabolism is discussed. PMID:8067730

Crowley, J H; Lorenz, R T; Parks, L W

1994-01-01

70

Herpes simplex encephalitis: treatment with surgical decompression and cytosine arabinoside.  

PubMed Central

Herpes simplex encephalitis in a 21-year-old man presented as a flu-like illness, followed by inappropriate behaviour, drowsiness and focal neurologic signs. Investigations indicated a lesion in the right temporal lobe. The diagnosis was confirmed by isolation of the virus from a cerebral biopsy. Pronounced clinical improvement was noted when cytosine arabinoside therapy was started in the postoperative period. This report supports the observation by some authors that cytosine arabinoside may be of value in the treatment of herpes simplex encephalitis. Images FIG. 1 FIG. 2 PMID:953885

Laha, R. K.; Saunders, F. W.; Huestis, W. S.

1976-01-01

71

Fabrication of duplex DNA microarrays incorporating methyl-5-cytosine  

PubMed Central

We synthesized customized double-stranded DNA microarrays including methyl-5-cytosine at CpG dinucleotides and produced all 163,555 possible 8-mers (un-, hemi-, and di-methylated) to gain insight into how methylation affects transcription factor binding. An antibody to methyl-5-cytidine showed greater binding to the methylated DNA, demonstrating efficient incorporation of methyl-5-cytosine into the synthesized DNA. In contrast, binding of the transcription factor CREB was inhibited by CpG methylation. This platform represents a powerful new technology to evaluate the effect of DNA methylation on protein binding in any sequence context. PMID:22139143

Warren, Christopher L.; Zhao, Jianfei; Glass, Kimberly; Rishi, Vikas; Ansari, Aseem Z.; Vinson, Charles

2014-01-01

72

The catalase activity of diiron adenine deaminase  

SciTech Connect

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.; Bagaria, A.; Kumaran, D.; Tichy, S. E.; Gheyi, T.; Zheng, X.; Bain, K.; Groshong, C.; Emtage, S.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

2011-12-01

73

[Molecular taxonomy techniques used for yeast identification].  

PubMed

Due to the major impact of yeasts in human life based on the existence of pathogen yeast species and of species with biotechnological abilities, in the last few years new molecular techniques are performed for an accurate identification of natural isolates. Our study is aimed to review some of these techniques such as electrokariotyping by PFGE (Pulsed Field Gel Electrophoresis), estimation of the molar percentage of guanine and cytosine, the applications of PCR reaction in yeast identification using RAPD (Random amplified polymorphic DNA), UP-PCR (Universally Primed Polymerase Chain Reaction), MLST (Multilocus sequence typing) techniques, mtDNA and rDNA homology studies. Such molecular techniques complete the phenotypical characterization based on classical taxonomical tests allowing thus the polyphasic identification of the microorganisms. PMID:16938931

Ghindea, Raluca; Csutak, Ortansa; Stoica, Ileana; Ionescu, Robertina; Soare, Simona; Pelinescu, Diana; Nohit, Ana-Maria; Creang?, Oana; Vassu, Tatiana

2004-01-01

74

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1Carboxylate Deaminase Promotes Nodulation of Pea Plants  

Microsoft Academic Search

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and charac- terized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria

Wenbo Ma; Frederique C. Guinel; Bernard R. Glick

2003-01-01

75

Folic acid deaminase activity during development in Dictyostelium discoideum.  

PubMed Central

Folic acid attracts vegetative amoebae of Dictyostelium discoideum. Secreted by bacteria, it may act as a food-seeking device. The inactivation of this attractant is catalyzed by a deaminase. As assay has been developed to measure the folic acid deaminase activity. In addition to cell-surface an intracellular deaminase, the amoebae of D. discoideum release the enzyme into the medium. The pH optimum of the extracellular enzyme was 6.0, and higher for the cell-associated deaminases. The extracellular enzyme was secreted maximally by vegetative amoebae, and its activity diminished during cell differentiation. The cell-surface bound enzyme was less active than the extracellular enzyme, and its activity decreased twofold during a 6-h starvation period. The enzyme activity of homogenates and 48,000 x g pellets diminished during this period 35 to 40%. The supernatant of a homogenate had a higher deaminase activity than the homogenate itself or its pellet; this suggests the presence of an inhibitor in the particulate fraction. The underlying mechanism for inactivation of folic acid has similar characteristics as that for inactivation of cyclic adenosine monophosphate. PMID:7400095

Kakebeeke, P I; de Wit, R J; Konijn, T M

1980-01-01

76

Concentration by Evaporation and the Prebiotic Synthesis of Cytosine  

NASA Technical Reports Server (NTRS)

The efficient prebiotic synthesis of cytosine from urea and cyanoacetaldehyde (CA) has recently been claimed to be invalid on the basis of possible side reactions of the starting materials and the inapplicability of prebiotic syntheses using drying beach conditions. We therefore have investigated the synthesis of cytosine and uracil from urea and cyanoacetaldehyde at 100 C under dry-down conditions, and in solution at 4 C and -20 C. We find that cytosine is produced from the low temperature experiments more efficiently than calculated from the Arrhenius extrapolation from higher temperatures, i.e., 60-120 C. In addition, we find that CA dimer is as efficient as the monomer in cytosine synthesis. We also studied whether evaporating very dilute solutions of nonvolatile organic compounds will concentrate according to theory. Solutions as dilute as 10(exp -4) M concentrate from pure water approximately according to theory. Similar solutions in 0.5 M NaCl have less than theoretical concentrations due to absorption, but concentrations near dryness were very high.

Nelson, Kevin E.; Robertson, Michael P.; Levy, Matthew; Miller, Stanley L.

2001-01-01

77

On the stability of the guanine-cytosine hydrogen bond  

Microsoft Academic Search

Zusammenfassung Die Änderung des molaren Extinktionskoeffizienten natürlicher Desoxyribonucleinsäuren wurde untersucht in Abhängigkeit der Basenzusammensetzung. Aus den experimentellen Befunden wird geschlossen, dass die Wasserstoffbindung zwischen Cytosin und Guanin im Doppelhelix schwach ist und in wässriger Lösung ohne die stabilisierende Wirkung benachbarter Adenin-Tyminbindungen nicht vorliegt. Die mit der Nucleinsäure verbundenen Proteine liefern einen zusätzlichen Stabilisationsfaktor zur Guanin- und Cytosinbindung.

S. L. Bunch; B. V. Siegel

1964-01-01

78

High-Resolution Analysis of Cytosine Methylation in Ancient DNA  

PubMed Central

Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution. PMID:22276161

Cropley, Jennifer E.; Cooper, Alan; Suter, Catherine M.

2012-01-01

79

The Diagnostic Efficacy of Adenosine Deaminase in Tubercular Effusion  

PubMed Central

Objective This study aims to evaluate the diagnostic efficacy of adenosine deaminase in tubercular effusions. Methods This study was conducted at the Department of General Medicine and Cardiovascular and Thoracic Surgery, SKIMS, for a period of two years between November 2008 and November 2010. A total of 57 patients presenting with pleural effusions during the two-year study period, who presented with clinical manifestations suggestive of tuberculosis (i.e., the presence of productive cough, low-grade fever, night sweats, weight loss, and chest pain, especially if these symptoms last 34 weeks) were included in the study. If the patients presented with less than two of these symptoms, and especially if the clinical manifestations were of <4 weeks duration, they were excluded from the study. Results The mean adenosine deaminase activity level in all the 57 patients was 109 U/L while the mean adenosine deaminase activity levels in pleural TB patients was 80 U/, and 64 U/L in the controls (p=0.381). Considering 40 U/L as the cut off, the results were positive in 35 out of 39 tuberculosis patients and 9 out of 18 controls. The sensitivity of adenosine deaminase for tubercular effusions worked out to be 90%, with only 50% specificity. Conclusion This study suggests that the estimation of adenosine deaminase activity in pleural fluid is a rapid diagnostic tool for differentiation of tubercular and non tubercular-effusions. The sensitivity and specificity of adenosine deaminase for tubercular effusions in this study was 90% and 50% respectively. PMID:24223245

Kelam, Mohd Arif; Ganie, Farooq Ahmad; Shah, Bashir Ahmad; Ganie, Shabir Ahmad; Wani, Mohd Lateef; Wani, Nasir-U-Din; Gani, Masaratul

2013-01-01

80

The Curious Chemical Biology of Cytosine: Deamination, Methylation and Oxidation as Modulators of Genomic Potential  

PubMed Central

A multitude of functions have evolved around cytosine within DNA, endowing the base with physiological significance beyond simple information storage. This versatility arises from enzymes that chemically modify cytosine to expand the potential of the genome. Some modifications alter coding sequences, such as deamination of cytosine by AID/APOBEC enzymes to generate immunologic or virologic diversity. Other modifications are critical to epigenetic control, altering gene expression or cellular identity. Of these, cytosine methylation is well understood, in contrast to recently discovered modifications, such as oxidation by TET enzymes to 5-hydroxymethylcytosine. Further complexity results from cytosine demethylation, an enigmatic process that impacts cellular pluripotency. Recent insights help us to propose an integrated DNA demethylation model, accounting for contributions from cytosine oxidation, deamination and base excision repair. Taken together, this rich medley of alterations renders cytosine a genomic “wild card”, whose context-dependent functions make the base far more than a static letter in the code of life. PMID:22004246

Nabel, Christopher S.; Manning, Sara A.; Kohli, Rahul M.

2011-01-01

81

Guanine Deaminase Functions as Dihydropterin Deaminase in the Biosynthesis of Aurodrosopterin, a Minor Red Eye Pigment of Drosophila*  

PubMed Central

Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 °C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (kcat/Km) for guanine (8.6 × 106 m?1·s?1) was 860-fold higher than that for 7,8-dihydropterin (1.0 × 104 m?1·s?1). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism. PMID:19567870

Kim, Jaekwang; Park, Sang Ick; Ahn, Chiyoung; Kim, Heuijong; Yim, Jeongbin

2009-01-01

82

The control of natural variation in cytosine methylation in Arabidopsis.  

PubMed Central

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

Riddle, Nicole C; Richards, Eric J

2002-01-01

83

Plant cytosine-5 DNA methyltransferases: Structure, function, and molecular evolution  

Microsoft Academic Search

A detailed analysis of the structure and function, along with evolutionary aspects, of the main plant cytosine-5 DNA methyltransferases (C5-MTases) is presented. The evolutionary relationships between the already known and four candidate plant C5-MTases identified in this work were investigated using the distance, maximum-parsimony, and maximum-likelihood approaches. The topologies of the trees were overall congruent: four monophyletic groups corresponding to

Athanasia Pavlopoulou; Sophia Kossida

2007-01-01

84

High-throughput sequencing of cytosine methylation in plant DNA  

PubMed Central

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

2013-01-01

85

Adenosine-5'-phosphate deaminase. A novel herbicide target.  

PubMed Central

The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool. PMID:9159944

Dancer, J E; Hughes, R G; Lindell, S D

1997-01-01

86

A Role for Adenosine Deaminase in Drosophila Larval Development  

Microsoft Academic Search

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of

Tomas Dolezal; Eva Dolezelova; Michal Zurovec; Peter J. Bryant

2005-01-01

87

in Adenosine Deaminase Deficiency and Severe Combined Immunodeficienc y  

Microsoft Academic Search

T HE SYNDROME of severe combined immune deficiency (SCID) with adeno- sine deaminase (ADA) deficiency was first described in 1972.' A 4-mo-old infant with this syndrome was referred to us for diagnosis and management. ADA activity was found to be undetectable in his erythrocytes and serum. He was commenced on a course of red cell transfusion therapy similar to that

Choon H. Lee; Susan P. Evans; Maurice C. Rozenberg; Aldo S. Bagnara; John B. Ziegler

2010-01-01

88

Activation-Induced Cytidine Deaminase Expression in Gastric Cancer  

Microsoft Academic Search

Helicobacter pylori increases the risk of gastric cancer development and triggers aberrant expression of activation-induced cytidine deaminase (AID). The goal of the present study was to investigate whether AID expression is involved in the development or progression of gastric cancer and the nuclear expression of p53 protein in cancer cells. We examined the expression pattern of the AID and p53

Chang Jae Kim; Jae Hwi Song; Yong Gu Cho; Zhang Cao; Su Young Kim; Suk Woo Nam; Jung Young Lee; Won Sang Park

2007-01-01

89

Effects of cytosine methylation on transcription factor binding sites  

PubMed Central

Background DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important. Results We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines “traffic lights”. We observed a strong selection against CpG “traffic lights” within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions. Conclusions Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. PMID:24669864

2014-01-01

90

An efficient prebiotic synthesis of cytosine and uracil  

NASA Technical Reports Server (NTRS)

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.

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

1995-01-01

91

Interaction of Cyclic Cytosine, Guanine, Thymine, Uracil and Mixed GuanineCytosine Base Tetrads with K, Na and Li Ions—A Density Functional Study  

Microsoft Academic Search

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

Michael Meyer; Jürgen Sühnel

2003-01-01

92

Haploinsufficiency of activation-induced deaminase for antibody diversification and chromosome translocations both in vitro and in vivo.  

PubMed

The humoral immune response critically relies on the secondary diversification of antibodies. This diversification takes places through somatic remodelling of the antibody genes by two molecular mechanisms, Class Switch Recombination (CSR) and Somatic Hypermutation (SHM). The enzyme Activation Induced Cytidine Deaminase (AID) initiates both SHM and CSR by deaminating cytosine residues on the DNA of immunoglobulin genes. While crucial for immunity, AID-catalysed deamination is also the triggering event for the generation of lymphomagenic chromosome translocations. To address whether restricting the levels of AID expression in vivo contributes to the regulation of its function, we analysed mice harbouring a single copy of the AID gene (AID(+/-)). AID(+/-) mice express roughly 50% of normal AID levels, and display a mild hyperplasia, reminiscent of AID deficient mice and humans. Moreover, we found that AID(+/-) cells have an impaired competence for CSR and SHM, which indicates that AID gene dose is limiting for its physiologic function. We next evaluated the impact of AID reduction in AID(+/-) mice on the generation of chromosome translocations. Our results show that the frequency of AID-promoted c-myc/IgH translocations is reduced in AID(+/-) mice, both in vivo and in vitro. Therefore, AID is haploinsufficient for antibody diversification and chromosome translocations. These findings suggest that limiting the physiologic levels of AID expression can be a regulatory mechanism that ensures an optimal balance between immune proficiency and genome integrity. PMID:19079594

Sernández, Isora V; de Yébenes, Virginia G; Dorsett, Yair; Ramiro, Almudena R

2008-01-01

93

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase  

SciTech Connect

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps.

S Kamat; A Bagaria; D Kumaran; G Holmes-Hampton; H Fan; A Sali; J Sauder; S Burley; P Lindahl; et. al.

2011-12-31

94

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase  

SciTech Connect

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps.

Kamat, S.S.; Swaminathan, S.; Bagaria, A.; Kumaran, D.; Holmes-Hampton, G. P.; Fan, H.; Sali, A.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

2011-03-22

95

Yeast Infections  

MedlinePLUS

Candida is the scientific name for yeast. It is a fungus that lives almost everywhere, including in ... infection that causes white patches in your mouth Candida esophagitis is thrush that spreads to your esophagus, ...

96

Difference between deoxyribose- and tetrahydrofuran-type abasic sites in the in vivo mutagenic responses in yeast  

Microsoft Academic Search

We have analyzed the mutagenic specificity of an abasic site in DNA using the yeast oligonucleotide transformation assay. Oligonucleotides containing an abasic site or its analog were introduced into B7528 or its derivatives, and nucleotide incorpor- ation opposite abasic sites was analyzed. Cytosine was most frequently incorporated opposite a natural abasic site (O) ('C-rule'), followed by thymine. Deletion of REV1

Chie Otsuka; Sachi Sanadai; Yasuhiro Hata; Hisanori Okuto; Vladimir N. Noskov; David Loakes; Kazuo Negishi

2002-01-01

97

Regulation of adenosine receptor engagement by ecto-adenosine deaminase  

Microsoft Academic Search

Adenosine deaminase (ADA) can localize to the cell surface through its interaction with CD26. Using CD26-transfected cells, we demonstrate that cell surface ADA (ecto-ADA) can regulate adenosine receptor engagement by degrading extracellular adenosine (Ado) to inosine. This ability was dependent upon CD26 expression, the extent of CD26 saturation with ecto-ADA, and the kinetics of the cAMP response. Thus, the cAMP

Tomoko Hashikawa; Scott W. Hooker; Jerzy G. Maj; Christopher J. Knott-Craig; Masahide Takedachi; Shinya Murakami; Linda F. Thompson

2003-01-01

98

Adenosine Deaminases Acting on RNA, RNA Editing, and Interferon Action  

PubMed Central

Adenosine deaminases acting on RNA (ADARs) catalyze adenosine (A) to inosine (I) editing of RNA that possesses double-stranded (ds) structure. A-to-I RNA editing results in nucleotide substitution, because I is recognized as G instead of A both by ribosomes and by RNA polymerases. A-to-I substitution can also cause dsRNA destabilization, as I:U mismatch base pairs are less stable than A:U base pairs. Three mammalian ADAR genes are known, of which two encode active deaminases (ADAR1 and ADAR2). Alternative promoters together with alternative splicing give rise to two protein size forms of ADAR1: an interferon-inducible ADAR1-p150 deaminase that binds dsRNA and Z-DNA, and a constitutively expressed ADAR1-p110 deaminase. ADAR2, like ADAR1-p110, is constitutively expressed and binds dsRNA. A-to-I editing occurs with both viral and cellular RNAs, and affects a broad range of biological processes. These include virus growth and persistence, apoptosis and embryogenesis, neurotransmitter receptor and ion channel function, pancreatic cell function, and post-transcriptional gene regulation by microRNAs. Biochemical processes that provide a framework for understanding the physiologic changes following ADAR-catalyzed A-to-I (?=?G) editing events include mRNA translation by changing codons and hence the amino acid sequence of proteins; pre-mRNA splicing by altering splice site recognition sequences; RNA stability by changing sequences involved in nuclease recognition; genetic stability in the case of RNA virus genomes by changing sequences during viral RNA replication; and RNA-structure-dependent activities such as microRNA production or targeting or protein–RNA interactions. PMID:21182352

George, Cyril X.; Gan, Zhenji; Liu, Yong

2011-01-01

99

Adenosine deaminase from camel tick Hyalomma dromedarii: purification and characterization.  

PubMed

Adenosine deaminase is involved in purine metabolism and is a key enzyme for the control of the cellular levels of adenosine. Adenosine deaminase activity showed significant changes during embryogenesis of the camel tick Hyalomma dromedarii. From the elution profile of chromatography on DEAE-sepharose, three forms of enzyme (ADAI, ADAII and ADAIII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass of adenosine deaminase ADAII was 42 kDa for the native enzyme and represented a monomer of 42 kDa by SDS-PAGE. The enzyme had a pH optimum at 7.5 and temperature optimum at 40 degrees C with heat stability up to 40 degrees C. ADAII had a K (m) of 0.5 mM adenosine with higher affinity toward deoxyadenosine and adenosine than other purines. Ni(2+), Ba(2+), Zn(2+), Li(2+), Hg(2+) and Mg(2+) partially inhibited the ADAII. Mg(2+) was the strongest inhibitor by 91% of the enzyme's activity. PMID:17089216

Mohamed, Tarek M

2006-01-01

100

DNA deaminases induce break-associated mutation showers with implication of APOBEC3B and 3A in breast cancer kataegis.  

PubMed

Breast cancer genomes have revealed a novel form of mutation showers (kataegis) in which multiple same-strand substitutions at C:G pairs spaced one to several hundred nucleotides apart are clustered over kilobase-sized regions, often associated with sites of DNA rearrangement. We show kataegis can result from AID/APOBEC-catalysed cytidine deamination in the vicinity of DNA breaks, likely through action on single-stranded DNA exposed during resection. Cancer-like kataegis can be recapitulated by expression of AID/APOBEC family deaminases in yeast where it largely depends on uracil excision, which generates an abasic site for strand breakage. Localized kataegis can also be nucleated by an I-SceI-induced break. Genome-wide patterns of APOBEC3-catalyzed deamination in yeast reveal APOBEC3B and 3A as the deaminases whose mutational signatures are most similar to those of breast cancer kataegic mutations. Together with expression and functional assays, the results implicate APOBEC3B/A in breast cancer hypermutation and give insight into the mechanism of kataegis. DOI:http://dx.doi.org/10.7554/eLife.00534.001. PMID:23599896

Taylor, Benjamin Jm; Nik-Zainal, Serena; Wu, Yee Ling; Stebbings, Lucy A; Raine, Keiran; Campbell, Peter J; Rada, Cristina; Stratton, Michael R; Neuberger, Michael S

2013-01-01

101

DNA deaminases induce break-associated mutation showers with implication of APOBEC3B and 3A in breast cancer kataegis  

PubMed Central

Breast cancer genomes have revealed a novel form of mutation showers (kataegis) in which multiple same-strand substitutions at C:G pairs spaced one to several hundred nucleotides apart are clustered over kilobase-sized regions, often associated with sites of DNA rearrangement. We show kataegis can result from AID/APOBEC-catalysed cytidine deamination in the vicinity of DNA breaks, likely through action on single-stranded DNA exposed during resection. Cancer-like kataegis can be recapitulated by expression of AID/APOBEC family deaminases in yeast where it largely depends on uracil excision, which generates an abasic site for strand breakage. Localized kataegis can also be nucleated by an I-SceI-induced break. Genome-wide patterns of APOBEC3-catalyzed deamination in yeast reveal APOBEC3B and 3A as the deaminases whose mutational signatures are most similar to those of breast cancer kataegic mutations. Together with expression and functional assays, the results implicate APOBEC3B/A in breast cancer hypermutation and give insight into the mechanism of kataegis. DOI: http://dx.doi.org/10.7554/eLife.00534.001 PMID:23599896

Taylor, Benjamin JM; Nik-Zainal, Serena; Wu, Yee Ling; Stebbings, Lucy A; Raine, Keiran; Campbell, Peter J; Rada, Cristina; Stratton, Michael R; Neuberger, Michael S

2013-01-01

102

Anion photoelectron imaging of deprotonated thymine and cytosine Bradley F. Parsons,a  

E-print Network

.1039/b703045b We report the anion photoelectron spectra of deprotonated thymine and cytosine at 3.496 eV photodetachment energy using velocity-mapped imaging. The photoelectron spectra of both species exhibit bands photoelectron spectrum. For both thymine and cytosine, the photoelectron spectra are consistent with anions

Neumark, Daniel M.

103

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant  

E-print Network

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven

Boyer, Edmond

104

Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants.  

PubMed

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes. PMID:12902221

Ma, Wenbo; Guinel, Frèdèrique C; Glick, Bernard R

2003-08-01

105

Evidence for Horizontal Transfer of 1-Aminocyclopropane-1-Carboxylate Deaminase Genes  

PubMed Central

PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT. PMID:16269802

Hontzeas, N.; Richardson, A. O.; Belimov, A.; Safronova, V.; Abu-Omar, M. M.; Glick, B. R.

2005-01-01

106

Double-Stranded RNA Adenosine Deaminases ADAR1 and ADAR2 Have Overlapping Specificities  

E-print Network

Double-Stranded RNA Adenosine Deaminases ADAR1 and ADAR2 Have Overlapping Specificities Katrina A, 2000 ABSTRACT: Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to produce inosines adenosines more efficiently than others. Most of what is known about the intrinsic deamination specificity

Bass, Brenda L.

107

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila  

E-print Network

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine Michal Zurovec* , Tomas Dolezal* , Michal Gazi* , Eva Pavlova* , and Peter J. Bryant-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum

Â?urovec, Michal

108

ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616  

EPA Science Inventory

D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

109

Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III  

SciTech Connect

A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO/sub 4/-damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants.

Gossett, J.; Lee, K.; Cunningham, R.P.; Doetsch, P.W.

1988-04-05

110

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

PubMed

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

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

2014-01-23

111

A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically-Modified Neural Stem Cells Expressing E.Coli Cytosine Deaminase for Treatment of Recurrent High Grade Gliomas  

ClinicalTrials.gov

Adult Anaplastic Astrocytoma; Recurrent Grade III Glioma; Recurrent Grade IV Glioma; Adult Anaplastic Oligodendroglioma; Adult Brain Tumor; Adult Giant Cell Glioblastoma; Adult Glioblastoma; Adult Gliosarcoma; Adult Mixed Glioma; Recurrent Adult Brain Tumor; Adult Anaplastic Oligoastrocytoma; Recurrent High Grade Glioma

2014-05-06

112

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

PubMed Central

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

Shapiro, Robert

1999-01-01

113

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

PubMed

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

Fryxell, K J; Zuckerkandl, E

2000-09-01

114

Alkali metal cation binding affinities of cytosine in the gas phase: revisited.  

PubMed

Binding of metal cations to the nucleobases can influence base pairing, base stacking and nucleobase tautomerism. Gas-phase condensation of dc discharge generated alkali metal cations and thermally vaporized cytosine (DC/FT) has been found to produce kinetically trapped excited tautomeric conformations of the M(+)(cytosine) complexes, which influences the threshold collision-induced dissociation (TCID) behavior. In order to elucidate the effects of the size of alkali metal cation on the strength of binding to the canonical form of cytosine, the binding affinities of Na(+) and K(+) to cytosine are re-examined here, and studies are extended to include Rb(+) and Cs(+) again using TCID techniques. The M(+)(cytosine) complexes are generated in an electrospray ionization source, which has been shown to produce ground-state tautomeric conformations of M(+)(cytosine). The energy-dependent cross sections are interpreted to yield bond dissociation energies (BDEs) using an analysis that includes consideration of unimolecular decay rates, the kinetic and internal energy distributions of the reactants, and multiple M(+)(cytosine)-Xe collisions. Revised BDEs for the Na(+)(cytosine) and K(+)(cytosine) complexes exceed those previously measured by 31.9 and 25.5 kJ mol(-1), respectively, consistent with the hypothesis proposed by Yang and Rodgers that excited tautomeric conformations are accessed when the complexes are generated by DC/FT ionization. Experimentally measured BDEs are compared to theoretical values calculated at the B3LYP and MP2(full) levels of theory using the 6-311+G(2d,2p)_HW* and def2-TZVPPD basis sets. The B3LYP/def2-TZVPPD level of theory is found to provide the best agreement with the measured BDEs, suggesting that this level of theory can be employed to provide reliable energetics for similar metal-ligand systems. PMID:24967574

Yang, Bo; Rodgers, M T

2014-08-14

115

Single-stranded DNA Scanning and Deamination by APOBEC3G Cytidine Deaminase at Single Molecule Resolution*?  

PubMed Central

APOBEC3G (Apo3G) is a single-stranded (ss)DNA cytosine deaminase that eliminates HIV-1 infectivity by converting C ? U in numerous small target motifs on the minus viral cDNA. Apo3G deaminates linear ssDNA in vitro with pronounced spatial asymmetry favoring the 3? ? 5? direction. A similar polarity observed in vivo is believed responsible for initiating localized C ? T mutational gradients that inactivate the virus. When compared with double-stranded (ds)DNA scanning enzymes, e.g. DNA glycosylases that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. Here, we investigate ssDNA scanning and motif-targeting mechanisms for Apo3G using single molecule Förster resonance energy transfer. We address the specific issue of deamination asymmetry within the general context of ssDNA scanning mechanisms and show that Apo3G scanning trajectories, ssDNA contraction, and deamination efficiencies depend on motif sequence, location, and ionic strength. Notably, we observe the presence of bidirectional quasi-localized scanning of Apo3G occurring proximal to a 5? hot motif, a motif-dependent DNA contraction greatest for 5? hot > 3? hot > 5? cold motifs, and diminished mobility at low salt. We discuss the single molecule Förster resonance energy transfer data in terms of a model in which deamination polarity occurs as a consequence of Apo3G binding to ssDNA in two orientations, one that is catalytically favorable, with the other disfavorable. PMID:22362763

Senavirathne, Gayan; Jaszczur, Malgorzata; Auerbach, Paul A.; Upton, Thomas G.; Chelico, Linda; Goodman, Myron F.; Rueda, David

2012-01-01

116

Detection of single methylated cytosine using junction-forming DNA probes.  

PubMed

DNA methylation is an epigenetic mechanism for transcriptional regulation. The methylation process controls cellular differentiation and is defective in many diseases including cancer. Therefore, the development of a simple method for analysing cytosine methylation in a target gene is required. Here we report a conceptually new method for sequence-selective chemical modification of a single cytosine in single-stranded DNA (ssDNA) using two DNA probes to form a DNA three-way junction with the ssDNA. The method was successfully used in a simple quantitative polymerase-chain-reaction-based assay for discrimination of a single methylated cytosine. PMID:24614975

Takanashi, Kenta; Kato, Teru

2014-05-01

117

A Role for Adenosine Deaminase in Drosophila Larval Development  

PubMed Central

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals. PMID:15907156

2005-01-01

118

Bioluminescent assay for serum adenosine deaminase with immobilized bacterial luciferase.  

PubMed

We describe a bioluminescence method for measuring adenosine deaminase activity in serum. The method involves use of batchwise enzyme reaction containing adenosine, alpha-ketoglutarate, glutamic dehydrogenase and NADH. The resulting solution is injected to the continuous-flow bioluminescence system. In the system, a bacterial luciferase and NAD(P)H:FMN oxidoreductase are covalently co-immobilized on Sepharose 4B. Carrier solution (pH 6.8) for bioluminescence reaction contains FMN and decanal. The continuous-flow light-emitting system, in which the reactor (flow cell packed with immobilized enzyme) is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. Concentration and response are linearly related from 1.2 to 92.5 pmol per injection of ammonia. The precision of the method is satisfactory (coefficient of variation 3.9-6.8%). We validated the technique by comparing results with conventional assay method (UV method). Normal values for adenosine deaminase activity of serum ranged from 7.0 to 22.0 U/l in agreement with those obtained by other method. The Sepharose 4B-immobilized enzymes are stable for more than one year. This assay system could be used as a routine clinical laboratory test in the diagnosis of liver damage. PMID:2620450

Oda, K; Yoshida, S; Hirose, S; Takeda, T

1989-10-31

119

Laser photobleaching leads to a fluorescence grade adenosine deaminase  

SciTech Connect

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

Parola, A.H.; Caiolfa, V.R.; Bar, I.; Rosenwaks, S. (Ben Gurion Univ. of the Negev, Beer Sheva (Israel))

1989-09-01

120

Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases.  

PubMed Central

The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group. PMID:2690010

Klimasauskas, S; Timinskas, A; Menkevicius, S; Butkiene, D; Butkus, V; Janulaitis, A

1989-01-01

121

A Mutant of Uracil DNA Glycosylase That Distinguishes between Cytosine and 5-Methylcytosine  

PubMed Central

We demonstrate that a mutant of uracil DNA glycosylase (N123D:L191A) distinguishes between cytosine and methylcytosine. Uracil DNA glycosylase (UDG) efficiently removes uracil from DNA in a reaction in which the base is flipped into the enzyme’s active site. Uracil is selected over cytosine by a pattern of specific hydrogen bonds, and thymine is excluded by steric clash of its 5-methyl group with Y66. The N123D mutation generates an enzyme that excises cytosine. This N123D:L191A mutant excises C when it is mispaired with A or opposite an abasic site, but not when it is paired with G. In contrast no cleavage is observed with any substrates that contain 5-methylcytosine. This enzyme may offer a new approach for discriminating between cytosine and 5-methylcytosine. PMID:24740413

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

2014-01-01

122

Induction of guanine deaminase and its inhibitor in rodent liver and brain  

PubMed Central

1. Guanine deaminase activities in homogenates and supernatant fractions of liver and brain of rat and mouse were elevated by administration of guanine to the animals. The maximum induction in mouse tissues occurred within 24h and in rat tissues within 48h. 2. Mitochondria of rat (but not mouse) liver and brain contain an inhibitor of supernatant guanine deaminase, and this was also increased by guanine treatment. 3. Administration of ethionine, cycloheximide or actinomycin D prevented the guanine-dependent increase in deaminase activity and also the increase in mitochondrial inhibitory activity; chloramphenicol suppressed only the latter. PMID:4822729

Sitaramayya, A.; Ali, Shahid; Kumar, K. Sree; Krishnan, P. S.

1974-01-01

123

Methylation of cytosines in nonconventional methylation acceptor sites can contribute to reduced gene expression  

Microsoft Academic Search

Epigenetic silencing of gene expression is often correlated with extensive DNA methylation at cytosine residues in the promoter\\u000a and the coding region of silenced genes. Increasing evidence indicates that, in such cases, DNA methylation can also occur\\u000a in sequence contexts other than CG and CNG, resulting in genomic regions with almost complete modification of cytosines. Whether\\u000a this nonconventional methylation at

M. J. Diéguez; M. Bellotto; K. Afsar; O. Mittelsten Scheid; J. Paszkowski

1997-01-01

124

Yeast 14, 14531469 (1998) Expanding Yeast Knowledge Online  

E-print Network

YEAST Yeast 14, 1453­1469 (1998) Expanding Yeast Knowledge Online KARA DOLINSKI1 , CATHERINE A in the amount of new yeast genetics and molecular biology data. Efficient organization, presentation Sequences; Yeast Protein Database CONTENTS Introduction

Botstein, David

125

TET proteins: on the frenetic hunt for new cytosine modifications  

PubMed Central

Epigenetic genome marking and chromatin regulation are central to establishing tissue-specific gene expression programs, and hence to several biological processes. Until recently, the only known epigenetic mark on DNA in mammals was 5-methylcytosine, established and propagated by DNA methyltransferases and generally associated with gene repression. All of a sudden, a host of new actors—novel cytosine modifications and the ten eleven translocation (TET) enzymes—has appeared on the scene, sparking great interest. The challenge is now to uncover the roles they play and how they relate to DNA demethylation. Knowledge is accumulating at a frantic pace, linking these new players to essential biological processes (e.g. cell pluripotency and development) and also to cancerogenesis. Here, we review the recent progress in this exciting field, highlighting the TET enzymes as epigenetic DNA modifiers, their physiological roles, and their functions in health and disease. We also discuss the need to find relevant TET interactants and the newly discovered TET–O-linked N-acetylglucosamine transferase (OGT) pathway. PMID:23625996

Delatte, Benjamin

2013-01-01

126

DNA cytosine methylation: Structural and thermodynamic characterization of the epigenetic marking mechanism  

PubMed Central

DNA cytosine methyltransferases regulate the expression of the genome through the precise epigenetic marking of certain cytosines with a methyl group, and aberrant methylation is a hallmark of human diseases including cancer. Targeting these enzymes for drug design is currently a high priority. We have utilized ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations to extensively investigate the reaction mechanism of the representative DNA methyltransferase HhaI (M.HhaI) from prokaryotes, whose overall mechanism is shared with the mammalian enzymes. We obtain for the first time full free energy profiles for the complete reaction, together with reaction dynamics in atomistic detail. Our results show an energetically preferred mechanism in which nucleophilic attack of cytosine C5 on the S-adenosyl-L-methionine (AdoMet) methyl group is concerted with formation of the Michael adduct between a conserved Cys in the active site with cytosine C6. Spontaneous and reversible proton transfer between a conserved Glu in the active site and cytosine N3 at the transition state was observed in our simulations, revealing the chemical participation of this Glu residue in the catalytic mechanism. Subsequently, the ?-elimination of the C5 proton utilizes as base an OH? derived from a conserved crystal water that is part of a proton wire water channel, and this syn ?-elimination reaction is the rate-limiting step. Design of novel cytosine methylation inhibitors would be advanced by our structural and thermodynamic characterization of the reaction mechanism. PMID:23528166

Yang, Jin; Lior-Hoffmann, Lee; Wang, Shenglong; Zhang, Yingkai; Broyde, Suse

2013-01-01

127

Adenosine deaminase gene amplification in deoxycoformycin-resistant mammalian cells.  

PubMed

Deoxycoformycin (dCF)-resistant mutants of rat hepatoma, mouse LMTK-, and Chinese hamster ovary (CHO) cells have been isolated and shown to overproduce adenosine deaminase (ADA). The overproduction of ADA was found to be due to ADA-gene amplification in rat and mouse cells but not in CHO cells. Deoxycoformycin-resistant rat hepatoma cells have large HSRs (homogeneously staining regions), mouse cells carry DMs (Double minutes), and CHO cells do not appear to have any gross chromosomal anomalies. When dCF-resistant rat hepatoma and mouse cells are selected by increasing the concentration of the inhibitor in small increments, there is a good correlation between the increase in ADA gene copy number and the increase in the level of expression of ADA, suggesting that all of the amplified genes are equally active in the expression of ADA. PMID:3873908

Rowland, P; Pfeilsticker, J; Hoffee, P A

1985-06-01

128

Comparative studies on heart and skeletal muscle AMP-deaminase from rainbow trout (Salmo gairdneri).  

PubMed

1. AMP-deaminases from fish heart and skeletal muscle have been isolated, and their kinetic and regulatory properties compared. 2. The results obtained indicate that the enzyme variants present in fish heart and skeletal muscle, in contrast to their mammalian counterparts, show very similar chromatographic, kinetic and regulatory characteristics. 3. The above may reflect evolutionary programmed differences in AMP-deaminase gene(s) organization. PMID:1790670

Kaletha, K; Thebault, M; Raffin, J P

1991-01-01

129

Toxicity of Cordycepin in Combination with the Adenosine Deaminase Inhibitor 2?Deoxycoformycin in Beagle Dogs  

Microsoft Academic Search

For 3 consecutive days, the nucleoside cordycepin (3?-deoxyadenosine) was administered as 1-hr iv infusions (0, 1, 4, 8, 10, or 20 mg\\/kg\\/day) to dogs. These doses were given 1 hr after a bolus iv injection (0.25 mg\\/kg\\/day) of 2?-deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase. The hypothesis was that dCF would affect the toxicity of cordycepin. Plasma adenosine deaminase

Larry E. Rodman; Daniel R. Farnell; John M. Coyne; Paula W. Allan; Donald L. Hill; Kimberly L. K. Duncan; Joseph E. Tomaszewski; Adaline C. Smith; John G. Page

1997-01-01

130

Cloning of cDNAs Encoding Mammalian Double-Stranded RNA-Specific Adenosine Deaminase  

Microsoft Academic Search

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the aminoacidlevelexceptattheirNterminiandcontainthreedsRNAbindingmotifs,aputativenucleartargeting signal, and a possible deaminase motif. Antibodies raised

MARY A. O'CONNELL; SABINE KRAUSE; MIYOKO HIGUCHI; J. JUSTIN HSUAN; NICHOLAS F. TOTTY; ANDREAS JENNY; ANDWALTER KELLER

1995-01-01

131

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors  

Microsoft Academic Search

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA\\/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A1 adenosine receptor (A1R)

Francisco Ciruela; Carles Saura; Enric I. Canela; Josefa Mallol; Carmen Lluis; Rafael Franco

1996-01-01

132

1-aminocyclopropane-1-carboxylate (ACC) deaminase genes in rhizobia from southern Saskatchewan.  

PubMed

A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain reaction (PCR) was used to rapidly isolate ACC deaminase structural genes from the above-mentioned 27 strains; 17 of them have 99% identities with the previously characterized ACC deaminase structural gene (acdS) from R. leguminosarum bv. viciae 128C53K, whereas the other ten strains are 84% identical (864-866/1,020 bp) compared to the acdS from strain 128C53K. Southern hybridization showed that each strain has only one ACC deaminase gene. Using inverse PCR, the region upstream of the ACC deaminase structural genes was characterized for all 27 strains, and 17 of these strains were shown to encode a leucine-responsive regulatory protein. The results are discussed in the context of a previously proposed model for the regulation of bacterial ACC deaminase in R. leguminosarum 128C53K. PMID:18548183

Duan, Jin; Müller, Kirsten M; Charles, Trevor C; Vesely, Susanne; Glick, Bernard R

2009-04-01

133

Expression of an Exogenous 1-Aminocyclopropane-1Carboxylate Deaminase Gene in Sinorhizobium meliloti Increases Its Ability To Nodulate Alfalfa  

Microsoft Academic Search

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its

Wenbo Ma; Trevor C. Charles; Bernard R. Glick

2004-01-01

134

Recombinant AAV-Mediated Gene Transfer for the Potential Therapy of Adenosine Deaminase Deficient Severe Combined Immune Deficiency.  

E-print Network

??Adenosine deaminase (ADA) deficiency fosters a rare, but devastating pediatric severe combined immune deficiency (SCID) with a T- B- NK- phenotype, concomitant opportunistic infections including… (more)

Silver, Jared

2008-01-01

135

Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription.  

PubMed

DNA cytosine methylation regulates gene expression in mammals. In bacteria, its role in gene expression and genome architecture is less understood. Here we perform high-throughput sequencing of bisulfite-treated genomic DNA from Escherichia coli K12 to describe, for the first time, the extent of cytosine methylation of bacterial DNA at single-base resolution. Whereas most target sites (C(m)CWGG) are fully methylated in stationary phase cells, many sites with an extended CC(m)CWGG motif are only partially methylated in exponentially growing cells. We speculate that these partially methylated sites may be selected, as these are slightly correlated with the risk of spontaneous, non-synonymous conversion of methylated cytosines to thymines. Microarray analysis in a cytosine methylation-deficient mutant of E. coli shows increased expression of the stress response sigma factor RpoS and many of its targets in stationary phase. Thus, DNA cytosine methylation is a regulator of stationary phase gene expression in E. coli. PMID:22673913

Kahramanoglou, Christina; Prieto, Ana I; Khedkar, Supriya; Haase, Bettina; Gupta, Ankur; Benes, Vladimir; Fraser, Gillian M; Luscombe, Nicholas M; Seshasayee, Aswin S N

2012-01-01

136

Marked influences on the adenine-cytosine base pairs by electron attachment and ionization.  

PubMed

The reverse wobble and the reverse Hoogsteen adenine-cytosine mispairs regarding their radical cations and anions are studied with the hybrid three-parameter B3LYP density functional method and 6-31+G(d), 6-311+G(2df,2p) basis sets. Hydrogen bonding mispairs are remarkably influenced by electron attachment and ionization. Only one stronger hydrogen bond N6-H (in adenine)...N3 (in cytosine) exists in the radical pair, while the strengths of two N-H...N hydrogen bonds in the neutral pair are comparable. Geometrical coplanarity is found for the neutral and cationic pairs, in contrast to the anionic pairs in which the cytosine moiety exhibits significant deformation due to electron attachment. Dissociation energies for the neutral and radical pairs are slightly higher than those of the adenine-thymine pairs but much smaller than those of the guanine-cytosine pairs. Valence-bound anions of these two adenine-cytosine pairs are thermodynamically stable by 0.1-0.2 eV with respect to the neutral pairs. On the basis of the comparison between the experimental data of the solvated clusters and the calculated values, these two pairs can be quantitatively equivalent to the clusters in which each base is solvated by five water molecules. PMID:16833870

Tian, Shan Xi

2005-06-16

137

Influence of cytosine methylation on ultraviolet-induced cyclobutane pyrimidine dimer formation in genomic DNA.  

PubMed

The ultraviolet (UV) component of sunlight is the main cause of skin cancer. More than 50% of all non-melanoma skin cancers and >90% of squamous cell carcinomas in the US carry a sunlight-induced mutation in the p53 tumor suppressor gene. These mutations have a strong tendency to occur at methylated cytosines. Ligation-mediated PCR (LMPCR) was used to compare at nucleotide resolution DNA photoproduct formation at dipyrimidine sites either containing or lacking a methylated cytosine. For this purpose, we exploited the fact that the X chromosome is methylated in females only on the inactive X chromosome, and that the FMR1 (fragile-X mental retardation 1) gene is methylated only in fragile-X syndrome male patients. Purified genomic DNA was irradiated with UVC (254nm), UVB (290-320nm) or monochromatic UVB (302 and 313nm) to determine the effect of different wavelengths on cyclobutane pyrimidine dimer (CPD) formation along the X-linked PGK1 (phosphoglycerate kinase 1) and FMR1 genes. We show that constitutive methylation of cytosine increases the frequency of UVB-induced CPD formation by 1.7-fold, confirming that methylation per se is influencing the probability of damage formation. This was true for both UVB sources used, either broadband or monochromatic, but not for UVC. Our data prove unequivocally that following UVB exposure methylated cytosines are significantly more susceptible to CPD formation compared with unmethylated cytosines. PMID:19427505

Rochette, Patrick J; Lacoste, Sandrine; Therrien, Jean-Philippe; Bastien, Nathalie; Brash, Douglas E; Drouin, Régen

2009-06-01

138

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

SciTech Connect

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.

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

2009-12-14

139

Electrochemical and fluorescence detection of cytosine-related SNPs using a ferrocenyl naphthyridine derivative.  

PubMed

A novel hydrogen bond-forming ligand for cytosine-related single nucleotide polymorphism, which contains both a fluorescent naphthyridine moiety and a ferrocene group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase within an abasic site-containing DNA duplex was confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1: 1 complex with the target nucleobase opposite an AP site, and the selectivity was in the order of cytosine > thymine > adenine, guanine, reflecting the stability of hydrogen bond formation. PMID:18029703

Morita, Kotaro; Sato, Yusuke; Seino, Takehiro; Nishizawa, Seiichi; Teramae, Norio

2007-01-01

140

Plasma adenosine deaminase isoform 2 in cancer patients undergoing chemotherapy.  

PubMed

Adenosine deaminase (AD), a purine salvage enzyme, exists as AD isoform 1 (AD1) and AD isoform 2 (AD2). Plasma AD has been advocated for the screening and monitoring of cancer, as AD2 activity is increased in conditions associated with tumour growth. Plasma AD2 was measured before and seven to 10 days after the first dose of chemotherapy in patients with different tumours. A 'tumour regression score' was assessed independently based on radiological changes seen in the tumour following completion of chemotherapy. Changes in plasma AD2 were then compared with the tumour regression score. Following first-dose chemotherapy, plasma AD2 decreased on average from 22.7 +/- 10.5 U/L to 15.0 +/- 4.6 U/L. The percentage decrease in plasma AD2 correlated with the tumour regression score (r=0.5, P=0.028). These data suggest plasma AD2 may have a role in determining tumour response to treatment. PMID:22558798

Roberts, E L; Roberts, O T

2012-01-01

141

ADA (adenosine deaminase) gene therapy enters the competition  

SciTech Connect

Around the world, some 70 children are members of a select and deadly club. Born with an immune deficiency so severe that they will die of infection unless their immune systems can be repaired, they have captured the attention of would-be gene therapists who believe that a handful of these kids--the 15 or 20 who lack functioning levels of the enzyme adenosine deaminase (ADA)--could be saved by a healthy ADA gene. A team of gene therapists is ready to put the theory to the test. In April 1987, a team of NIH researchers headed by R. Michael Blaese and W. French Anderson came up with the first formal protocol to introduce a healthy ADA gene into an unhealthy human. After 3 years of line-by-line scrutiny by five review committees, they have permission to go ahead. Two or three children will be treated in the next year, and will be infused with T lymphocytes carrying the gene for ADA. If the experiment works, the ADA gene will begin producing normal amounts of ADA. An interesting feature of ADA deficiency, that makes it ideal for initial gene studies, is that the amount of ADA one needs for a healthy immune system is quite variable. Hence, once inside a patient's T cells, the new ADA gene needs only to express the enzyme in moderate amounts. No precise gene regulation is necessary.

Culliton, B.J.

1990-08-31

142

The ONIOM molecular dynamics method for biochemical applications: cytidine deaminase  

SciTech Connect

Abstract We derived and implemented the ONIOM-molecular dynamics (MD) method for biochemical applications. The implementation allows the characterization of the functions of the real enzymes taking account of their thermal motion. In this method, the direct MD is performed by calculating the ONIOM energy and gradients of the system on the fly. We describe the first application of this ONOM-MD method to cytidine deaminase. The environmental effects on the substrate in the active site are examined. The ONIOM-MD simulations show that the product uridine is strongly perturbed by the thermal motion of the environment and dissociates easily from the active site. TM and MA were supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2007-03-22

143

Activity regulation of adenosine deaminases acting on RNA (ADARs).  

PubMed

Adenosine deaminases acting on RNA (ADARs) are the enzymes that are responsible for the A to I RNA editing process in mammals, which is an important mechanism that increases molecular diversity. A to I RNA editing consists of an enzymatic conversion of specific adenosine in pre-mRNA, leading to alteration of the properties of both the RNA itself and the translated protein. Currently, the importance of this phenomenon is increasingly recognized as it affects a diverse set of cellular pathways. ADAR function within the cell, especially in the neurons, is to diversify the features of a limited set of unique transcripts, mostly neurotransmitter receptors; however, a growing set of target is going to be discovered, increasing the importance of the RNA editing event in the proper physiology of the cell. Despite the functional relevance of these enzymes, there is a gap of knowledge in the mechanisms that regulate ADAR activity and consequently about the modulation of RNA editing process. This review summarizes ongoing investigations of ADAR regulation at the transcriptional, post-transcriptional and post-translational level and addresses new hypothetical mechanisms that are capable of modulating ADAR activity, including subcellular localization, dimerization and interaction with trans-acting factors. PMID:22113393

Orlandi, Cesare; Barbon, Alessandro; Barlati, Sergio

2012-02-01

144

Functions and Regulation of RNA Editing by ADAR Deaminases  

PubMed Central

One type of RNA editing converts adenosines to inosines (A?I editing) in double-stranded RNA (dsRNA) substrates. A?I RNA editing is mediated by adenosine deaminase acting on RNA (ADAR) enzymes. A?I RNA editing of protein-coding sequences of a limited number of mammalian genes results in recoding and subsequent alterations of their functions. However, A?I RNA editing most frequently targets repetitive RNA sequences located within introns and 5? and 3? untranslated regions (UTRs). Although the biological significance of noncoding RNA editing remains largely unknown, several possibilities, including its role in the control of endogenous short interfering RNAs (esiRNAs), have been proposed. Furthermore, recent studies have revealed that the biogenesis and functions of certain microRNAs (miRNAs) are regulated by the editing of their precursors. Here, I review the recent findings that indicate new functions for A?I editing in the regulation of noncoding RNAs and for interactions between RNA editing and RNA interference mechanisms. PMID:20192758

Nishikura, Kazuko

2010-01-01

145

ACC deaminase genes are conserved among Mesorhizobium species able to nodulate the same host plant.  

PubMed

Rhizobia strains expressing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase have been reported to display an augmented symbiotic performance as a consequence of lowering the plant ethylene levels that inhibit the nodulation process. Genes encoding ACC deaminase (acdS) have been studied in Rhizobium spp.; however, not much is known about the presence of acdS genes in Mesorhizobium spp. The aim of this study was to assess the prevalence and phylogeny of acdS genes in Mesorhizobium strains including a collection of chickpea-nodulating mesorhizobia from Portugal. ACC deaminase genes were detected in 10 of 12 mesorhizobia type strains as well as in 18 of 18 chickpea Mesorhizobium isolates studied in this work. No ACC deaminase activity was detected in any Mesorhizobium strain tested under free-living conditions. Despite the lack of ACC deaminase activity, it was possible to demonstrate that in Mesorhizobium ciceri UPM-Ca7(T) , the acdS gene is transcribed under symbiotic conditions. Phylogenetic analysis indicates that strains belonging to different species of Mesorhizobium, but nodulating the same host plant, have similar acdS genes, suggesting that acdS genes are horizontally acquired by transfer of the symbiosis island. This data, together with analysis of the symbiosis islands from completely sequenced Mesorhizobium genomes, suggest the presence of the acdS gene in a Mesorhizobium common ancestor that possessed this gene in a unique symbiosis island. PMID:22846039

Nascimento, Francisco X; Brígido, Clarisse; Glick, Bernard R; Oliveira, Solange

2012-11-01

146

Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture.  

PubMed

Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into alpha-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species. PMID:17665234

Saleem, Muhammad; Arshad, Muhammad; Hussain, Sarfraz; Bhatti, Ahmad Saeed

2007-10-01

147

Protein expression-yeast.  

PubMed

Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline. PMID:24423273

Nielsen, Klaus H

2014-01-01

148

Yeast Based Sensors  

NASA Astrophysics Data System (ADS)

Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.

Shimomura-Shimizu, Mifumi; Karube, Isao

149

Regulation of ethylene levels in canola ( Brassica campestris ) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense  

Microsoft Academic Search

We report the presence of ACC deaminase in Methylobacterium fujisawaense and its lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions. To test a part of the previous model proposed for ACC deaminase producing bacteria with Methylobacterium, ACC levels and various enzyme activities were monitored in canola. Lower amounts of ACC were present in

Munusamy Madhaiyan; Selvaraj Poonguzhali; Jeounghyun Ryu; Tongmin Sa

2006-01-01

150

CLONTECHInnovative Yeast Protocols Handbook  

E-print Network

CLONTECHInnovative Tools to Accelerate Discovery Yeast Protocols Handbook PT3024-1 (PR13103 FOR RESEARCH USE ONLY #12;Yeast Protocols Handbook CLONTECH Laboratories, Inc. www.clontech.com Protocol # PT3024-1 2 Version # PR13103 I. Introduction 4 II. Introduction to Yeast Promoters 5 III. Culturing

Erickson, F. Les

151

[Yeasts contaminating salmon roe].  

PubMed

Quantitative and species compositions of yeast contaminating eggs, fry and fingerlings of Salmo gairdneri Rich under artificial breeding have been studied. Prevalence of species of genera Candida, Rhodotorula, Cryptococcus and Debaryomyces is noted. Yeast isolated from perished eggs and sick fry do not possess pathogenic properties. Certain strains of yeast make stimulating effect on the studied microorganisms. PMID:8983527

Nagornaia, S S; Ignatova, E A; Isaeva, N M; Davydov, O N; Podgorski?, V S

1996-01-01

152

YEAST GENETICS Fred Winston  

E-print Network

YEAST GENETICS Fred Winston 7.1 Introduction Key Concepts · Genetic studies of the yeast. The yeast Saccharomyces cerevisiae is an ideal experimental organism. It is a microorganism that has a fast rate of growth, with a generation time of only ninety minutes under optimal conditions. Genetic methods

Winston, Fred

153

Conversion of viramidine to ribavirin in vivo by adenosine deaminase and its inhibition by 2'-deoxycoformycin.  

PubMed

Previously we reported that viramidine is a prodrug of ribavirin and that adenosine deaminase catalyses viramidine deamination to ribavirin in vivo. This in vivo study explores this prodrug conversion in rats and inhibition by a potent adenosine deaminase inhibitor, 2'-deoxycoformycin. We found that conversion of viramidine to ribavirin was viramidine dose-dependent in rat plasma. A single intravenous dose of 0.25 mg/kg 2'-deoxycoformycin suppressed orally administered viramidine conversion to ribavirin in plasma by 50%. The inhibition was 2'-deoxycoformycin dose-dependent and a single dose of 2 mg/kg decreased the ribavirin/viramidine area under the concentration-time curve between 0 h and 6 h ratio by 2.5-fold. These findings provide strong evidence that adenosine deaminase plays a major role in converting viramidine to ribavirin in vivo. PMID:16542004

Wu, Jim Zhen; Yeh, Li-Tain; Lin, Chin-Chung; Hong, Zhi

2006-01-01

154

Rat cerebral-cortex adenosine deaminase activity and its subcellular distribution  

PubMed Central

A radioisotopic assay for adenosine deaminase (EC 3.5.4.4) is described together with its application in investigating the activity of the enzyme in rat cerebral cortex. Activity of the adenosine deaminase was determined to be 115nmol/min per g of tissue, measured in isoosmotic sucrose dispersions of the neocortex, and to be 170nmol/min per g of tissue after treatment with Triton X-100. The enzyme was concluded to be largely cytoplasmic, with a Km of 54–57?m for adenosine. Action of the deaminase, and other aspects of the metabolism of adenosine in intact neocortical tissue, were quantitatively appraised on the basis of the newly determined characteristics. PMID:4462574

Pull, Ian; McIlwain, Henry

1974-01-01

155

Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations  

Microsoft Academic Search

The chemical reactivity of thymine (T), when mismatched with the bases cytosine, guanine, and thymine, and of cytosine (C), when mismatched with thymine, adenine, and cytosine, has been examined. Heteroduplex DNAs containing such mismatched base pairs were first incubated with osmium tetroxide (for T and C mismatches) or hydroxylamine (for C mismatches) and then incubated with piperidine to cleave the

R. G. H. Cotton; N. R. Rodrigues; R. D. Campbell

1988-01-01

156

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

NASA Astrophysics Data System (ADS)

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'.

Shapiro, Robert

2002-06-01

157

Effects of Cytosine Arabinoside, Daunomycin, Mithramycin, Azacytidine, Adriamycin, and Camptothecin on Mammalian Cell Cycle Traverse1  

Microsoft Academic Search

Six chemotherapeutic agents were tested for effects on mammalian cell cycle traverse in synchronized cultures of Chinese hamster cells. Cytosine arabinoside was found to inhibit DNA synthesis, as well as to reduce grossly the rate of progression from Gj into S. At low dosage levels, daunomycin and adriamycin had only a slight effect upon entry of cells into S, but

Robert A. Tobey

158

Guanine deaminase inhibitor from rat liver. Isolation and characterization  

PubMed Central

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver `heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH4)2SO4 and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH4)2SO4 in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor–phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested. PMID:4821397

Ali, Shahid; Sitaramayya, A.; Kumar, K. Sree; Krishnan, Padmanabhan S.

1974-01-01

159

Biological function of activation-induced cytidine deaminase (AID).  

PubMed

Activation-induced Cytidine Deaminase (AID) is an essential regulator of B cell diversification, but its full range of action has until recently been an enigma. Based on homology, it was originally proposed to be an RNA-editing enzyme, but so far, no RNA substrates are known. Rather, it functions by deaminating cytidine, and in this manner, coupled with base-excision repair or mismatch repair machinery, it is a natural mutator. This allows it to play a central role in adaptive immunity, whereby it initiates the processes of class switch recombination and somatic hypermutation to help generate a diverse and high-affinity repertoire of immunoglobulin isotypes. More recently, it has been appreciated that methylated cytidine, already known as a key epigenetic mark on DNA controlling gene expression, can also be a target for AID modification. Coupled with repair machinery, this can facilitate the active removal of methylated DNA. This activity can impact the process of cellular reprogramming, including transition of a somatic cell to pluripotency, which requires major reshuffling of epigenetic memory. Thus, seemingly disparate roles for AID in controlling immune diversity and epigenetic memory have a common mechanistic basis. However, the very activity that is so useful for B cell diversity and cellular reprogramming is dangerous for the integrity of the genome. Thus, AID expression and activity is tightly regulated, and deregulation is associated with diseases including cancer. Here, we review the range of AID functions with a focus on its mechanisms of action and regulation. Major questions remain to be answered concerning how and when AID is targeted to specific loci and how this impacts development and disease. PMID:25163501

Kumar, Ritu; DiMenna, Lauren J; Chaudhuri, Jayanta; Evans, Todd

2014-01-01

160

Structural and Metabolic Specificity of Methylthiocoformycin for Malarial Adenosine Deaminases  

SciTech Connect

Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5?-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5?-methylthioribosyl groups are rotated 130 degrees. A hydrogen bonding network between Asp172 and the 3?-hydroxyl of MT-coformycin is essential for recognition of the 5?-methylthioribosyl group. Water occupies the 5?-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

Ho, M.; Cassera, M; Madrid, D; Ting, L; Tyler, P; Kim, K; Almo, S; Schramm, V

2009-01-01

161

Autoimmune Dysregulation and Purine Metabolism in Adenosine Deaminase Deficiency  

PubMed Central

Genetic defects in the adenosine deaminase (ADA) gene are among the most common causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive, and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-ADA), or hematopoietic stem cell gene therapy (HSC-GT). Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment. A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T- and B-cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties. PMID:22969765

Sauer, Aisha Vanessa; Brigida, Immacolata; Carriglio, Nicola; Aiuti, Alessandro

2012-01-01

162

CONTRASTING BEHAVIOR OF CONFORMATIONALLY LOCKED CARBOCYCLIC NUCLEOSIDES OF ADENOSINE AND CYTIDINE AS SUBSTRATES FOR DEAMINASES  

PubMed Central

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn+2 coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4’) oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold. PMID:20183605

Schroeder, Gottfried K.; Ludek, Olaf R.; Siddiqui, Maqbool A.; Ezzitouni, Abdallah; Wolfenden, Richard

2010-01-01

163

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

PubMed Central

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 the affected plant populations to the changed environments. PMID:23418457

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

2013-01-01

164

Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation  

Microsoft Academic Search

Activation-induced cytidine deaminase (AID) is a protein required for B cells to undergo class switch recombination and somatic hypermutation (SHM)-two processes essential for producing high-affinity antibodies. Purified AID catalyses the deamination of C to U on single-stranded (ss)DNA. Here, we show in vitro that AID-catalysed C deaminations occur preferentially on 5' WRC sequences in accord with SHM spectra observed in

Phuong Pham; Ronda Bransteitter; John Petruska; Myron F. Goodman

2003-01-01

165

Contributory role of adenosine deaminase in metabolic syndrome.  

PubMed

Adenosine deaminase (ADA) is an enzyme of purine metabolism commonly associated with severe combined immunodeficiency disease and believed to modulate bioactivity of insulin. Its contributory role in patients with metabolic syndrome (having features such as obesity, insulin resistance, fasting hyperglycaemia, lipid abnormalities and hypertension) in South Eastern Nigeria was studied. Body mass index (BMI), fasting blood glucose (FBG), Glycated haemoglobin (GHbA1c), total cholesterol, HDL-cholesterol, LDL-cholesterol (usually impaired in metabolic syndrome) and total serum ADA activity were measured in different groups of patients with metabolic syndrome (test subjects) and apparently healthy subjects (controls). The test subjects comprised six subgroups made up of the following; obese diabetic (N=25), obese non-diabetic (N=25), Non-obese diabetic (N=25), patients with hypercholesterolaemia (N=25), LDL-cholesterolaemia (N=25) and HDL-cholesterolaemia (N=25). The results showed that the mean values of all the parameters studied (BMI, FBG, GHbA1c, total cholesterol, HDL-cholesterol and LDL-cholesterol) were higher in the test subjects than their controls. BMI did not correlate significantly with FBG, GHbA1c, and ADA in the test and control subjects respectively. The mean serum ADA activity in the test subjects of obese diabetic, obese non-diabetic and non-obese diabetic subjects was higher than in controls (p< 0.001). ADA activity was also higher in the test subjects of hypercholesterolaemia, HDL-cholesterolaemia and LDL-cholesterolaemia than in control (p< 0.001). ADA activity also correlated positively with hypercholesterolemia (r = 0.640; p<0.001), HDL-cholesterolaemia (r = 0.646; p<0.001) and LDL-cholesterolaemia (r = 0.932; p<0.001), with the highest correlation in the LDL-cholesterolaemia. In conclusion, ADA activity is increased significantly in all parameters of metabolic syndrome studied and showed a significant correlation with all the three groups of dyslipidaemic subjects studied. ADA could therefore be used in daily routine laboratory assessment of most metabolic diseases especially in obese and diabetic patients. PMID:23955411

Nwankwo, A A; Osim, E E; Bisong, S A

2013-01-01

166

IMMUNE FUNCTION IN MICE EXPOSED TO THE ADENOSINE DEAMINASE INHIBITOR 2'-DEOXYCOFORMYCIN DURING IMMUNE SYSTEM DEVELOPMENT  

EPA Science Inventory

Pregnant mice were administered 2'-deoxycoformycin (2dCF), a potent inhibitor of adensoine deaminase activity, by intraperitoneal injection on day 7 or 15 of gestation or from day 8-12 or 14-18 of gestation. A total dose of 0, 0.5 or 2.0 micrograms 2dCF/g of maternal body weight ...

167

Adaptive evolution of threonine deaminase in plant defense against insect herbivores  

Microsoft Academic Search

Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to -ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and

Eliana Gonzales-Vigil; Christopher M. Bianchetti; G. N. Phillips; Gregg A. Howe

2011-01-01

168

SELECTIVE IMMUNOTOXIC EFFECTS IN MICE TREATED WITH THE ADENOSINE DEAMINASE INHIBITOR 2-DEOXYCOFORMYCIN (JOURNAL VERSION)  

EPA Science Inventory

Mice given the adenosine deaminase inhibitor 2-deoxycoformycin, for five days were evaluated 24 h, 72 h and 6 days after the final dose. Spleen weight was decreased for up to 6 days after treatment. The number and relative percentage of circulating lymphocytes were decreased 24 a...

169

Isolation and characterization of the threonine deaminase promoter in Nicotiana attenuata  

Microsoft Academic Search

The enzyme encoded by the threonine deaminase (TD) gene catalyzes the conversion of threonine to ?-keto butyrate in the biosynthesis of isoleucine (Ile). In Nicotiana attenuata, TD transcripts accumulate constitutively in cotyledons and flowers and are elicited in leaves by wounding, herbivore attack, and methyl jasmonic acid (MeJA) treatment. To understand TD's unique pattern of expression, we isolated a genomic

Jin-Ho Kang; Ian T. Baldwin

2006-01-01

170

Characterization of the adenosine deaminase-related growth factor (ADGF) gene family in Drosophila  

Microsoft Academic Search

A novel family of growth factors, with sequence similarity to adenosine deaminase, has been identified in various organisms including flesh fly, tsetse fly, sand fly, mollusk and human. The human homologue, CECR1, is a candidate gene for the genetic disorder cat eye syndrome. Here, we describe six members of this growth factor family in Drosophila and two in vertebrates. The

Stephanie A. Maier; Lynn Podemski; Sean W. Graham; Heather E. McDermid; John Locke

2001-01-01

171

Transgenerational epigenetic effects of the Apobec1 cytidine deaminase deficiency on testicular germ  

E-print Network

for maternal estrogens, birth order, birth weight, and other factors (15­17) together raise the possibilityTransgenerational epigenetic effects of the Apobec1 cytidine deaminase deficiency on testicular. These transgenerational effects challenge conventional understanding about the modes and mechanisms of inheritance

172

Rapid determination of adenosine deaminase kinetics using fast-scan cyclic voltammetry.  

PubMed

Adenosine deaminase is an enzyme involved in purine metabolism and its inhibitors are used as anticancer and antiviral drugs. In this study, we show that fast-scan cyclic voltammetry at carbon-fiber microelectrodes can be used to study the kinetics of adenosine deaminase by electrochemically monitoring decreases in adenosine concentration. Buffer and salt concentrations were shown to affect the enzyme kinetics and the inhibition by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin (DCF). In a Tris buffer containing salts that mimic cerebrospinal fluid, EHNA and DCF showed non-competitive inhibition with a K(i) of 1.7 +/- 0.6 nM and 1.2 +/- 0.2 nM, respectively. However, removing the divalent cations from the Tris buffer caused the inhibition to be competitive and reduced the K(i) for DCF by two orders of magnitude. In phosphate-buffered saline, the K(i) was 1.0 +/- 0.2 nM for EHNA and 3.6 +/- 0.3 pM for DCF, similar to literature values. Adenosine deaminase was also competitively inhibited by AgNO(3), showing it is susceptible to silver toxicity. Caffeine was found to increase adenosine deaminase activity. This is a fast, easy method for screening drug effects on enzyme kinetics and could be applied to other enzymatic reactions where there is a significant difference in the electroactivity of the reactant and product. PMID:20577678

Xu, Yida; Venton, B Jill

2010-09-14

173

A coming-of-age story: activation-induced cytidine deaminase turns 10  

Microsoft Academic Search

The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the

Rebecca K Delker; Sebastian D Fugmann; F Nina Papavasiliou

2009-01-01

174

Chloroviruses encode a bifunctional dCMP-dCTP deaminase that produces two key intermediates in dTTP formation.  

PubMed

The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP. Unexpectedly, however, the viral enzyme also has dCTP deaminase activity, producing dUTP. Typically, these two reactions are catalyzed by proteins in separate enzyme classes; to our knowledge, this is the first example of a protein having both deaminase activities. Kinetic experiments established that (i) the PBCV-1 enzyme has a higher affinity for dCTP than for dCMP, (ii) dCTP serves as a positive heterotropic effector for the dCMP deaminase activity and a positive homotropic effector for the dCTP deaminase activity, and (iii) the enzymatic efficiency of the dCMP deaminase activity is about four times higher than that of the dCTP deaminase activity. Inhibitor studies suggest that the same active site is involved in both dCMP and dCTP deaminations. The discovery that the PBCV-1 dCMP deaminase has two activities, together with a previous report that the virus also encodes a functional dUTP triphosphatase (Y. Zhang, H. Moriyama, K. Homma, and J. L. Van Etten, J. Virol. 79:9945-9953, 2005), means that PBCV-1 is the first virus to encode enzymes involved in all three known pathways to form dUMP. PMID:17475641

Zhang, Yuanzheng; Maley, Frank; Maley, Gladys F; Duncan, Garry; Dunigan, David D; Van Etten, James L

2007-07-01

175

Chloroviruses Encode a Bifunctional dCMP-dCTP Deaminase That Produces Two Key Intermediates in dTTP Formation?  

PubMed Central

The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP. Unexpectedly, however, the viral enzyme also has dCTP deaminase activity, producing dUTP. Typically, these two reactions are catalyzed by proteins in separate enzyme classes; to our knowledge, this is the first example of a protein having both deaminase activities. Kinetic experiments established that (i) the PBCV-1 enzyme has a higher affinity for dCTP than for dCMP, (ii) dCTP serves as a positive heterotropic effector for the dCMP deaminase activity and a positive homotropic effector for the dCTP deaminase activity, and (iii) the enzymatic efficiency of the dCMP deaminase activity is about four times higher than that of the dCTP deaminase activity. Inhibitor studies suggest that the same active site is involved in both dCMP and dCTP deaminations. The discovery that the PBCV-1 dCMP deaminase has two activities, together with a previous report that the virus also encodes a functional dUTP triphosphatase (Y. Zhang, H. Moriyama, K. Homma, and J. L. Van Etten, J. Virol. 79:9945-9953, 2005), means that PBCV-1 is the first virus to encode enzymes involved in all three known pathways to form dUMP. PMID:17475641

Zhang, Yuanzheng; Maley, Frank; Maley, Gladys F.; Duncan, Garry; Dunigan, David D.; Van Etten, James L.

2007-01-01

176

Precise estimates of mutation rate and spectrum in yeast  

PubMed Central

Mutation is the ultimate source of genetic variation. The most direct and unbiased method of studying spontaneous mutations is via mutation accumulation (MA) lines. Until recently, MA experiments were limited by the cost of sequencing and thus provided us with small numbers of mutational events and therefore imprecise estimates of rates and patterns of mutation. We used whole-genome sequencing to identify nearly 1,000 spontaneous mutation events accumulated over ?311,000 generations in 145 diploid MA lines of the budding yeast Saccharomyces cerevisiae. MA experiments are usually assumed to have negligible levels of selection, but even mild selection will remove strongly deleterious events. We take advantage of such patterns of selection and show that mutation classes such as indels and aneuploidies (especially monosomies) are proportionately much more likely to contribute mutations of large effect. We also provide conservative estimates of indel, aneuploidy, environment-dependent dominant lethal, and recessive lethal mutation rates. To our knowledge, for the first time in yeast MA data, we identified a sufficiently large number of single-nucleotide mutations to measure context-dependent mutation rates and were able to (i) confirm strong AT bias of mutation in yeast driven by high rate of mutations from C/G to T/A and (ii) detect a higher rate of mutation at C/G nucleotides in two specific contexts consistent with cytosine methylation in S. cerevisiae. PMID:24847077

Zhu, Yuan O.; Siegal, Mark L.; Hall, David W.; Petrov, Dmitri A.

2014-01-01

177

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

NASA Technical Reports Server (NTRS)

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.

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

1995-01-01

178

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

SciTech Connect

In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-{angstrom} resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C-C{sup +} 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{prime} thymine Hoogsteen-base-paired to the third adenine. The 5{prime} end of this loop is in close proximity to the 3{prime} 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. 25 refs., 5 figs.

Kang, C.H.; Lockshin, C.; Rich, A. [Massachusetts Institute of Technology, Cambridge, MA (United States)] [and others

1995-04-25

179

Acute Oral Administration of Cytosine Arabinoside Triacetate to Male Albino Rats and Male and Female Rhesus Monkeys.  

National Technical Information Service (NTIS)

The acute toxicity of cytosine arabinoside triacetate was evaluated following single oral administration in male albino rats and in male and female rhesus monkeys. Methyl cellulose suspensions of the compound were prepared at concentrations ranging from 4...

H. Feinman, T. W. Tusing, E. R. Homan, D. P. Rall

1966-01-01

180

Potential energy surfaces of the microhydrated guanine...cytosine base pair and its methylated analogue.  

PubMed

A complete scan of the potential and free-energy surfaces of monohydrated and dihydrated guanine...cytosine and 9-methylguanine...1-methylcytosine base pairs was realized by the molecular dynamics/quenching technique using the force field of Cornell et al. implemented in the AMBER7 program. The most stable and populated structures localized were further fully reoptimized at the correlated ab initio level employing the resolution of identity Møller-Plesset method with a large basis set. A systematic study of microhydration of these systems using a high-level correlated ab initio approach is presented for the first time. The different behavior of guanine...cytosine and adenine...thymine complexes is also discussed. These studies of nucleic acid base pairs are important for finding binding sites of water molecules around bases and for better understanding of the influence of the solvent on the stability of the structure of DNA. PMID:16463334

Zendlová, Lucie; Hobza, Pavel; Kabelác, Martin

2006-02-13

181

Thiocytosine as a radiation energy trap in a single crystal of cytosine hydrochloride.  

PubMed

Single crystals of cytosine hydrochloride with thiocytosine as an impurity were found suitable for the study of a possible new mechanism of long-range migration of energy deposited by ionizing radiation. In a crystal containing thiocytosine, two kinds of chlorine-containing paramagnetic centres are present that are completely absent in the pure cytosine. HCl crystals irradiated under the same condition. The thiocytosine molecules in conjunction with Cl- ions behave as hole traps. The centres have been characterized by EPR spectroscopy. One of the centres is derived from the cationic thiocytosine radical by interaction with a Cl- ion, and the other centre is formed by interaction of Cl. with a thiocytosine molecule. It is suggested that the transfer of an electron-loss site (a hole) in the Cl- network is the actual mechanism of the long-range energy transfer. PMID:8027610

Herak, J N; Sankovic, K; Hütterman, J

1994-07-01

182

A theoretical study of structures and electron affinities of radical anions of guanine-cytosine, adenine-thymine, and hypoxanthine-cytosine base pairs.  

PubMed

Adiabatic electron affinities (AEA) and structural perturbations due to addition of an excess electron to each of the neutral guanine-cytosine (G-C), adenine-thymine (A-T), and hypoxanthine-cytosine (HX-C) base pairs were studied using the self-consistent charge, density functional tight-binding (SCC-DFTB-D) method, augmented by the empirical London dispersion energy term. Performance of the SCC-DFTB-D method was examined by comparing the calculated results using it with those obtained from experiment as well as ab initio and other different density functional theoretical studies. An excellent agreement between the SCC-DFTB-D results and those obtained by the other calculations regarding the structural modifications, hydrogen bonding, and dissociation energies of the neutral and radical anion base pairs was found. It is shown that adiabatic electron affinity can be better predicted by considering reaction enthalpies of formation of the respective neutral and anionic base pairs from their respective molecular components instead of taking the difference between their total energies. The calculated AEAs of the base pairs were compared with those obtained by the bracketing method from Schaefer and coworkers, where a satisfactory agreement was found. It shows applicability of the SCC-DFTB-D method to study charged DNA models at a highly economical computational cost. PMID:15067680

Kumar, Anil; Knapp-Mohammady, Michaela; Mishra, P C; Suhai, Sándor

2004-06-01

183

Cytosine-bulge-dependent fluorescence quenching for the real-time hairpin primer PCR.  

PubMed

The progress of a polymerase chain reaction (PCR) was sensitively monitored based on the increase in fluorescence of N,N'-bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine, which was covalently anchored on the cytosine bulge directly neighbouring the 5'-T_G-3'/5'-CCA-3' sequence in the hairpin tag at the 5' end of the PCR primer. PMID:25338232

Takei, F; Chen, X; Yu, G; Shibata, T; Dohno, C; Nakatani, K

2014-11-01

184

Treatment of relapsed and refractory acute leukaemia with high-dose cytosine arabinoside and etoposide  

Microsoft Academic Search

A total of 65 patients under the age of 55 with acute leukaemia received high-dose cytosine arabinoside (Ara-C) in combination with high-dose etoposide without an anthracycline. Complete remission rates for patients with relapsed or refractory acute myelogenous leukaemia (AML) were 15\\/25 (60%) and 11\\/16 (69%), respectively. The complete remission rate for patients with refractory or relapsed acute lymphoblastic leukaemia (ALL)

Martin Gore; Ray Powles; Anil Lakhani; Sarah Milan; Jennifer Maitland; Glen Goss; Ann Nandi; Timothy Perren; Garry Forgeson; Jennifer Treleaven; Ayed Zuiable; Fulvio Porta

1989-01-01

185

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

PubMed

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

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

2007-08-01

186

Cytosine substituted calix[4]pyrroles: Neutral receptors for 5?-guanosine monophosphate  

PubMed Central

The synthesis and characterization of two cytosine-substituted calix[4]pyrrole conjugates, bearing the appended cytosine attached at either a ?- or meso-pyrrolic position, is described. These systems were tested as nucleotide-selective carriers and as active components of nucleotide-sensing ion-selective electrodes at pH 6.6. Studies of carrier selectivity were made using a Pressman-type model membrane system consisting of an initial pH 6.0 aqueous phase, an intervening dichloromethane barrier containing the calix[4]pyrrole conjugate, and a receiving basic aqueous phase. Good selectivity for the Watson–Crick complementary nucleotide, 5?-guanosine monophosphate (5?-GMP), was seen in the case of the meso-linked conjugate with the relative rates of through-membrane transport being 7.7:4.1:1 for 5?-GMP, 5?-AMP, and 5?-CMP, respectively. By contrast, the ?-substituted conjugate, while showing a selectivity for 5?-GMP that was enhanced relative to unsubstituted calix[4]pyrrole, was found to transport 5?-CMP roughly 4.5 times more quickly than 5?-GMP. Higher selectivities were also found for 5?-CMP when both the ?- and meso-substituted conjugates were incorporated into polyvinyl chloride membranes and tested as ion selective electrodes at pH 6.6, whereas near-equal selectivities were observed for 5?-CMP and 5?-GMP in the case of unsubstituted calix[4]pyrroles. These seemingly disparate results are consistent with a picture wherein the meso-substituted cytosine calix[4]pyrrole conjugate, but not its ?-linked congener, is capable of acting as a ditopic receptor, binding concurrently both the phosphate anion and nucleobase portions of 5?-GMP to the calixpyrrole core and cytosine “tails” of the molecule, respectively, with the effect of this binding being most apparent under the conditions of the transport experiments. PMID:11929967

Sessler, Jonathan L.; Kral, Vladimir; Shishkanova, Tatiana V.; Gale, Philip A.

2002-01-01

187

Murine DNA (Cytosine-5-)-methyltransferase: Steady-State and Substrate Trapping Analyses of the Kinetic Mechanism  

E-print Network

-poly(dI,dC-dI,dC) complex was observed to be competent for catalysis. Product inhibition studies also support a sequential-adenosyl-L-methionine- (AdoMet-)1 dependent DNA (cytosine-5-)-methyltransferase (DCMTase, EC 2.1.1.37). Methyl group transfer localization to DNA replication foci during S-phase (14), metal binding by zinc finger domains, and DNA binding

Reich, Norbert O.

188

Influence of cytosine methylation on ultraviolet-induced cyclobutane pyrimidine dimer formation in genomic DNA  

Microsoft Academic Search

The ultraviolet (UV) component of sunlight is the main cause of skin cancer. More than 50% of all non-melanoma skin cancers and >90% of squamous cell carcinomas in the US carry a sunlight-induced mutation in the p53 tumor suppressor gene. These mutations have a strong tendency to occur at methylated cytosines. Ligation-mediated PCR (LMPCR) was used to compare at nucleotide

Patrick J. Rochette; Sandrine Lacoste; Jean-Philippe Therrien; Nathalie Bastien; Douglas E. Brash; Régen Drouin

2009-01-01

189

Covalent Bond Formation between a DNA-Cytosine Methyltransferase and DNA Containing 5-azacytosine  

Microsoft Academic Search

DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable

Daniel V. Santi; Anne Norment; Charles E. Garrett

1984-01-01

190

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

PubMed

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

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

2014-11-15

191

Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage  

Microsoft Academic Search

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical

Miral Dizdaroglu; Asuman Karakaya; Pawel Jaruga; Geir Slupphaug; Hans E. Krokan

1996-01-01

192

Supramolecular Chemistry And Self-assembly Special Feature: Cytosine substituted calix[4]pyrroles: Neutral receptors for 5'-guanosine monophosphate  

Microsoft Academic Search

The synthesis and characterization of two cytosine-substituted calix[4]pyrrole conjugates, bearing the appended cytosine attached at either a - or meso-pyrrolic position, is described. These systems were tested as nucleotide-selective carriers and as active components of nucleotide-sensing ion-selective electrodes at pH 6.6. Studies of carrier selectivity were made using a Pressman-type model membrane system consisting of an initial pH 6.0 aqueous

Jonathan L. Sessler; Vladimír Král; Tatiana V. Shishkanova; Philip A. Gale

2002-01-01

193

Study of the Hydrogen Bonding in the Adenine-Thymine, Adenine-Cytosine and Guanine-Thymine Base Pairs  

Microsoft Academic Search

The motion of the protons in the hydrogen bonds of the guanine—cytosine base pair has recently been studied by Rein and Harris, using a semiempirical LCAO MO SCF method. These investigations are extended to the adenine—thymine base pair and the ``rare'' base pairs adenine—cytosine and guanine—thymine. The total electronic energy of the ground states of the neutral base pairs and

Sten Lunell; Gunnar Sperber

1967-01-01

194

VUV photoionization of gas phase adenine and cytosine: a comparison between oven and aerosol vaporization.  

PubMed

We studied the single photon ionization of gas phase adenine and cytosine by means of vacuum ultraviolet synchrotron radiation coupled to a velocity map imaging electron?ion coincidence spectrometer. Both in-vacuum temperature-controlled oven and aerosol thermodesorption were successfully applied to promote the intact neutral biological species into the gas phase. The photoion yields are consistent with previous measurements. In addition, we deduced the threshold photoelectron spectra and the slow photoelectron spectra for both species, where the close to zero kinetic energy photoelectrons and the corresponding photoions are measured in coincidence. The photoionization close and above the ionization energies are found to occur mainly via direct processes. Both vaporization techniques lead to similar electronic spectra for the two molecules, which consist of broadbands due to the complex electronic structure of the cationic species and to the possible contribution of several neutral tautomers for cytosine prior to ionization. Accurate ionization energies are measured for adenine and cytosine at, respectively, 8.267 ± 0.005 eV and 8.66 ± 0.01 eV, and we deduce precise thermochemical data for the adenine radical cation. Finally, we performed an evaluation and a comparison of the two vaporization techniques addressing the following criteria: measurement precision, thermal fragmentation, sensitivity, and sample consumption. The aerosol thermodesorption technique appears as a promising alternative to vaporize large thermolabile biological compounds, where extended thermal decomposition or low sensitivity could be encountered when using a simple oven vaporization technique. PMID:23485287

Touboul, D; Gaie-Levrel, F; Garcia, G A; Nahon, L; Poisson, L; Schwell, M; Hochlaf, M

2013-03-01

195

Comprehensive gene expression analysis of the DNA (cytosine-5) methyltransferase family in rice (Oryza sativa L.).  

PubMed

Cytosine DNA methylation is a conserved epigenetic regulatory mechanism in both plants and animals. DNA methyltransferases (DNA MTases) not only initiate (de novo) but also maintain the process of DNA methylation. Here, we characterized the genome-wide expression profiles of 10 cytosine DNA MTase genes belonging to 4 subfamilies, MET1, CMT, DNMT2, and DRM, in rice. Tissue-specific gene expression analysis showed that all family members varied widely in their expression and specificities and might be involved in some basic metabolic pathways. Similarly, the expression of all rice cytosine DNA MTase genes was not regulated by plant hormones except OsDRM1a and OsDRM1b, which were downregulated by jasmonic acid. The transcription level of 10 genes in rice shoots and roots was also measured under salt and osmotic stress. Meanwhile, quantitative polymerase chain reaction data of the japonica and indica rice cultivars revealed that there is large variation in the expression activities of all genes. The results provide a foundation to further explore the roles of DNA MTases and the epigenetic regulation of abiotic stress responses in rice. PMID:25061741

Ahmad, F; Huang, X; Lan, H X; Huma, T; Bao, Y M; Huang, J; Zhang, H S

2014-01-01

196

Mitochondrial assembly in yeast  

Microsoft Academic Search

The yeast Saccharomyces cerevisiae is likely to be the first organism for which a complete inventory of mitochondrial proteins and their functions can be drawn up. A survey of the 340 or so proteins currently known to be localised in yeast mitochondria reveals the considerable investment required to maintain the organelle’s own genetic system, which itself contributes seven key components

Les A Grivell; Marta Artal-Sanz; Gertjan Hakkaart; Liesbeth de Jong; Leo G. J Nijtmans; Katinka van Oosterum; Michel Siep; Hans van der Spek

1999-01-01

197

Yeasts: Neglected Pathogens  

Microsoft Academic Search

Background: Current research on Crohn’s disease (CD) concerns molecular events related to loss of tolerance to microbes that could trigger or maintain inflammation in genetically susceptible individuals. CD is also associated with antimicrobial antibodies, including the antibodies we described against yeast oligomannosides (ASCA). This prompted us to investigate a role for another yeast, Candida albicans, a very common commensal of

Daniel Poulain; Boualem Sendid; Annie Standaert-Vitse; Chantal Fradin; Thierry Jouault; Samir Jawhara; Jean-Frederic Colombel

2009-01-01

198

Prions in Yeast  

PubMed Central

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-? aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

Liebman, Susan W.; Chernoff, Yury O.

2012-01-01

199

Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis[W  

PubMed Central

Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2’-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed. PMID:24130159

Dahncke, Kathleen; Witte, Claus-Peter

2013-01-01

200

Oxidative stress-induced mutagenesis in single-strand DNA occurs primarily at cytosines and is DNA polymerase zeta-dependent only for adenines and guanines  

PubMed Central

Localized hyper-mutability caused by accumulation of lesions in persistent single-stranded (ss) DNA has been recently found in several types of cancers. An increase in endogenous levels of reactive oxygen species (ROS) is considered to be one of the hallmarks of cancers. Employing a yeast model system, we addressed the role of oxidative stress as a potential source of hyper-mutability in ssDNA by modulation of the endogenous ROS levels and by exposing cells to oxidative DNA-damaging agents. We report here that under oxidative stress conditions the majority of base substitution mutations in ssDNA are caused by erroneous, DNA polymerase (Pol) zeta-independent bypass of cytosines, resulting in C to T transitions. For all other DNA bases Pol zeta is essential for ROS-induced mutagenesis. The density of ROS-induced mutations in ssDNA is lower, compared to that caused by UV and MMS, which suggests that ssDNA could be actively protected from oxidative damage. These findings have important implications for understanding mechanisms of oxidative mutagenesis, and could be applied to development of anticancer therapies and cancer prevention. PMID:23925127

Degtyareva, Natalya P.; Heyburn, Lanier; Sterling, Joan; Resnick, Michael A.; Gordenin, Dmitry A.; Doetsch, Paul W.

2013-01-01

201

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala  

Microsoft Academic Search

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the

Panlada Tittabutr; Jonathan D. Awaya; Qing X. Li; Dulal Borthakur

2008-01-01

202

APOBEC3 Cytidine Deaminases in Double-Strand DNA Break Repair and Cancer Promotion  

PubMed Central

High frequency of cytidine to thymidine conversions were identified in the genome of several types of cancer cells. In breast cancer cells these mutations are clustered in long DNA regions associated with ssDNA, double-strand DNA breaks (DSBs) and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs and clustered mutations. PMID:23598277

Nowarski, Roni; Kotler, Moshe

2013-01-01

203

Yeast Proteome Analysis  

NASA Astrophysics Data System (ADS)

Yeast organisms, and specifically Saccharomyces cerevisiae, have become model systems for many aspects in fundamental and applied research. Consistently, many papers have been published applying proteome techniques to study these organisms. The review will give an overview on the proteome research performed on yeast systems so far; however, due to the large number of publications, only selected reports can be cited neglecting many more interesting ones in the interest of space. The review will focus on research involving mass spectrom-etry as a basic proteome technique, although many more approaches are relevant for the functional characterization of proteins in the cell, e.g. the yeast two-hybrid system. We will provide an overview on yeasts as models in the context of pro-teome analysis, and explain the basic techniques currently applied in proteome approaches. The main part of the review will deal with a survey on the current status of proteomic studies in yeasts. In a first part of this chapter, we will deal with the currently available proteome maps of yeasts, and in the following part we will discuss studies dealing with fundamental aspects, but also mention proteome studies related to applied microbiology. Finally, we will envisage future perspectives of the proteome technology for studying yeasts, and draw major conclusion on the current status reached in this field of functional genomics.

Matros, Andrea; Mock, Hans-Peter

204

Methylthioadenosine deaminase in an alternative quorum sensing pathway in Pseudomonas aeruginosa.  

PubMed

Pseudomonas aeruginosa possesses an unusual pathway for 5'-methylthioadenosine (MTA) metabolism involving deamination to 5'-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-?-d-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported [Guan, R., Ho, M. C., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 1247-1254], and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The k(cat)/K(m) value of 1.6 × 10(7) M(-1) s(-1) for MTA is the highest catalytic efficiency known for an MTA deaminase. 5'-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC(50) of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-?-d-ribose are involved in quorum sensing pathways; thus, PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8(R)-hydroxyl group in contact with a catalytic site Zn(2+), the 5'-methylthio group in a hydrophobic pocket, and the transition state mimic of the diazepine ring in contact with a catalytic site Glu. PMID:23050701

Guan, Rong; Ho, Meng-Chiao; Fröhlich, Richard F G; Tyler, Peter C; Almo, Steven C; Schramm, Vern L

2012-11-13

205

Myoadenylate deaminase deficiency: Successful symptomatic therapy by high dose oral administration of ribose  

Microsoft Academic Search

Summary A 55 years old patient suffering from exercise-induced muscle pain and stiffness due to primary myoadenylate deaminase deficiency has been successfully treated with D-ribose since 1984: single doses of 4 grams administered at the beginning of exercise prevented the symptoms completely; on continuation of exercise this dose had to be repeated all 10–30 min. Total doses of 50–60 g

N. Zöllner; S. Reiter; M. Gross; D. Pongratz; C. D. Reimers; K. Gerbitz; I. Paetzke; T. Deufel; G. Hübner

1986-01-01

206

Promotion of plant growth by ACC deaminase-producing soil bacteria  

Microsoft Academic Search

Plant growth-promoting bacteria that contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase facilitate plant\\u000a growth and development by decreasing plant ethylene levels, especially following a variety of environmental stresses. In this\\u000a review, the physiological basis for this growth-promotion effect is examined in some detail. In addition, models are presented\\u000a that endeavour to explain (i) the seemingly paradoxical effects of ethylene on a

Bernard R. Glick; Zhenyu Cheng; Jennifer Czarny; Jin Duan

2007-01-01

207

Specific Activity of Phenylalanine Deaminase in Extracts of the Proteus-Providence Group  

Microsoft Academic Search

THE possession of a phenylalanine deaminase which converts phenylalanine to phenylpyruvic acid is one of a combination of five properties unique for the Proteus-Providence group of organisms1. Of a total of 185 P. hauseri (P. mirabilis + P. vulgaris), 155 P. morganii, 29 P. rettgeri and 239 Providence strains qualitatively examined for the presence of this enzyme2-4, all but two

J. A. Smit

1966-01-01

208

1-Aminocyclopropane-1Carboxylate (ACC) Deaminase Genes in Rhizobia from Southern Saskatchewan  

Microsoft Academic Search

A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate\\u000a (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S\\u000a rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain

Jin Duan; Kirsten M. Müller; Trevor C. Charles; Susanne Vesely; Bernard R. Glick

2009-01-01

209

A novel zinc-binding motif found in two ubiquitous deaminase families.  

PubMed

Two families of deaminases, one specific for cytidine, the other for deoxycytidylate, are shown to possess a novel zinc-binding motif, here designated ZBS. We have (1) identified the protein members of these 2 families, (2) carried out sequence analyses that allow specification of this zinc-binding motif, and (3) determined signature sequences that will allow identification of additional members of these families as their sequences become available. PMID:8061614

Reizer, J; Buskirk, S; Bairoch, A; Reizer, A; Saier, M H

1994-05-01

210

A novel zinc-binding motif found in two ubiquitous deaminase families.  

PubMed Central

Two families of deaminases, one specific for cytidine, the other for deoxycytidylate, are shown to possess a novel zinc-binding motif, here designated ZBS. We have (1) identified the protein members of these 2 families, (2) carried out sequence analyses that allow specification of this zinc-binding motif, and (3) determined signature sequences that will allow identification of additional members of these families as their sequences become available. PMID:8061614

Reizer, J.; Buskirk, S.; Bairoch, A.; Reizer, A.; Saier, M. H.

1994-01-01

211

Partial Characterization of AMP Deaminase from Jumbo Squid (Dosidicus gigas) Mantle  

Microsoft Academic Search

AMP deaminase enzyme showed an optimum temperature at 35°C. It was relatively stable between 30 and 40°C but extremely unstable at higher temperatures. The optimum pH was 6.0 and the enzyme presented an excellent stability with pH between 5.5 and 6.5. The K ion acted as a negative effector when used in concentrations higher than 50 mM. The nucleotide ATP

R. Pacheco-Aguilar; J. M. Ezquerra-Brauer; J. Castillo-Yañez; M. E. Lugo-Sanchez; E. Marquez-Rios

2009-01-01

212

Yeast transcription factors Kevin Struhl  

E-print Network

Yeast transcription factors Kevin Struhl Harvard Medical School, Boston, USA Studies of yeast Transcriptional regulatory mechanisms are fundamentally similar in eukaryotic organisms from yeasts to humans (for reviews of yeast transcription, see [1,2]). Compo- nents of the chromatin template and the basic RNA

213

Yeast ecology of Kombucha fermentation  

Microsoft Academic Search

Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and

Ai Leng Teoh; Gillian Heard; Julian Cox

2004-01-01

214

Quantification of deaminase activity-dependent and -independent restriction of HIV-1 replication mediated by APOBEC3F and APOBEC3G through experimental-mathematical investigation.  

PubMed

APOBEC3F and APOBEC3G cytidine deaminases potently inhibit human immunodeficiency virus type 1 (HIV-1) replication by enzymatically inserting G-to-A mutations in viral DNA and/or impairing viral reverse transcription independently of their deaminase activity. Through experimental and mathematical investigation, here we quantitatively demonstrate that 99.3% of the antiviral effect of APOBEC3G is dependent on its deaminase activity, whereas 30.2% of the antiviral effect of APOBEC3F is attributed to deaminase-independent ability. This is the first report quantitatively elucidating how APOBEC3F and APOBEC3G differ in their anti-HIV-1 modes. PMID:24623435

Kobayashi, Tomoko; Koizumi, Yoshiki; Takeuchi, Junko S; Misawa, Naoko; Kimura, Yuichi; Morita, Satoru; Aihara, Kazuyuki; Koyanagi, Yoshio; Iwami, Shingo; Sato, Kei

2014-05-01

215

Quantification of Deaminase Activity-Dependent and -Independent Restriction of HIV-1 Replication Mediated by APOBEC3F and APOBEC3G through Experimental-Mathematical Investigation  

PubMed Central

APOBEC3F and APOBEC3G cytidine deaminases potently inhibit human immunodeficiency virus type 1 (HIV-1) replication by enzymatically inserting G-to-A mutations in viral DNA and/or impairing viral reverse transcription independently of their deaminase activity. Through experimental and mathematical investigation, here we quantitatively demonstrate that 99.3% of the antiviral effect of APOBEC3G is dependent on its deaminase activity, whereas 30.2% of the antiviral effect of APOBEC3F is attributed to deaminase-independent ability. This is the first report quantitatively elucidating how APOBEC3F and APOBEC3G differ in their anti-HIV-1 modes. PMID:24623435

Kobayashi, Tomoko; Koizumi, Yoshiki; Takeuchi, Junko S.; Misawa, Naoko; Kimura, Yuichi; Morita, Satoru; Aihara, Kazuyuki; Koyanagi, Yoshio

2014-01-01

216

Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.  

PubMed

Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants. PMID:16023358

Stearns, Jennifer C; Shah, Saleh; Greenberg, Bruce M; Dixon, D George; Glick, Bernard R

2005-07-01

217

Beyond SHM and CSR: AID and related cytidine deaminases in the host response to viral infection.  

PubMed

As the primary effector of immunoglobulin somatic hypermutation (SHM) and class switch recombination (CSR), activation-induced cytidine deaminase (AID) serves an important function in the adaptive immune response. Recent advances have demonstrated that AID and a group of closely related cytidine deaminases, the APOBEC3 proteins, also act in the innate host response to viral infection. Antiviral activity was first attributed to APOBEC3G as a potent inhibitor of HIV. It is now apparent that the targets of the APOBEC3 proteins extend beyond HIV, with family members acting against a wide variety of viruses as well as host-encoded retrotransposable genetic elements. Although it appears to function through a different mechanism, AID also possesses antiviral properties. Independent of its antibody diversification functions, AID protects against transformation by Abelson murine leukemia virus (Ab-MLV), an oncogenic retrovirus. Additionally, AID has been implicated in the host response to other pathogenic viruses. These emerging roles for the AID/APOBEC cytidine deaminases in viral infection suggest an intriguing evolutionary connection of innate and adaptive immune mechanisms. PMID:17560276

Rosenberg, Brad R; Papavasiliou, F Nina

2007-01-01

218

Genome-wide identification of cytosine-5 DNA methyltransferases and demethylases in Solanum lycopersicum.  

PubMed

Recent studies have reported that decreased level of DNA cytosine methylation in the global genome was closely related to the initiation of tomato (Solanum lycopersicum) fruit ripening. However, genome-scale analysis of cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in tomato has not been engaged. In this study, 7 C5-MTases and 3 demethylases were identified in tomato genome, which probably contributed to DNA cytosine methylation level in tomato. The 7 C5-MTases were categorized into 4 subgroups, and the 3 demethylases were classified into 2 subgroups based on phylogenetic analyses. Comprehensive analysis of their structure and genomic localization was also performed in this paper. According to online RNA-seq data, 4 S. lycopersicum C5-MTase (SlC5-MTase) genes (SlMET, SlDRM1L1, SlDRM5, SlMET3L) were expressed higher than others, and one DNA demethylase gene (SlDML) was significantly changed during tomato fruit development and ripening. Furthermore, all these five gene expressions at breaker (BK) stage changed with 1-methylcyclopropene (1-MCP) treatment, indicating that they were regulated by ethylene directly or indirectly in tomato fruit. In addition, subcellular localization analysis indicated that SlDRM1L1 and SlDRM5 located in the nucleus might have responsibility for RNA-directed DNA methylation (RdDM). Collectively, this paper provided a framework for gene discovery and functional characterization of C5-MTases and DNA demethylases in other Solanaceae species. PMID:25149677

Cao, Dongyan; Ju, Zheng; Gao, Chao; Mei, Xiaohong; Fu, Daqi; Zhu, Hongliang; Luo, Yunbo; Zhu, Benzhong

2014-10-25

219

DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition.  

PubMed

We followed the cytosine C(5) exchange reaction with Dnmt1 to characterize its preference for different DNA substrates, its allosteric regulation, and to provide a basis for comparison with the bacterial enzymes. We determined that the methyl transfer is rate-limiting, and steps up to and including the cysteine-cytosine covalent intermediate are in rapid equilibrium. Changes in these rapid equilibrium steps account for many of the previously described features of Dnmt1 catalysis and specificity including faster reactions with premethylated DNA versus unmethylated DNA, faster reactions with DNA in which guanine is replaced with inosine [poly(dC-dG) vs poly(dI-dC)], and 10-100-fold slower catalytic rates with Dnmt1 relative to the bacterial enzyme M.HhaI. Dnmt1 interactions with the guanine within the CpG recognition site can prevent the premature release of the target base and solvent access to the active site that could lead to mutagenic deamination. Our results suggest that the beta-elimination step following methyl transfer is not mediated by free solvent. Dnmt1 shows a kinetic lag in product formation and allosteric inhibition with unmethylated DNA that is not observed with premethylated DNA. Thus, we suggest the enzyme undergoes a slow relief from allosteric inhibition upon initiation of catalysis on unmethylated DNA. Notably, this relief from allosteric inhibition is not caused by self-activation through the initial methylation reaction, as the same effect is observed during the cytosine C(5) exchange reaction in the absence of AdoMet. We describe limitations in the Michaelis-Menten kinetic analysis of Dnmt1 and suggest alternative approaches. PMID:15996102

Svedruzi?, Zeljko M; Reich, Norbert O

2005-07-12

220

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

NASA Astrophysics Data System (ADS)

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).

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

2013-03-01

221

The Application of High-Level Iterative Coupled-Cluster Methods to the Cytosine Molecule  

SciTech Connect

The need for inclusion higher-order correlation effects for adequate description of the excitation energies of the DNA bases became clear in the last few years. In particular, we demonstrated that there is a sizable effect of triply excited configurations estimated in a non-iterative manner on the coupled-cluster excitation energies of the cytosine molecule in DNA environment. In this paper we discuss the accuracies of the non-iterative methods for biologically relevant systems in realistic environment in comparison with interative formulations that explicitly include the effect of triply excited clusters.

Kowalski, Karol; Valiev, Marat

2008-06-19

222

Supramolecular hydrogen-bonding networks in cytosine salicylic acid hydrate (2 : 3 : 2) complex  

NASA Astrophysics Data System (ADS)

Cytosine-cytosinium base pairs are interconnected by triple hydrogen bonds thereby resembling a pseudo-Watson-Crick pattern and generates two characteristic R {2/2}(8)-motifs. Both molecules of the salicylic acids interconnect the base pair and lead to the formation of one dimensional supramolecular hexameric tape along b-axis. This hexameric tape are sandwiched by the water molecules, one of the salicylic acid and salicylate anion which form one dimensional and two dimensional supramolecular hydrogen bonded networks in the crystal packing. Macrocylic rings of cavities are also noticed in the crystal structure.

Sridhar, B.; Ravikumar, K.

2010-03-01

223

RNAi in Budding Yeast  

E-print Network

RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Saccharomyces cerevisiae. Here, we show that RNAi ...

Drinnenberg, Ines A.

224

RNAi in budding yeast.  

PubMed

RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' messenger RNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a previously unknown class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi. PMID:19745116

Drinnenberg, Ines A; Weinberg, David E; Xie, Kathleen T; Mower, Jeffrey P; Wolfe, Kenneth H; Fink, Gerald R; Bartel, David P

2009-10-23

225

[Penicillium-inhibiting yeasts].  

PubMed

The objective of this work was to establish the in vitro and in vivo inhibition of post-harvest pathogenic moulds by yeasts in order to make a biocontrol product. Post-harvest pathogenic moulds Penicillium digitatum, P. italicum, P. ulaiense, Phyllosticta sp., Galactomyces geotrichum and yeasts belonging to genera Brettanomyces, Candida, Cryptococcus, Kloeckera, Pichia, Rhodotorula were isolated from citrus fruits. Some yeasts strains were also isolated from other sources. The yeasts were identified by their macro and micro-morphology and physiological tests. The in vitro and in vivo activities against P. digitatum or P. ulaiense were different. Candida cantarellii and one strain of Pichia subpelliculosa produced a significant reduction of the lesion area caused by the pathogenic moulds P. digitatum and P. ulaiense, and could be used in a biocontrol product formulation. PMID:15786872

Benítez Ahrendts, M R; Carrillo, L

2004-01-01

226

Nitrile Metabolizing Yeasts  

NASA Astrophysics Data System (ADS)

Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing enzymes of yeasts.

Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

227

Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study  

PubMed Central

Using 13C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C–H vectors in all cytosine ribose moieties within the Dickerson–Drew dodecamer (5?-CGCGAATTCGCG-3?) are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase. PMID:18579564

Duchardt, Elke; Nilsson, Lennart

2008-01-01

228

Base Flip in DNA Studied by Molecular Dynamics Simulations of Differently-Oxidized Forms of Methyl-Cytosine  

PubMed Central

Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme’s active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition. PMID:24995694

Helabad, Mahdi Bagherpoor; Kanaan, Natalia; Imhof, Petra

2014-01-01

229

Tautomerism in cytosine and uracil: a theoretical and experimental X-ray absorption and resonant auger study.  

PubMed

The core level photoabsorption spectra of the nucleobases cytosine and uracil in the gas phase have been measured and the results interpreted with theoretical calculations using an ab initio Green’s function approach. A single tautomer of uracil is populated, in agreement with previous work, while three tautomers of cytosine are clearly identified, whose identity and relative populations at the temperature of the experiment were reported previously. The second-order ADC approach to polarization propagator was employed in calculations of X-ray photoabsorption energies and intensities. The theoretical spectra have been constructed as Boltzmann-factor-weighted sums of individual tautomer spectra. These theoretical spectra are in good agreement with the experimental photoabsorption results at the oxygen, nitrogen, and carbon edges. In addition we report resonant Auger spectra of the valence band of cytosine, which support previous assignments of the character of the valence band states. PMID:20795686

Feyer, Vitaliy; Plekan, Oksana; Richter, Robert; Coreno, Marcello; de Simone, Monica; Prince, Kevin C; Trofimov, Alexander B; Zaytseva, Irina L; Schirmer, Jochen

2010-09-23

230

Base flip in DNA studied by molecular dynamics simulationsof differently-oxidized forms of methyl-Cytosine.  

PubMed

Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme's active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition. PMID:24995694

Helabad, Mahdi Bagherpoor; Kanaan, Natalia; Imhof, Petra

2014-01-01

231

Methylation of DNA in early development: 5-methyl cytosine content of DNA in sea urchin sperm and embryos  

PubMed Central

By separating formic acid hydrolysates with high pressure chromatography on an Aminex-10 column, we determined the ratio of 5-methyl cytosine to cytosine and other bases of DNA from sea urchin sperm and nuclei of embryos from early cleavage through pluteus stages. Contrary to several previous reports, we could not find any measurable changes in the methylation levels of embryonic nuclear DNAs at different stages of development. We also found no consistent differences between the methylation levels of sea urchin sperm and embryonic nuclei or the 5-methyl cytosine content of fish (Mugilcephalus) sperm and liver nuclei. While these measurements would not have detected subtle variations associated with differentiation, they would have indicated the gross changes previously reported for embryos or between sperm and somatic nuclei had those changes been present. PMID:745996

Pollock, James M.; Swihart, Marcia; Taylor, J.Herbert

1978-01-01

232

Electron impact fragmentation of cytosine: partial ionization cross sections for positive fragments  

NASA Astrophysics Data System (ADS)

We have measured mass spectra for positive ions produced by low-energy electron impact on cytosine using a reflectron time-of-flight mass spectrometer. The electron impact energy has been varied from 0 to 100 eV in steps of 0.5 eV. Ion yield curves of most of the fragment ions have been determined by fitting groups of adjacent peaks in the mass spectra with sequences of normalized Gaussians. The ion yield curves have been normalized by comparing the sum of the ion yields to the average of calculated total ionization cross sections. Appearance energies of the fragment ions have been determined, showing that the fragments 68 u-84 u have appearance energies between 10 and 11 eV, whereas fragments of 55 u and lower mass all have appearance energies above 12 eV. Most of the ion yields of 55 u and smaller show multiple onsets. Several groups of fragments have ion yield curves with nearly the same shape, clearly indicating the relevance of tautomerization in the fragmentation of cytosine.

van der Burgt, Peter J. M.

2014-05-01

233

Solid phase synthesis and restriction endonuclease cleavage of oligodeoxynucleotides containing 5-(hydroxymethyl)-cytosine.  

PubMed Central

Emerging data suggest an important role for cytosine methylation in tumorigenesis. Simultaneously, recent studies indicate a significant contribution of endogenous oxidative DNA damage to the development of human disease. Oxidation of the 5-methyl group of 5-methylcytosine (5mC) residues in DNA results in the formation of 5-(hydroxymethyl)cytosine (hmC). The biological consequences ofhmC residues in vertebrate DNA are as yet unknown; however, conversion of the hydrophobic methyl group to the hydrophilic hydroxymethyl group may substantially alter the interaction of sequence-specific binding proteins with DNA. Central to both biophysical and biochemical studies on the potential consequences of specific DNA damage products such as hmC are efficient methods for the synthesis of oligodeoxynucleotides containing such modified bases at selected positions. In this paper, we describe a method for the placement of hmC residues in oligodeoxynucleotides using established phosphoramidite chemistry. In addition, we have examined the influence of specific hmC residues on enzymatic cleavage of oligodeoxynucleotides by the methylation-sensitive restriction endonucleases MspI and HpaII. PMID:9016595

Tardy-Planechaud, S; Fujimoto, J; Lin, S S; Sowers, L C

1997-01-01

234

Endogenous cytosine methylation and the formation of carcinogen carcinogen-DNA adducts.  

PubMed

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine ((Me)C, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O(6)-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of (Me)C were prepared, and their effects on reactivity of base the paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N(2)-BPDE-dG adducts. PMID:18776247

Tretyakova, Natalia; Guza, Rebecca; Matter, Brock

2008-01-01

235

Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate alfalfa.  

PubMed

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads. PMID:15466529

Ma, Wenbo; Charles, Trevor C; Glick, Bernard R

2004-10-01

236

Determination of Serum Adenosine Deaminase and Xanthine Oxidase Levels in Patients with Crimean-Congo Hemorrhagic Fever  

PubMed Central

OBJECTIVE: Crimean–Congo hemorrhagic fever is an acute viral hemorrhagic fever with a high mortality rate. Despite increasing knowledge about hemorrhagic fever viruses, little is known about the pathogenesis of Crimean–Congo hemorrhagic fever. In this study, we measured serum adenosine deaminase and xanthine oxidase levels in Crimean–Congo hemorrhagic fever patients. METHODS: Serum adenosine deaminase levels were measured with a sensitive colorimetric method described by Giusti and xanthine oxidase levels by the method of Worthington in 30 consecutive hospitalized patients (mean age 42.6 ± 21.0). Laboratory tests confirmed their diagnoses of Crimean–Congo hemorrhagic fever. Thirty-five subjects (mean age 42.9 ± 19.1) served as the control group. RESULTS: There was a significant difference in adenosine deaminase and xanthine oxidase levels between cases and controls (p<0.05). However, neither adenosine deaminase nor xanthine oxidase levels varied with the severity of disease in the cases assessed (p>0.05). CONCLUSION: Adenosine deaminase and xanthine oxidase levels were increased in patients with Crimean–Congo hemorrhagic fever. Elevated serum xanthine oxidase activity in patients with Crimean–Congo hemorrhagic fever may be associated with reactive oxygen species generated by the xanthine/xanthine oxidase system during inflammatory responses. In addition, elevated lipid peroxidation may contribute to cell damage and hemorrhage. The association of cell damage and hemorrhage with xanthine oxidase activity should be further investigated in large-scale studies. PMID:20668627

Celik, V. Kenan; Sari, Ismail; Engin, Aynur; Yildiz, Gursel; Aydin, Huseyin; Bakir, Sevtap

2010-01-01

237

Mapping Yeast Transcriptional Networks  

PubMed Central

The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms. PMID:24018767

Hughes, Timothy R.; de Boer, Carl G.

2013-01-01

238

Oxygen requirements of yeasts.  

PubMed Central

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth. Images PMID:2082825

Visser, W; Scheffers, W A; Batenburg-van der Vegte, W H; van Dijken, J P

1990-01-01

239

Yeast killer systems.  

PubMed Central

The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed. PMID:9227858

Magliani, W; Conti, S; Gerloni, M; Bertolotti, D; Polonelli, L

1997-01-01

240

Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.  

PubMed

Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ?dsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ?dsdA mutant was attenuated in virulence murine model of urinary tract infection. PMID:24082071

Korte-Berwanger, Miriam; Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sören G

2013-12-01

241

Significance of the d-Serine-Deaminase and d-Serine Metabolism of Staphylococcus saprophyticus for Virulence  

PubMed Central

Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a d-serine-deaminase (DsdA). As d-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the d-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that d-serine-deaminase or the ability to respond to or to metabolize d-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular d-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high d-serine concentrations; however, its d-serine metabolism has not been described. The activity of the d-serine-deaminase was verified by analyzing the formation of pyruvate from d-serine in different strains with and without d-serine-deaminase. Cocultivation experiments were performed to show that d-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of d-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ?dsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of d-serine. In addition, we show that S. saprophyticus is able to use d-serine as the sole carbon source, but interestingly, d-serine had a negative effect on growth when glucose was also present. Taken together, d-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of d-serine and a ?dsdA mutant was attenuated in virulence murine model of urinary tract infection. PMID:24082071

Sakinc, Turkan; Kline, Kimberly; Nielsen, Hailyn V.; Hultgren, Scott; Gatermann, Soren G.

2013-01-01

242

L-arabinose fermenting yeast  

DOEpatents

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

Zhang, Min (Lakewood, CO); Singh, Arjun (Lakewood, CO); Knoshaug, Eric (Golden, CO); Franden, Mary Ann (Centennial, CO); Jarvis, Eric (Boulder, CO); Suominen, Pirkko (Maple Grove, MN)

2010-12-07

243

Genotype is an important determinant of phenotype in adenosine deaminase deficiency.  

PubMed

Adenosine deaminase (ADA) deficiency is associated with a broad clinical and mutational spectrum. Defining the relationship of genotype to phenotype among patients with different degrees of immunodeficiency has been complicated because the disease is rare, most mutations are 'private' and patients are often heteroallelic. In recent years, knowledge of ADA structure and systematic expression of mutant alleles have revealed that phenotype is strongly associated with the sum of ADA activity provided by both alleles. A scale for ranking novel ADA alleles based on expression may have utility if newborn screening for primary immunodeficiency disorders is initiated. PMID:14499267

Hershfield, Michael S

2003-10-01

244

Mitochondrial assembly in yeast.  

PubMed

The yeast Saccharomyces cerevisiae is likely to be the first organism for which a complete inventory of mitochondrial proteins and their functions can be drawn up. A survey of the 340 or so proteins currently known to be localised in yeast mitochondria reveals the considerable investment required to maintain the organelle's own genetic system, which itself contributes seven key components of the electron transport chain. Translation and respiratory complex assembly are particularly expensive processes, together requiring around 150 of the proteins so far known. Recent developments in both areas are reviewed and approaches to the identification of novel mitochondrial proteins are discussed. PMID:10376678

Grivell, L A; Artal-Sanz, M; Hakkaart, G; de Jong, L; Nijtmans, L G; van Oosterum, K; Siep, M; van der Spek, H

1999-06-01

245

Energetics of the lattice: packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules  

NASA Technical Reports Server (NTRS)

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin.

Berger, I.; Cai, L.; Chen, L.; Rich, A.

1997-01-01

246

Intermolecular Proton Transfer in Microhydrated Guanine-Cytosine Base Pairs: a New Mechanism for Spontaneous Mutation in DNA  

Microsoft Academic Search

Accurate calculations of the double proton transfer (DPT) in the adenine-thymine base pair (AT) were presented in a previous work [ J. Phys. Chem. A 2009, 113, 7892. ] where we demonstrated that the mechanism of the reaction in solution is strongly affected by surrounding water. Here we extend our methodology to the guanine-cytosine base pair (GC), for which it

J. P. Cerón-Carrasco; A. Requena; J. Zúñiga; C. Michaux; E. A. Perpête; D. Jacquemin

2009-01-01

247

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

Microsoft Academic Search

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

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

2009-01-01

248

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

PubMed

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 polycrystalline 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) cm(2) 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 NH(2) 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) cm(2), 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) cm(2) 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 are ascribed to shape resonances associated with the electron occupation of the second and third antibonding ?-orbitals of the molecule in its ground state, the correspondence with DEA features suggests the existence of common precursor anion states decaying with certain probabilities into the vibrationally excited ground state. PMID:22998289

Michaud, M; Bazin, M; Sanche, L

2012-09-21

249

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

PubMed Central

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

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

2013-01-01

250

Proton-coupled hole transfer in X-irradiated doped crystalline cytosine.H2O.  

PubMed

Following exposure to X-irradiation at low temperatures, the main reactions taking place in single crystals of cytosine monohydrate doped with minute amounts of 2-thiocytosine are hole transfer (HT) from the electron-loss centers to the dopant and recombination of oxidation and reduction products, assumedly by electron transfer. A huge deuterium kinetic isotope effect (KIE; >102-103) at 100 K, together with the kinetic curves obtained and density functional theory (DFT) calculations of equilibrium energy changes, indicates that these reactions proceed through a concerted proton-coupled electron/hole transfer where the proton transfer occurs between hydrogen-bonded cytosine molecules. The temperature dependence of these reaction rates between 10 and 150 K in normal and partially deuterated samples was investigated by monitoring the growth and decay of the various radical species over time using electron paramagnetic resonance (EPR) spectroscopy. By assuming a random distribution of the hole donors and acceptors in the crystals, the data are consistent with an exponential distance-dependent rate, giving a distance decay constant (beta) around 1 A-1 for the HT, which indicates that a long-range single-step superexchange mechanism mediates the charge transfer. The reactions undergo a transition from a slow, weakly temperature-dependent rate to an Arrhenius-type rate at 40-50 K, presumably being activated by excitation of low-frequency intermolecular vibrations that couple to the process. Below this transition temperature, the transfer probability might be dominated by temperature-independent nuclear tunneling. A similar beta value in both temperature regions suggests that hopping is not activated. PMID:18341308

Krivokapi?, André; Herak, Janko N; Sagstuen, Einar

2008-04-24

251

Nitrosative Cytosine Deamination. An Exploration of the Chemistry Emanating from Deamination with Pyrimidine Ring-Opening  

PubMed Central

A discussion of nitrosative deamination of cytosine 1 is presented that argues for the formation of 6 by diazotization of 1 to cytosinediazonium ion 2 and its electrostatic complex 3, dediazoniation to 4 ? 5, and amide-bond cleavage to 6. The reaction channels available to 6 include hydrolytic deglycation to 3-isocyanatoacrylonitrile 7, water addition to carbamic acid 9 with the possibility for re-closure to uracil 13, and water addition to carbamic acid 9 and decarboxylation to 3-aminoacrylonitrile 10. With a view to the instability of the carbamic acid 9, the carbamate models ethyl (Z)-2-cyanovinyl-carbamate 14 and (Z)-2-cyano-1-t-butylvinylcarbamate 20 were studied. Acid-catalyzed hydrolysis of 14 leads to 2-amino-carbonylphenylcarbamate 15 and its cyclization yields the benzo-fused uracil quinazoline-2,4-dione 16. In contrast to the aromatic system 14, acid-catalyzed cyclization cannot compete with oligomerization in the case of 20 and 5-tert-butyluracil 22 is accessible only with base-catalysis. It is shown that 23, the parent of 10, also easily polymerizes. The experimental results provide a rational as to why 9, 10 and 12 would have escaped detection in in vitro studies: they would have oligomerized. In contrast to the in vitro experiments, the oligomerizations of 9, 10 or 12 clearly are not relevant in vivo because of low monomer concentrations. With the exclusion of recyclization and of oligomerization in vivo, attention thus needs to focus on (Z)-3-aminoacrylonitrile 10 as the most likely deamination product of cytosine aside from uracil. PMID:16097794

Rayat, Sundeep; Qian, Ming; Glaser, Rainer

2008-01-01

252

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

PubMed Central

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

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

2011-01-01

253

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair  

PubMed Central

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy. PMID:22645179

Nowarski, Roni; Wilner, Ofer I.; Cheshin, Ori; Shahar, Or D.; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S.; Goldberg, Michal; Willner, Itamar

2012-01-01

254

The Role of Cytidine Deaminases on Innate Immune Responses against Human Viral Infections  

PubMed Central

The APOBEC family of proteins comprises deaminase enzymes that edit DNA and/or RNA sequences. The APOBEC3 subgroup plays an important role on the innate immune system, acting on host defense against exogenous viruses and endogenous retroelements. The role of APOBEC3 proteins in the inhibition of viral infection was firstly described for HIV-1. However, in the past few years many studies have also shown evidence of APOBEC3 action on other viruses associated with human diseases, including HTLV, HCV, HBV, HPV, HSV-1, and EBV. APOBEC3 inhibits these viruses through a series of editing-dependent and independent mechanisms. Many viruses have evolved mechanisms to counteract APOBEC effects, and strategies that enhance APOBEC3 activity constitute a new approach for antiviral drug development. On the other hand, novel evidence that editing by APOBEC3 constitutes a source for viral genetic diversification and evolution has emerged. Furthermore, a possible role in cancer development has been shown for these host enzymes. Therefore, understanding the role of deaminases on the immune response against infectious agents, as well as their role in human disease, has become pivotal. This review summarizes the state-of-the-art knowledge of the impact of APOBEC enzymes on human viruses of distinct families and harboring disparate replication strategies. PMID:23865062

Vieira, Valdimara C.; Soares, Marcelo A.

2013-01-01

255

Ab Initio ONIOM-Molecular Dynamics (MD) Study on the Deamination Reaction by Cytidine Deaminase  

SciTech Connect

We applied the ONIOM-molecular dynamics (MD) method to the hydrolytic deamination of cytidine by cytidine deaminase, which is an essential step of the activation process of the anticancer drug inside the human body. The direct MD simulations were performed for the realistic model of cytidine deaminase calculating the energy and its gradient by the ab initio ONIOM method on the fly. The ONIOM-MD calculations including the thermal motion show that the neighboring amino acid residue is an important factor of the environmental effects and significantly affects not only the geometry and energy of the substrate trapped in the pocket of the active site but also the elementary step of the catalytic reaction. We successfully simulate the second half of the catalytic cycle, which has been considered to involve the rate-determining step, and reveal that the rate-determing step is the release of the NH3 molecule. TM and MA were supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2007-08-23

256

128 Cure of HBV infection: can APOBEC3 deaminases mediate it?  

PubMed Central

Current antivirals cannot efficiently control but not eliminate hepatitis B virus (HBV) in the 300 million carriers at risk to develop liver diseases and cancer, because HBV establishes a stable nuclear cccDNA in infected hepatocytes. Interferon can clear HBV but therapeutic dosing is limited by side effects. We found a mechanism by which nuclear viral DNA can specifically be degraded. In HBV-infected cells and primary human hepatocytes, human liver-needle biopsies and in vivo mouse models, interferon and even at much lower doses lymphotoxin—receptor activation up-regulated nuclear APOBEC3 family deaminases. Cytidine-deamination resulted in apurinic/apyrimidinic site formation and finally cccDNA degradation. No deamination was detected in genomic DNA since HBV-core protein targeted the APOBEC3 protein to cccDNA. Inducing nuclear APOBEC3 deaminases eg, by triggering the lymphotoxin—receptor pathway will be of high interest for the development of new therapeutics. The combination with existing antivirals shows great promise to eliminate the virus in chronic hepatitis B.

Protzer, Ulrike

2014-01-01

257

ACC deaminase and IAA producing growth promoting bacteria from the rhizosphere soil of tropical rice plants.  

PubMed

Beneficial plant-associated bacteria play a key role in supporting and/or promoting plant growth and health. Plant growth promoting bacteria present in the rhizosphere of crop plants can directly affect plant metabolism or modulate phytohormone production or degradation. We isolated 355 bacteria from the rhizosphere of rice plants grown in the farmers' fields in the coastal rice field soil from five different locations of the Ganjam district of Odisha, India. Six bacteria producing both ACC deaminase (ranging from 603.94 to 1350.02?nmol ?-ketobutyrate mg(-1) ?h(-1) ) and indole acetic acid (IAA; ranging from 10.54 to 37.65??M?ml(-1) ) in pure cultures were further identified using polyphasic taxonomy including BIOLOG((R)) , FAME analysis and the 16S rRNA gene sequencing. Phylogenetic analyses of the isolates resulted into five major clusters to include members of the genera Bacillus, Microbacterium, Methylophaga, Agromyces, and Paenibacillus. Seed inoculation of rice (cv. Naveen) by the six individual PGPR isolates had a considerable impact on different growth parameters including root elongation that was positively correlated with ACC deaminase activity and IAA production. The cultures also had other plant growth attributes including ammonia production and at least two isolates produced siderophores. Study indicates that presence of diverse rhizobacteria with effective growth-promoting traits, in the rice rhizosphere, may be exploited for a sustainable crop management under field conditions. PMID:23681643

Bal, Himadri Bhusan; Das, Subhasis; Dangar, Tushar K; Adhya, Tapan K

2013-12-01

258

Rapid degradation of Pseudomonas fluorescens 1-aminocyclopropane-1-carboxylic acid deaminase proteins expressed in transgenic Arabidopsis.  

PubMed

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is commonly produced by plant growth-promoting rhizobacteria (PGPR) and has been suggested to facilitate the growth and stress tolerance of hosts via a reduction in levels of ethylene. However, the regulatory mechanism of ACC deaminase (AcdS) protein within host plant cells is largely unknown. Here, we demonstrated beneficial effects and post-translational modification of PGPR-originated AcdS proteins in plants. Compared with the wild-type, transgenic Arabidopsis expressing the Pseudomonas fluorescens acdS (PfacdS) gene displayed increased root elongation and reduced sensitivity to 10 ?M exogenous ACC, an ethylene precursor. Arabidopsis expressing PfacdS also showed increased tolerance to high salinity (150 mM NaCl). PfAcdS proteins accumulated in transgenic Arabidopsis were rapidly degraded, which was potentially mediated by the 26S proteasome pathway. The degradation of PfAcdS was alleviated in the presence of exogenous ACC. In conclusion, our data suggest that the plant growth-promoting effects of bacterial AcdS proteins are potentially modulated via protein turnover inside the host plant cells. Such post-translational modification plays a physiological role in the mutualistic interactions between microorganisms and plants in the rhizospheric and/or endospheric niche. PMID:24801274

Kim, Kangmin; Park, Sung-Hee; Chae, Jong-Chan; Soh, Byoung Yul; Lee, Kui-Jae

2014-06-01

259

Non-viral delivery of the porphobilinogen deaminase cDNA into a mouse model of acute intermittent porphyria  

Microsoft Academic Search

Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deficient activity of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD). Clinical symptoms of this autosomal dominant hepatic porphyria include episodic acute attacks of abdominal pain, neuropathy, and psychiatric disturbances. Current therapy based on intravenous heme administration is palliative and there is no way to

Annika Johansson; Grzegorz Nowak; Christer Möller; Pauline Harper

2004-01-01

260

Elongation of Shoot and Root in Wheat by ACC Deaminase of Rhizobium Spp. Indigenous to Soils of Iran  

Microsoft Academic Search

This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium strains indigenous to Iranian soils and their ability to reduce stress ethylene and promote the elongation of the roots of wheat seedlings. One hundred different rhizobial strains isolated from some Iranian soils were cultured on Rhizobial Minimal Medium (RMM) with two different sources of nitrogen (ACC

ATEFEH RAMEZANIAN BAJGIRAN; AMIR LAKZIAN; NAHID SALEH-RASTIN

261

Silencing Threonine Deaminase and JAR4 in Nicotiana attenuata Impairs Jasmonic Acid-Isoleucine-Mediated Defenses against Manduca sexta  

Microsoft Academic Search

Threonine deaminase (TD) catalyzes the conversion of Thr to a-keto butyrate in Ile biosynthesis; however, its dramatic upregulation in leaves after herbivore attack suggests a role in defense. In Nicotiana attenuata, strongly silenced TD transgenic plants were stunted, whereas mildly silenced TD transgenic plants had normal growth but were highly susceptible to Manduca sexta attack. The herbivore susceptibility was associated

Jin-Ho Kang; Lei Wang; Ashok Giri; Ian T. Baldwin

2006-01-01

262

Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase  

SciTech Connect

The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. (Texas A M Univ., College Station (USA))

1988-10-18

263

5-methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni  

PubMed Central

Background Biomphalaria glabrata is the mollusc intermediate host for Schistosoma mansoni, a digenean flatworm parasite that causes human intestinal schistosomiasis. An estimated 200 million people in 74 countries suffer from schistosomiasis, in terms of morbidity this is the most severe tropical disease after malaria. Epigenetic information informs on the status of gene activity that is heritable, for which changes are reversible and that is not based on the DNA sequence. Epigenetic mechanisms generate variability that provides a source for potentially heritable phenotypic variation and therefore could be involved in the adaptation to environmental constraint. Phenotypic variations are particularly important in host-parasite interactions in which both selective pressure and rate of evolution are high. In this context, epigenetic changes are expected to be major drivers of phenotypic plasticity and co-adaptation between host and parasite. Consequently, with characterization of the genomes of invertebrates that are parasite vectors or intermediate hosts, it is also essential to understand how the epigenetic machinery functions to better decipher the interplay between host and parasite. Methods The CpGo/e ratios were used as a proxy to investigate the occurrence of CpG methylation in B. glabrata coding regions. The presence of DNA methylation in B. glabrata was also confirmed by several experimental approaches: restriction enzymatic digestion with isoschizomers, bisulfite conversion based techniques and LC-MS/MS analysis. Results In this work, we report that DNA methylation, which is one of the carriers of epigenetic information, occurs in B. glabrata; approximately 2% of cytosine nucleotides are methylated. We describe the methylation machinery of B. glabrata. Methylation occurs predominantly at CpG sites, present at high ratios in coding regions of genes associated with housekeeping functions. We also demonstrate by bisulfite treatment that methylation occurs in multiple copies of Nimbus, a transposable element. Conclusions This study details DNA methylation for the first time, one of the carriers of epigenetic information in B. glabrata. The general characteristics of DNA methylation that we observed in the B. glabrata genome conform to what epigenetic studies have reported from other invertebrate species. PMID:23742053

2013-01-01

264

Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi  

NASA Astrophysics Data System (ADS)

Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

265

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

NASA Astrophysics Data System (ADS)

The absolute cross sections (CSs) 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'(?-->?*) and 2 3A'(?-->?*) valence states of the molecule. Their energy dependent CSs 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'(?-->?*) 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'(?-->?*), 4 1A'(?-->?*), 5 1A'(?-->?*), and 6 1A'(?-->?*) valence states, respectively. The CSs 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-->?*) 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 the four highest occupied molecular orbitals of cytosine. The sum of the ionization CS for these four excitation regions reaches a maximum of 8.1×10-16 cm2 at the incident energy of 13 eV.

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

2010-10-01

266

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

PubMed Central

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?(???*) and 2 3A?(???*) 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?(???*) 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?(???*), 4 1A?(???*), 5 1A?(???*), and 6 1A?(???*) 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??*) 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 the four highest occupied molecular orbitals of cytosine. The sum of the ionization CS for these four excitation regions reaches a maximum of 8.1 × 10?16 cm2 at the incident energy of 13 eV. PMID:20969430

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

2013-01-01

267

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

PubMed

The absolute cross sections (CSs) 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?(3)A(')(???(?)) and 2?(3)A(')(???(?)) valence states of the molecule. Their energy dependent CSs exhibit essentially a common maximum at about 6 eV with a value of 1.84×10(-17)?cm(2) for the former and 4.94×10(-17)?cm(2) 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?(1)A(')(???(?)) valence state, shows only a steep rise to about 1.04×10(-16)?cm(2) 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?(3,1)A(')(???(?)), 4?(1)A(')(???(?)), 5?(1)A(')(???(?)), and 6?(1)A(')(???(?)) valence states, respectively. The CSs for the 3?(3,1)A(') and 4?(1)A(') 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)?cm(2), 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)?cm(2) near its excitation threshold is attributed to transitions from the ground state to the 1?(3,1)A(")(n??(?)) 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 the four highest occupied molecular orbitals of cytosine. The sum of the ionization CS for these four excitation regions reaches a maximum of 8.1×10(-16)?cm(2) at the incident energy of 13 eV. PMID:20969430

Bazin, M; Michaud, M; Sanche, L

2010-10-21

268

Yeast artificial chromosome cloning  

Microsoft Academic Search

Yeast artificial chromosome (YAC) cloning systems enable the cloning of DNA stretches of 50 to well over 2000 kb. This makes\\u000a it possible to study large intact regions of DNA in detail, by restriction mapping the YAC to produce a physical map and by\\u000a examining the YAC for coding sequences or genes. YACs are important for their ability to clone

Michele Ramsay

1994-01-01

269

Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4.  

PubMed Central

The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. We examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D+ or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D+ or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D+ strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with [gamma-32P]ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly. It thus appears likely that gpalc inhibits transcript elongation on cytosine-containing DNA by interacting with actively transcribing core polymerase as a complex with the enzyme and cytosine-rich stretches of the template. Images PMID:2185231

Drivdahl, R H; Kutter, E M

1990-01-01

270

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

Microsoft Academic Search

The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine,\\u000a and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under\\u000a temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of\\u000a the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics

Douglas E. LaRowe; Pierre Regnier

2008-01-01

271

Intramolecular flexibility of DNA bases in adenine–thymine and guanine–cytosine Watson–Crick base pairs  

Microsoft Academic Search

The conformational flexibility of pyrimidine rings in adenine (A)–thymine (T) and guanine (G)–cytosine (C) Watson–Crick base pairs was investigated at the ab initio Hartree–Fock (HF) level using 6-31G** basis set. Transition of these rings from the planar equilibrium conformation to a distorted sofa conformation with torsion angles of 20° results in a marginal energy increase of approximately 1.3kcal\\/mol for the

OlegV Shishkin; Ji??? Šponer; Pavel Hobza

1999-01-01

272

Structure and the energy of base pairing in non-natural bases of nucleic acids: the azaguanine–cytosine and azaadenine–thymine base pairs  

Microsoft Academic Search

Watson–Crick optimized geometries and the energies of base pairing for the natural pairs of nucleic bases: adenine–thymine (AT) and guanine–cytosine (GC) have been recalculated by ab initio methods in order to compare results to those found for the non-natural azaadenine–thymine (AAT) and azaguanine–cytosine (AGC) pairs. Geometry optimizations carried out at the HF\\/6-31G** level and energies obtained at MP2\\/6-31G**, show that

J. Guillermo Contreras; Sandra T. Madariaga

2003-01-01

273

Strand displacement recognition of mixed adenine-cytosine sequences in double stranded DNA by thymine-guanine PNA (peptide nucleic acid).  

PubMed

Mixed pyrimidine-purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA(2)-DNA triplex with Watson-Crick complementary adenine-cytosine oligonucleotides and to bind complementary adenine-cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine-purine target in double stranded DNA and illustrate a novel binding mode of PNA. PMID:11553484

Nielsen, P E; Egholm, M

2001-09-01

274

Strand displacement recognition of mixed adenine–cytosine sequences in double stranded DNA by thymine–Guanine PNA (Peptide nucleic acid)  

Microsoft Academic Search

Mixed pyrimidine–purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA2–DNA triplex with Watson–Crick complementary adenine–cytosine oligonucleotides and to bind complementary adenine–cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine–purine target in double stranded DNA and illustrate

Peter E Nielsen; Michael Egholm

2001-01-01

275

Double-Stranded RNA Adenosine Deaminases Enhance Expression of Human Immunodeficiency Virus Type 1 Proteins?  

PubMed Central

ADARs (adenosine deaminases that act on double-stranded RNA) are RNA editing enzymes that catalyze a change from adenosine to inosine, which is then recognized as guanosine by translational machinery. We demonstrate here that overexpression of ADARs but not of an ADAR mutant lacking editing activity could upregulate human immunodeficiency virus type 1 (HIV-1) structural protein expression and viral production. Knockdown of ADAR1 by RNA silencing inhibited HIV-1 production. Viral RNA harvested from transfected ADAR1-knocked-down cells showed a decrease in the level of unspliced RNA transcripts. Overexpression of ADAR1 induced editing at a specific site in the env gene, and a mutant with the edited sequence was expressed more efficiently than the wild-type viral genome. These data suggested the role of ADAR in modulation of HIV-1 replication. Our data demonstrate a novel mechanism in which HIV-1 employs host RNA modification machinery for posttranscriptional regulation of viral protein expression. PMID:18753201

Phuphuakrat, Angsana; Kraiwong, Romchat; Boonarkart, Chompunuch; Lauhakirti, Darat; Lee, Tun-Hou; Auewarakul, Prasert

2008-01-01

276

Targeting Activation Induced Cytidine Deaminase Overcome Tumor Evasion of Immunotherapy by Cytotoxic T Lymphocytes  

PubMed Central

Activation induced cytidine deaminase (AID) is an enzyme essential for the generation of antibody diversity in B cells and is considered to be a general gene mutator. In addition, AID expression was also implicated in the pathogenesis of human B cell malignancies and associated with poor prognosis. Here we report that siRNA silencing of AID in plasmacytoma dramatically increased its susceptibility to immunotherapy by cytotoxic T lymphocytes. AID silencing did not decrease the mutation frequencies of tumor antigen gene P1A. Gene-array analysis showed dramatically altered expression of a number of genes in AID-silenced plasmacytoma cells and upregulation of CD200 was shown to be in favor of tumor eradication by CTL. Taken together, we demonstrate a novel function of AID in tumor evasion of CTL therapy and that targeting AID should be beneficial in the immunotherapy of AID positive tumors. PMID:20404277

Liu, Jin-Qing; Joshi, Pramod S.; Wang, Chuansong; El-Omrani, Hani Y.; Xiao, Yi; Liu, Xiuping; Hagan, John P.; Liu, Chang-Gong; Wu, Lai-Chu; Bai, Xue-Feng

2010-01-01

277

Maternal and fetal plasma adenosine deaminase, xanthine oxidase and malondialdehyde levels in pre-eclampsia.  

PubMed

The aim of this study was to evaluate maternal-fetal plasma adenosine deaminase, xanthine oxidase (ADA, XO) activity and malondialdehyde (MDA) levels and the relationship between them in pre-eclampsia. Maternal and umbilical cord whole blood samples were taken from 29 pre-eclamptic and 33 normal pregnants. The plasma ADA, XO activities as well as MDA levels were assayed by spectrophotometric methods. MDA levels and ADA, XO activities were found to be higher in maternal and fetal plasma in pre-eclamptics than in normal pregnancy. The differences were statistically significant between groups (p < 0.05). Increased maternal-fetal plasma XO and ADA activities, as a marker of immunological disorder, may be related to the pathogenesis of pre-eclampsia. In addition, increased MDA levels may be a reflection of increased oxidative stress in pre-eclamptics and their fetuses. PMID:15515122

Karabulut, Aysun Bay; Kafkasli, Ayse; Burak, Feza; Gozukara, Engin M

2005-01-01

278

Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein  

PubMed Central

The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells. DOI: http://dx.doi.org/10.7554/eLife.03110.001 PMID:25006166

Buerstedde, Jean-Marie; Lowndes, Noel; Schatz, David G

2014-01-01

279

Somatic Hypermutation Is Limited by CRM1-dependent Nuclear Export of Activation-induced Deaminase  

PubMed Central

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated in activated B lymphocytes by activation-induced deaminase (AID). AID is thought to make lesions in DNA by deaminating cytidine residues in single-stranded DNA exposed by RNA polymerase during transcription. Although this must occur in the nucleus, AID is found primarily in the cytoplasm. Here we show that AID is actively excluded from the nucleus by an exportin CRM1-dependent pathway. The AID nuclear export signal (NES) is found at the carboxyl terminus of AID in a region that overlaps a sequence required for CSR but not SHM. We find that AID lacking a functional NES causes more hypermutation of a nonphysiologic target gene in transfected fibroblasts. However, the NES does not impact on the rate of mutation of immunoglobulin genes in B lymphocytes, suggesting that the AID NES does not limit AID activity in these cells. PMID:15117971

McBride, Kevin M.; Barreto, Vasco; Ramiro, Almudena R.; Stavropoulos, Pete; Nussenzweig, Michel C.

2004-01-01

280

Acquisition of Genetic Aberrations by Activation-Induced Cytidine Deaminase (AID) during Inflammation-Associated Carcinogenesis  

PubMed Central

Genetic abnormalities such as nucleotide alterations and chromosomal disorders that accumulate in various tumor-related genes have an important role in cancer development. The precise mechanism of the acquisition of genetic aberrations, however, remains unclear. Activation-induced cytidine deaminase (AID), a nucleotide editing enzyme, is essential for the diversification of antibody production. AID is expressed only in activated B lymphocytes under physiologic conditions and induces somatic hypermutation and class switch recombination in immunoglobulin genes. Inflammation leads to aberrant AID expression in various gastrointestinal organs and increased AID expression contributes to cancer development by inducing genetic alterations in epithelial cells. Studies of how AID induces genetic disorders are expected to elucidate the mechanism of inflammation-associated carcinogenesis. PMID:24212831

Takai, Atsushi; Marusawa, Hiroyuki; Chiba, Tsutomu

2011-01-01

281

Somatic hypermutation is limited by CRM1-dependent nuclear export of activation-induced deaminase.  

PubMed

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated in activated B lymphocytes by activation-induced deaminase (AID). AID is thought to make lesions in DNA by deaminating cytidine residues in single-stranded DNA exposed by RNA polymerase during transcription. Although this must occur in the nucleus, AID is found primarily in the cytoplasm. Here we show that AID is actively excluded from the nucleus by an exportin CRM1-dependent pathway. The AID nuclear export signal (NES) is found at the carboxyl terminus of AID in a region that overlaps a sequence required for CSR but not SHM. We find that AID lacking a functional NES causes more hypermutation of a nonphysiologic target gene in transfected fibroblasts. However, the NES does not impact on the rate of mutation of immunoglobulin genes in B lymphocytes, suggesting that the AID NES does not limit AID activity in these cells. PMID:15117971

McBride, Kevin M; Barreto, Vasco; Ramiro, Almudena R; Stavropoulos, Pete; Nussenzweig, Michel C

2004-05-01

282

YY1 controls immunoglobulin class switch recombination and nuclear activation-induced deaminase levels.  

PubMed

Activation-induced deaminase (AID) is an enzyme required for class switch recombination (CSR) and somatic hypermutation (SHM), processes that ensure antibody maturation and expression of different immunoglobulin isotypes. AID function is tightly regulated by tissue- and stage-specific expression, nuclear localization, and protein stability. Transcription factor YY1 is crucial for early B cell development, but its function at late B cell stages is unknown. Here, we show that YY1 conditional knockout in activated splenic B cells interferes with CSR. Knockout of YY1 did not affect B cell proliferation, transcription of the AID and IgM genes, or levels of various switch region germ line transcripts. However, we show that YY1 physically interacts with AID and controls the accumulation of nuclear AID, at least in part, by increasing nuclear AID stability. We show for the first time that YY1 plays a novel role in CSR and controls nuclear AID protein levels. PMID:22290437

Zaprazna, Kristina; Atchison, Michael L

2012-04-01

283

Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix  

PubMed Central

Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts. We succeeded in identifying and locating AID protein in cells by immunohistochemical and immunofluorescent staining techniques and examined colocalization of AID and other molecules important for Ab diversification. This report extends our knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged Ig genes. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics. PMID:16280388

Yang, Guibin; Obiakor, Harold; Sinha, Rajesh K.; Newman, Barbara A.; Hood, Brian L.; Conrads, Thomas P.; Veenstra, Timothy D.; Mage, Rose G.

2005-01-01

284

Mechanisms regulating the targeting and activity of activation induced cytidine deaminase.  

PubMed

Activation induced cytidine deaminase (AID) plays a central role in the vertebrate adaptive immune response, initiating immunoglobulin (Ig) somatic hypermutation (SHM) and class-switch recombination (CSR). AID converts deoxycytosine (dC) in the DNA to deoxyuridine (dU), causing a DNA base-pairing mismatch. How this mismatch is recognised and resolved determines whether the site will undergo mutation, recombination or high-fidelity repair. Although AID action is essential for antibody diversification it is also known to act upon many non-Ig genes where it can cause tumourigenic mutations and translocations. Although much is known about the pathways of Ig diversification, there is still very little known about the mechanisms that target AID to its sites of action and regulate the different repair processes that can participate at these sites. PMID:24209594

Fear, David J

2013-10-01

285

Motion of the hydrogen bond proton in cytosine and the transition between its normal and imino states  

NASA Astrophysics Data System (ADS)

The potential energy surface of the H13 proton in base cytosine of the DNA molecules is calculated ab initio at the Gaussian98 MP2/6-311G(d,p) level. Two potential wells are found. One corresponds to the normal cytosine, while the other corresponds to its imino tautomer. The bindings of the proton in these wells are stable enough against the thermo-disturbance. The motions of the proton in these wells are oscillations around the nearest nitrogen atom like the pendula, and may move far away from the nitrogen atom to form the hydrogen bond with other bases. The estimated tunneling probability of the H13 proton from one well to another well shows that the life time of the proton staying in one of these wells is about 6×10 yr. It is too long to let tautomers of cytosine be in thermodynamical equilibrium in a room temperature gas phase experiment. The biological significance of this result is discussed.

Zhao, Zhen-Min; Zhang, Qi-Ren; Gao, Chun-Yuan; Zhuo, Yi-Zhong

2006-11-01

286

NGSmethDB: an updated genome resource for high quality, single-cytosine resolution methylomes  

PubMed Central

The updated release of ‘NGSmethDB’ (http://bioinfo2.ugr.es/NGSmethDB) is a repository for single-base whole-genome methylome maps for the best-assembled eukaryotic genomes. Short-read data sets from NGS bisulfite-sequencing projects of cell lines, fresh and pathological tissues are first pre-processed and aligned to the corresponding reference genome, and then the cytosine methylation levels are profiled. One major improvement is the application of a unique bioinformatics protocol to all data sets, thereby assuring the comparability of all values with each other. We implemented stringent quality controls to minimize important error sources, such as sequencing errors, bisulfite failures, clonal reads or single nucleotide variants (SNVs). This leads to reliable and high-quality methylomes, all obtained under uniform settings. Another significant improvement is the detection in parallel of SNVs, which might be crucial for many downstream analyses (e.g. SNVs and differential-methylation relationships). A next-generation methylation browser allows fast and smooth scrolling and zooming, thus speeding data download/upload, at the same time requiring fewer server resources. Several data mining tools allow the comparison/retrieval of methylation levels in different tissues or genome regions. NGSmethDB methylomes are also available as native tracks through a UCSC hub, which allows comparison with a wide range of third-party annotations, in particular phenotype or disease annotations. PMID:24271385

Geisen, Stefanie; Barturen, Guillermo; Alganza, Angel M.; Hackenberg, Michael; Oliver, Jose L.

2014-01-01

287

EFFECTS OF CYTOSINE ARABINOSIDE ON DIFFERENTIAL GENE EXPRESSION IN EMBRYONIC NEURAL RETINA  

PubMed Central

The analogue of cytidine, cytosine arabinoside (Ara-C), elicited a significant increase in the level of glutamine synthetase (GS) in embryonic chick neural retina in the absence of the steroid inducer of the enzyme. The increase was due to de novo synthesis of GS and was mediated by RNA which accumulated in the presence of the effective concentration of Ara-C. Accumulation of GS did not result from the inhibition of DNA synthesis for which Ara-C is best known. This new effect of Ara-C involves differential suppression of macromolecular synthesis in this system: the concentration of Ara-C which caused maximum GS accumulation suppressed overall protein and RNA syntheses 65–75% without inhibiting the transcription and translation of templates essential for GS synthesis. Withdrawal of Ara-C resulted in restoration of RNA synthesis and cessation of GS accumulation, even though preformed templates for the enzyme were present; however, if all RNA synthesis was arrested with actinomycin D at the time of Ara-C withdrawal, GS continued to accumulate. The results are consistent with the hypothesis that Ara-C differentially affects the activity of structural and regulatory genes involved in the regulation of GS levels in the retina: Ara-C allows transcription of the enzyme-specific templates, but reversibly inhibits the expression of regulatory genes which limit the accumulation of GS. PMID:4151790

Jones, R. E.; Moscona, A. A.

1974-01-01

288

On the electron affinity of cytosine in bulk water and at hydrophobic aqueous interfaces.  

PubMed

In the past one possible mechanism of DNA damage in bulk water has been attributed to the presence of hydrated electrons in water. Recently, one important property of hydrated electrons, namely their binding energy, was reported to be smaller at hydrophobic interfaces than in bulk aqueous solution. This possibly opens up new reaction possibilities with different solutes such as the DNA at hydrophobic, aqueous interfaces. Here, we use QM/MM molecular dynamics simulation to study how the molecular environment at the vacuum-water interface and in the bulk alters the electron affinity of cytosine being a characteristic part of the DNA. The electron affinity at the interface is closer to the corresponding binding energy of the partially hydrated electron. The increased energy resonance makes the electron capture process more probable and suggests that hydrated electrons at hydrophobic interfaces may be more reactive than the fully hydrated ones. Additionally, we found that the relaxation of the anionic form after electron attachment also induces a proton transfer from the surrounding solvent that was confirmed by comparison with the experimental reduction potential. PMID:25300994

Vöhringer-Martinez, Esteban; Dörner, Ciro; Abel, Bernd

2014-10-01

289

Ultrafast two-dimensional infrared spectroscopy of guanine-cytosine base pairs in DNA oligomers.  

PubMed

NH and OH stretching excitations of hydrated double-stranded DNA oligomers containing guanine-cytosine (GC) base pairs in a Watson-Crick geometry are studied by two-dimensional (2D) infrared spectroscopy. The 2D spectra measured at a low hydration level (?4 water molecules/base pair) are dominated by NH stretch contributions from the NH2 groups of G and C and the NH group of G. Partially hydrated NH2 groups display red-shifted NH stretch frequencies and a mixing of the wave functions of the two local NH oscillators via the mechanical vibrational coupling. The NH stretch lifetimes are of the order of 200-300 fs. Weak couplings exist between NH stretch oscillators within a base pair, while interactions between neighboring GC pairs in the double helix are negligible. The absence of spectral diffusion on a 1 ps time scale suggests a relatively rigid structure of the hydrogen bonds between DNA and residual water molecules. 2D spectra recorded with fully hydrated DNA oligomers exhibit NH and OH stretch contributions with a weak influence of water fluctuations on the NH stretch lineshapes. The femtosecond spectral diffusion of OH stretch excitations is slower than that in bulk H2O and originates from structural fluctuations of the water shell and the formation of a vibrationally hot ground state by vibrational relaxation. We compare our findings with measurements on hydrated adenine-thymine DNA oligomers and anhydrous GC base pairs in solution. PMID:24127664

Greve, Christian; Elsaesser, Thomas

2013-11-14

290

Intramolecular flexibility of DNA bases in adenine thymine and guanine cytosine Watson Crick base pairs  

NASA Astrophysics Data System (ADS)

The conformational flexibility of pyrimidine rings in adenine (A)-thymine (T) and guanine (G)-cytosine (C) Watson-Crick base pairs was investigated at the ab initio Hartree-Fock (HF) level using 6-31G** basis set. Transition of these rings from the planar equilibrium conformation to a distorted sofa conformation with torsion angles of 20° results in a marginal energy increase of approximately 1.3 kcal/mol for the A-T pair and 3.5 kcal/mol for the G-C pair. In the GC pair, the simultaneous deformation of both pyrimidine rings was applied. Comparison of ring deformation energies calculated at the HF, with correlated levels indicates significant overestimation (up to 30%) of ring rigidity by the HF approximation. Reasonable correlations were found between the out-of-plane ring vibrational frequencies and the ring deformation energies. Formation of Watson-Crick base pairs is manifested in prolongation of the N-H bonds within the bases which results in significant reduction of the N-H stretching frequencies. On the basis of the calculated increase in the C-H stretching frequency of adenine, the existence of a third hydrogen bond of the C-H⋯O type in the A-T pair is ruled out.

Shishkin, OlegV; Šponer, Ji?í; Hobza, Pavel

1999-03-01

291

Molecular evolution of DNA-(cytosine-N4) methyltransferases: evidence for their polyphyletic origin.  

PubMed

DNA N4-cytosine methyltransferases (N4mC MTases) are a family of S-adenosyl-L-methionine (AdoMet)-dependent MTases. Members of this family were previously found to share nine conserved sequence motifs, but the evolutionary basis of these similarities has never been studied in detail. We performed phylogenetic analysis of 37 known and potential new family members from the multiple sequence alignment using distance matrix, parsimony and maximum likelihood approaches to infer the evolutionary relationship among the N4mC MTases and classify them into groups of orthologs. All the treeing algorithms employed as well as results of exhaustive sequence database searching support a scenario, in which the majority of N4mC MTases, except for M. Bal I and M. Bam HI, arose by divergence from a common ancestor. Interestingly, MTases M. Bal I and M. Bam HI apparently originated from N6-adenine MTases and represent the most recent addendum to the N4mC MTase family. In addition to the previously reported nine sequence motifs, two more conserved sequence patches were detected. Phylogenetic analysis also provided the evidence for massive horizontal transfer of MTase genes, presumably with the whole restriction-modification systems, between Bacteria and Archaea. PMID:10536161

Bujnicki, J M; Radlinska, M

1999-11-15

292

A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa  

PubMed Central

During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281–290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains. PMID:12072568

Freitag, Michael; Williams, Rebecca L.; Kothe, Gregory O.; Selker, Eric U.

2002-01-01

293

Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine  

PubMed Central

Covalent modification by methylation of cytosine residues represents an important epigenetic hallmark. While sequence analysis after bisulphite conversion allows correlative analyses with single-base resolution, functional analysis by interference with DNA methylation is less precise, due to the complexity of methylation enzymes and their targets. A cytidine analogue, 5-azacytidine, is frequently used as an inhibitor of DNA methyltransferases, but its rapid degradation in aqueous solution is problematic for culture periods of longer than a few hours. Application of zebularine, a more stable cytidine analogue with a similar mode of action that is successfully used as a methylation inhibitor in Neurospora and mammalian tumour cell lines, can significantly reduce DNA methylation in plants in a dose-dependent and transient manner independent of sequence context. Demethylation is connected with transcriptional reactivation and partial decondensation of heterochromatin. Zebularine represents a promising new and versatile tool for investigating the role of DNA methylation in plants with regard to transcriptional control, maintenance and formation of (hetero-) chromatin. PMID:18826433

Baubec, Tuncay; Pecinka, Ales; Rozhon, Wilfried; Mittelsten Scheid, Ortrun

2009-01-01

294

Production of food yeast from starchy substrates  

Microsoft Academic Search

Fifteen yeast strains were selected for the production of food yeast from starchy substrates. From comparison with the amylolytic yeasts, a strain of Schwanniomyces castellii was selected and its characteristics are described.

A. Touzi; J. P. Prebois; G. Moulin; F. Deschamps; P. Galzy

1982-01-01

295

21 CFR 172.896 - Dried yeasts.  

...FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis ) and dried torula yeast (Candida utilis ) may be safely used in food provided the...

2014-04-01

296

Activation-Induced Cytidine Deaminase (AID) Deficiency Causes the Autosomal Recessive Form of the Hyper-IgM Syndrome (HIGM2)  

Microsoft Academic Search

The activation-induced cytidine deaminase (AID) gene, specifically expressed in germinal center B cells in mice, is a member of the cytidine deaminase family. We herein report mutations in the human counterpart of AID in patients with the autosomal recessive form of hyper-IgM syndrome (HIGM2). Three major abnormalities characterize AID deficiency: (1) the absence of immunoglobulin class switch recombination, (2) the

Patrick Revy; Taro Muto; Yves Levy; Frédéric Geissmann; Alessandro Plebani; Ozden Sanal; Nadia Catalan; Monique Forveille; Rémi Dufourcq-Lagelouse; Andrew Gennery; Ilhan Tezcan; Fugen Ersoy; Hulya Kayserili; Alberto G Ugazio; Nicole Brousse; Masamichi Muramatsu; Luigi D Notarangelo; Kazuo Kinoshita; Tasuku Honjo; Alain Fischer; Anne Durandy

2000-01-01

297

Promotion of tomato ( Lycopersicon esculentum Mill.) plant growth by rhizosphere competent 1-aminocyclopropane-1-carboxylic acid deaminase-producing streptomycete actinomycetes  

Microsoft Academic Search

The ability of streptomycete actinomycetes to promote growth of tomato through the production of 1-aminocyclopropane-1-carboxylic\\u000a acid (ACC) deaminase was evaluated under gnotobiotic and greenhouse conditions. To achieve this, 64 isolates of Streptomyces spp. obtained from a tomato rhizosphere in the United Arab Emirates were initially selected for their ability to produce\\u000a ACC deaminase as well as indole-3-acetic acid (IAA) and

Khaled A. El-Tarabily

2008-01-01

298

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

PubMed Central

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 change. Conclusion Our results documented that tissue culture-induced mPing activity in rice ssp. indica is correlated with alteration in cytosine methylation patterns at both random genomic loci and the elements' flanks, while the stability of mPing positively correlates with enhanced methylation levels of both the flanks and probably the elements per se. Thus, our results implicate a possible role of cytosine methylation in maintaining mPing stability under normal conditions, and in releasing the element's activity as a consequence of epigenetic perturbation in a locus-specific manner under certain stress conditions. PMID:19604382

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

2009-01-01

299

New and emerging yeast pathogens.  

PubMed Central

The most common yeast species that act as agents of human disease are Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans. The incidence of infections by other yeasts has increased during the past decade. The most evident emerging pathogens are Malassezia furfur, Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Candida lusitaniae, and Candida krusei. Organisms once considered environmental contaminants or only industrially important, such as Candida utilis and Candida lipolytica, have now been implicated as agents of fungemia, onychomycosis, and systemic disease. The unusual yeasts primarily infect immunocompromised patients, newborns, and the elderly. The role of central venous catheter removal and antifungal therapy in patient management is controversial. The antibiograms of the unusual yeasts range from resistant to the most recent azoles and amphotericin B to highly susceptible to all antifungal agents. Current routine methods for yeast identification may be insufficient to identify the unusual yeasts within 2 days after isolation. The recognition of unusual yeasts as agents of sometimes life-threatening infection and their unpredictable antifungal susceptibilities increase the burden on the clinical mycology laboratory to pursue complete species identification and MIC determinations. Given the current and evolving medical practices for management of seriously ill patients, further evaluations of the clinically important data about these yeasts are needed. PMID:8665465

Hazen, K C

1995-01-01

300

Recent developments in use of 1-aminocyclopropane-1-carboxylate (ACC) deaminase for conferring tolerance to biotic and abiotic stress.  

PubMed

Ethylene is an essential plant hormone also known as a stress hormone because its synthesis is accelerated by induction of a variety of biotic and abiotic stress. The plant growth promoting bacteria containing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase enhances plant growth by decreasing plant ethylene levels under stress conditions. The expression of ACC deaminase (acdS) gene in transgenic plants is an alternative approach to overcome the ethylene-induced stress. Several transgenic plants have been engineered to express both bacterial/plant acdS genes which then lowers the stress-induced ethylene levels, thus efficiently combating the deleterious effects of environmental stresses. This review summarizes the current knowledge of various transgenic plants overexpressing microbial and plant acdS genes and their potential under diverse biotic and abiotic stresses. Transcription regulation mechanism of acdS gene from different bacteria, with special emphasis to nitrogen fixing bacteria is also discussed in this review. PMID:24563292

Gontia-Mishra, Iti; Sasidharan, Shaly; Tiwari, Sharad

2014-05-01

301

Regulation of Activation-Induced Cytidine Deaminase DNA Deamination Activity in B Cells by Serine-38 Phosphorylation  

PubMed Central

Human and mouse immunoglobulin (Ig) genes are diversified in mature B cells by distinct processes known as Ig heavy chain class switch recombination (CSR) and Ig variable region exon somatic hypermutation (SHM). These DNA modification processes are initiated by activation-induced cytidine deaminase (AID), a DNA cytidine deaminase predominantly expressed in activated B cells. AID is post-transcriptionally regulated via multiple mechanisms including microRNA regulation, nucleo-cytoplasmic shuttling, ubiquitination and phosphorylation. Among these regulatory processes, AID phosphorylation at Serine-38 (S38) has been a focus of particularly intense study and debate. Here, we discuss recent biochemical and mouse genetic studies that begin to elucidate the functional significance of AID S38 phosphorylation in the context of the evolution of this mode of AID regulation and the potential roles that it may play in activated B cells during a normal immune response. PMID:19442251

Basu, Uttiya; Franklin, Andrew; Schwer, Bjoern; Cheng, Hwei-Ling; Chaudhuri, Jayanta; Alt, Frederick W.

2012-01-01

302

McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues.  

PubMed Central

Restriction of DNA by the Escherichia coli K-12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation. From previous work by others it was known that restriction of 5-methylcytosine-containing DNA requires two methylated 5'-PuC sites separated by approximately 40-80 non-defined base pairs. Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit. McrB has a low affinity for non-methylated DNA, with which it forms low molecular weight complexes. The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi- or fully methylated 5'-PuC sites. Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules. Methylation at unique 5'-PuC sites can be sufficient to stimulate DNA binding by McrB. As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5'-PumC sites. The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non-sequence-specific manner with DNA containing methylated 5'-CpG sequences and might point to a common molecular origin of these proteins. In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC. McrC neither binds to DNA nor modulates the DNA binding potential of McrB. As McrC is essential for restriction it appears to predominantly function in catalysis. Images PMID:7781618

Kruger, T; Wild, C; Noyer-Weidner, M

1995-01-01

303

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

SciTech Connect

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.

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

2007-01-22

304

Evolution of complete proteomes: guanine-cytosine pressure, phylogeny and environmental influences blend the proteomic architecture  

PubMed Central

Background Guanine-cytosine (GC) composition is an important feature of genomes. Likewise, amino acid composition is a distinct, but less valued, feature of proteomes. A major concern is that it is not clear what valuable information can be acquired from amino acid composition data. To address this concern, in-depth analyses of the amino acid composition of the complete proteomes from 63 archaea, 270 bacteria, and 128 eukaryotes were performed. Results Principal component analysis of the amino acid matrices showed that the main contributors to proteomic architecture were genomic GC variation, phylogeny, and environmental influences. GC pressure drove positive selection on Ala, Arg, Gly, Pro, Trp, and Val, and adverse selection on Asn, Lys, Ile, Phe, and Tyr. The physico-chemical framework of the complete proteomes withstood GC pressure by frequency complementation of GC-dependent amino acid pairs with similar physico-chemical properties. Gln, His, Ser, and Val were responsible for phylogeny and their constituted components could differentiate archaea, bacteria, and eukaryotes. Environmental niche was also a significant factor in determining proteomic architecture, especially for archaea for which the main amino acids were Cys, Leu, and Thr. In archaea, hyperthermophiles, acidophiles, mesophiles, psychrophiles, and halophiles gathered successively along the environment-based principal component. Concordance between proteomic architecture and the genetic code was also related closely to genomic GC content, phylogeny, and lifestyles. Conclusions Large-scale analyses of the complete proteomes of a wide range of organisms suggested that amino acid composition retained the trace of GC variation, phylogeny, and environmental influences during evolution. The findings from this study will help in the development of a global understanding of proteome evolution, and even biological evolution. PMID:24088322

2013-01-01

305

Isolation and properties of AMP deaminase from jumbo squid ( Dosidicus gigas) mantle muscle from the Gulf of California, Mexico  

Microsoft Academic Search

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5?-AMP sepharose. Specific activity of 2.5U\\/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS–PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a

E. Marquez-Rios; R. Pacheco-Aguilar; F. J. Castillo-Yañez; C. G. Figueroa-Soto; J. M. Ezquerra-Brauer; T. Gollas-Galvan

2008-01-01

306

Characterization of 1-aminocyclopropane-1-carboxylate deaminase producing methylobacteria from phyllosphere of rice and their role in ethylene regulation  

Microsoft Academic Search

The presence of 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity among the phyllosphere methylobacteria of rice\\u000a was detected and its role in regulating plant ethylene level was assessed. Eighteen methylobacterial isolates from four different\\u000a cultivars of rice were isolated and screened for ACCD. The 16S rRNA homology of ACCD positive methylobacterial isolate closely\\u000a related to the species Methylobacterium radiotolerans. The accD gene sequence

C. Chinnadurai; D. Balachandar; S. P. Sundaram

2009-01-01

307

Brewer's yeast and sugarcane yeast as protein sources for dogs.  

PubMed

Brewer's yeast (BY), autolysed sugarcane yeast (ASCY) and integral sugar cane yeast (ISCY) were studied in two experiments as ingredients for dog diets. In the first experiment, 28 dogs were randomly assigned to four diets; one reference diet and three test diets containing 15% of BY, ASCY or ISCY and 85% of the reference diet (as-fed basis). The digestibilities of the yeasts were calculated by the substitution method. In the second experiment, 35 dogs were randomized to five diets with similar chemical composition but different levels of sugarcane yeast inclusion (0%, 7.5% ASCY, 15% ASCY, 7.5% ISCY and 15% ISCY). In both experiments, the coefficient of total tract apparent digestibility (CTTAD) of nutrients was determined through total collection of faeces. During experiment, two additional analyses of food palatability, nitrogen balance and urea postprandial responses were performed. The data were submitted to analysis of variance, and the means were compared by orthogonal or polynomial contrasts or Tukey's test (p < 0.05). In experiment 1, CTTAD of protein was lower for both sugarcane yeasts than for BY (p = 0.012), as was metabolizable energy content (p = 0.025). In experiment 2, a linear reduction in energy digestibility with ASCY inclusion (p = 0.05) was verified. Furthermore, faecal score and DM content were reduced with ISCY inclusion (p < 0.003). No effect of yeast inclusion on nitrogen balance or postprandial urea response was found. Also, the inclusion of 7.5% of ASCY or ISCY increased diet palatability (p < 0.01). Yeasts present adequate digestibility by dogs, but its effect on faecal formation needs to be considered. No clear advantage for the use of ASCY over ISCY was found. In conclusion, we find that sugarcane yeast is suitable for inclusion in dog food and can enhance the overall palatability of the diet. PMID:24304448

Martins, M S; Sakomura, N K; Souza, D F; Filho, F O R; Gomes, M O S; Vasconcellos, R S; Carciofi, A C

2014-10-01

308

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant Nature of APOBEC3G  

PubMed Central

Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10?2 to 10?5 in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR (?10?4 to 10?5). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1?vif efficiently. PMID:19169351

Henry, Michel; Guétard, Denise; Suspène, Rodolphe; Rusniok, Christophe; Wain-Hobson, Simon; Vartanian, Jean-Pierre

2009-01-01

309

Yeast ecology of Kombucha fermentation.  

PubMed

Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species. PMID:15282124

Teoh, Ai Leng; Heard, Gillian; Cox, Julian

2004-09-01

310

An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera.  

PubMed

Sequences hybridizing to mitochondrial DNA probes from Saccharomyces cerevisiae have been mapped in six mitochondrial genomes from the Dekkera/Brettanomyces yeasts and in mtDNA from the closely related Eeniella nana. Sequence order for the 34.5 kbp mtDNA of E. nana is identical to that for mtDNAs from B. custersianus (28.5 kbp) and B. naardenensis (41.7 kbp) thereby suggesting that the former yeast is affiliated with the latter two species. A closer relationship is suggested for D. intermedia and D. bruxellensis as mtDNAs from these yeasts, 73.2 and 85.0 kbp respectively, have the same sequence order and mostly common restriction endonuclease sites. Differences between the two molecules are reminiscent of those found in mtDNA polymorphisms of S. cerevisiae strains thereby suggesting that the two Dekkera yeasts are variants of a single species. An unusual feature of the Dekkera species mtDNA is an inversion of the cytochrome b hybridizable region relative to the LrRNA sequence. Likewise mtDNA from B. anomalus (57.7 kbp) has an inversion of the cytochrome oxidase subunit 1 sequence with respect to the LrRNA sequence. By contrast the largest mtDNA (101.1 kbp) from B. custersii has the cytochrome b and LrRNA sequences in the same orientation. In addition hybridizable regions in this mtDNA are found in three clusters that are separated by several thousand base pairs of sequence deficient in restriction endonuclease sites. This observation together with the low guanine and cytosine content of the mtDNA suggests that the regions separating the sequence clusters are mostly adenine and thymine residues. PMID:3442820

Hoeben, P; Clark-Walker, G D

1986-01-01

311

Characterizing the Protonation State of Cytosine in Transient GoC Hoogsteen Base Pairs in Duplex DNA  

PubMed Central

G•C Hoogsteen base pairs can form transiently in duplex DNA and play important roles in DNA recognition, replication and repair. G•C Hoogsteen base pairs are thought to be stabilized by protonation of cytosine N3, which affords a second key hydrogen bond, but experimental evidence for this is sparse because the proton cannot be directly visualized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Here, we combine NMR and constant pH molecular dynamics (MD) simulations to directly investigate the pKa of cytosine N3 in a chemically trapped N1-methyl-G•C Hoogsteen base pair within duplex DNA. Analysis of NMR chemical shift perturbations and NOESY data as a function of pH revealed that cytosine deprotonation is coupled to a syn-to-anti transition in N1-methyl-G, which results in a distorted Watson-Crick geometry at pH > 9. A four-state analysis of the pH titration profiles yields a lower bound pKa estimate of 7.2 ± 0.1 for the G•C Hoogsteen base pair, which is in good agreement with the pKa (7.1 ± 0.1) value calculated independently using constant pH MD simulations. Based on these results and pH dependent NMR relaxation dispersion measurements, we estimate that under physiological pH (pH 7 to 8), G•C Hoogsteen base pairs in naked DNA have a population of 0.02 to 0.002% as compared to 0.4% for A•T Hoogsteen base pairs and likely exist primarily as a protonated species. PMID:23506098

Nikolova, Evgenia N.; Goh, Garrett B.; Brooks, Charles L.; Al-Hashimi, Hashim M.

2013-01-01

312

Following Ultrafast Radiationless Relaxation Dynamics With Strong Field Dissociative Ionization: A Comparison Between Adenine, Uracil, and Cytosine  

SciTech Connect

We present the application of ultrafast time- and mass-resolved ion yield laser spectroscopy in conjunction with ab initio electronic structure calculations to track molecular excited-state dynamics. We discuss how molecular fragment ions can be associated with conformations the molecule assumes during its relaxation, and how various features of the pump-probe signal for those fragments can be used to infer details of the excited state dynamics. We present results for radiationless relaxation in DNA and RNA bases adenine, cytosine, and uracil in the gas phase, pumped near a one-photon resonance transition to an excited state, and probed via strong-field near-IR dissociative ionization.

Kotur, Marija; Weinacht, Thomas C.; Zhou, Congyi; Matsika, Spiridoula

2011-03-22

313

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

PubMed

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

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

1982-10-25

314

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

PubMed Central

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

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

1982-01-01

315

Cytosine Arabinoside Promotes Cytotoxic Effect of T Cells on Leukemia Cells Mediated by Bispecific Antibody  

PubMed Central

Abstract Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. Therefore, the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format that is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, anti-CD3×anti-CD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19+ human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could upregulate CD80 expressed on CD19+ Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by the anti-CD3×anti-CD19 diabody with or without a low dose of Ara-C was compared. The combination of the anti-CD3×anti-CD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2. Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could upregulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1–ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could upregulate CD80 expressed on two of five specimens of acute B lymphoblastic leukemia patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of the anti-CD3×anti-CD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects. PMID:23879717

Li, Wei; Yang, Ming; Yan, Yan; Shi, RuiZan; Cheng, JunPing; Li, ZhenZhen; Zhang, MengNan; Wang, JianXiang; Xiong, DongSheng

2013-01-01

316

Tetrahydrouridine Inhibits Cell Proliferation through Cell Cycle Regulation Regardless of Cytidine Deaminase Expression Levels  

PubMed Central

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells. PMID:22616006

Funamizu, Naotake; Lacy, Curtis Ray; Fujita, Kaori; Furukawa, Kenei; Misawa, Takeyuki; Yanaga, Katsuhiko; Manome, Yoshinobu

2012-01-01

317

Tipping the Balance: Antagonism of PKR Kinase and ADAR1 Deaminase Functions by Virus Gene Products  

PubMed Central

The protein kinase regulated by RNA (PKR) and the adenosine deaminase acting on RNA (ADAR1) are interferon-inducible enzymes that play important roles in biologic processes including the antiviral actions of interferons, signal transduction, and apoptosis. PKR catalyzes the RNA-dependent phosphorylation of protein synthesis initiation factor eIF-2?, thereby leading to altered translational patterns in interferon-treated and virus-infected cells. PKR also modulates signal transduction responses, including the induction of interferon. ADAR1 catalyzes the deamination of adenosine (A) to generate inosine (I) in RNAs with double-stranded character. Because I is recognized as G instead of A, A-to-I editing by ADAR1 can lead to genetic recoding and altered RNA structures. The importance of PKR and ADAR1 in innate antiviral immunity is illustrated by a number of viruses that encode either RNA or protein viral gene products that antagonize PKR and ADAR1 enzymatic activity, localization, or stability. PMID:19715457

George, Cyril X.; Li, Zhiqun; Okonski, Kristina M.; Toth, Ann M.; Wang, Ying

2009-01-01

318

Structural Insights into E. coli Porphobilinogen Deaminase during Synthesis and Exit of 1-Hydroxymethylbilane  

PubMed Central

Porphobilinogen deaminase (PBGD) catalyzes the formation of 1-hydroxymethylbilane (HMB), a crucial intermediate in tetrapyrrole biosynthesis, through a step-wise polymerization of four molecules of porphobilinogen (PBG), using a unique dipyrromethane (DPM) cofactor. Structural and biochemical studies have suggested residues with catalytic importance, but their specific role in the mechanism and the dynamic behavior of the protein with respect to the growing pyrrole chain remains unknown. Molecular dynamics simulations of the protein through the different stages of pyrrole chain elongation suggested that the compactness of the overall protein decreases progressively with addition of each pyrrole ring. Essential dynamics showed that domains move apart while the cofactor turn region moves towards the second domain, thus creating space for the pyrrole rings added at each stage. Residues of the flexible active site loop play a significant role in its modulation. Steered molecular dynamics was performed to predict the exit mechanism of HMB from PBGD at the end of the catalytic cycle. Based on the force profile and minimal structural changes the proposed path for the exit of HMB is through the space between the domains flanking the active site loop. Residues reported as catalytically important, also play an important role in the exit of HMB. Further, upon removal of HMB, the structure of PBGD gradually relaxes to resemble its initial stage structure, indicating its readiness to resume a new catalytic cycle. PMID:24603363

Bulusu, Gopalakrishnan

2014-01-01

319

Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages  

PubMed Central

While exploring the effects of aerosol IFN-? treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-?-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-? induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-? suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages. PMID:25272020

Levy, David N.; Li, Yonghua; Kumar, Rajnish; Burke, Sean A.; Dawson, Rodney; Hioe, Catarina E.; Borkowsky, William; Rom, William N.; Hoshino, Yoshihiko

2014-01-01

320

The Role of Zn2+ on the Structure and Stability of Murine Adenosine Deaminase  

PubMed Central

Adenosine deaminase (ADA) is a key enzyme in purine metabolism and crucial for normal immune competence. It is a 40 kDa-monomeric TIM-barrel protein containing a tightly bound Zn2+, which is required for activity. In this study, we have investigated the role of Zn2+with respect to ADA structure and stability. After removing Zn2+, the crystallographic structure of the protein remains highly ordered and similar to that of the holo protein with structural changes limited to regions capping the active site pocket. The stability of the protein, however, is decreased significantly in the absence of Zn2+. Denaturation with urea shows the midpoint to be about 3.5 M for the apo enzyme compared to 6.4 M for the holo enzyme. ADA contains four tryptophan residues distant from the Zn2+site. 19F-NMR studies in the presence and absence of Zn2+ were carried out after incorporation of 6-19F-tryptophan. Chemical shift differences were observed for three of the four tryptophan residues suggesting that, in contrast to the X-ray data, Zn2+-induced structural changes are propagated throughout the protein. Changes throughout the structure as suggested by the NMR data may explain the lower stability of the Zn2+-free protein. Real-time 19F-NMR spectroscopy measuring the loss of Zn2+ showed that structural changes correlated with the loss of enzymatic activity. PMID:20815357

Niu, Weiling; Shu, Qin; Chen, Zhiwei; Mathews, Scott; Cera, Enrico Di; Frieden, Carl

2010-01-01

321

Divergence of AMP Deaminase in the Ice Worm Mesenchytraeus solifugus (Annelida, Clitellata, Enchytraeidae)  

PubMed Central

Glacier ice worms, Mesenchytraeus solifugus and related species, are the largest glacially obligate metazoans. As one component of cold temperature adaptation, ice worms maintain atypically high energy levels in an apparent mechanism to offset cold temperature-induced lethargy and death. To explore this observation at a mechanistic level, we considered the putative contribution of 5? adenosine monophosphate deaminase (AMPD), a key regulator of energy metabolism in eukaryotes. We cloned cDNAs encoding ice worm AMPD, generating a fragment encoding 543 amino acids that included a short N-terminal region and complete C-terminal catalytic domain. The predicted ice worm AMPD amino acid sequence displayed conservation with homologues from other mesophilic eukaryotes with notable exceptions. In particular, an ice worm-specific K188E substitution proximal to the AMP binding site likely alters the architecture of the active site and negatively affects the enzyme's activity. Paradoxically, this would contribute to elevated intracellular ATP levels, which appears to be a signature of cold adapted taxa. PMID:21350654

Marotta, Roberto; Parry, Bradley R.; Shain, Daniel H.

2009-01-01

322

Kinetic characterization and gene expression of adenosine deaminase in intact trophozoites of Trichomonas vaginalis.  

PubMed

Trichomonas vaginalis is a parasite that resides in the human urogenital tract and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. Nucleoside triphosphate diphosphohydrolase (NTPDase), which hydrolyzes extracellular di- and triphosphate nucleotides, and ecto-5'-nucleotidase, which hydrolyzes AMP, have been characterized in T. vaginalis. The aim of this study was to characterize the adenosine deaminase (ADA) activity in intact trophozoites of T. vaginalis. A strong inhibition in adenosine deamination was observed in the presence of calcium and magnesium, which was prevented by EDTA. The apparent K(M) value for adenosine was 1.13 ± 0.07mM. The calculated V(max) was 2.61 ± 0.054 nmol NH(3) min(-1) mg(-1) protein. Adenosine deamination was inhibited in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine. Semi-quantitative reverse transcriptase-PCR experiments were performed and both ADA-related genes ada(125) and ada(231) mRNA were expressed, although ada(231) in higher quantity when compared with the ada(125) : ?-tubulin ratio. Furthermore, a phylogenetic analysis showed that the T. vaginalis sequences formed a clade with Entamoeba histolytica and Dictyostelium discoideum sequences, and it strongly suggests homologous functions in the T. vaginalis genome. The presence of ADA activity in T. vaginalis may be important to modulate the adenosine/inosine levels during infection and, consequently, to maintain the anti-inflammatory properties through different nucleoside-signalling mechanisms. PMID:21477257

Weizenmann, Marina; Frasson, Amanda Piccoli; de Barros, Muriel Primon; Vieira, Patrícia de Brum; Rosemberg, Denis Broock; De Carli, Geraldo Attilio; Bogo, Maurício Reis; Bonan, Carla Denise; Tasca, Tiana

2011-06-01

323

Activation-Induced Cytidine Deaminase and Aberrant Germinal Center Selection in the Development of Humoral Autoimmunities  

PubMed Central

Humoral immunity, which is the branch of the immune system governed by B cells, protects the body from extracellular pathogens through the secretion of immunoglobulins. Given the unpredictability of exogenous antigens, B cells must be accommodating to numerous genetic alterations to mold immunoglobulin specificity to recognize offending pathogens. Abnormalities in this process leave the host susceptible to permanent pathological modifications and in particular humoral autoimmunities in which secreted immunoglobulins mistake host proteins as pathogenic targets. Underlying the development of self-reactive immunoglobulins is activation-induced cytidine deaminase (AID), a mutagenic enzyme responsible for modifying the specificity of B cells by producing point mutations at the immunoglobulin gene locus. Ideally, these mutations result in an increased affinity for exogenous antigens. However, in pathological scenarios, these mutations produce or enhance a B cell's ability to target the host. AID-induced mutations occur in the germinal center microenvironment of peripheral lymphoid tissue, where pathogenic B-cell clones must evade overwhelming selection pressures to be released systemically. Recent research has revealed numerous genes and pathways responsible for eliminating self-reactive clones within the germinal center. On the basis of these studies, this review aims to clarify the link between AID and the generation of pathogenic immunoglobulins. Furthermore, it describes the selective pressures that pathogenic B cells must bypass within the germinal center to secrete immunoglobulins that ultimately result in disease. PMID:21281778

Zaheen, Ahmad; Martin, Alberto

2011-01-01

324

A Role for Host Activation-Induced Cytidine Deaminase in Innate Immune Defense against KSHV  

PubMed Central

Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens. PMID:24244169

Bekerman, Elena; Jeon, Diana; Ardolino, Michele; Coscoy, Laurent

2013-01-01

325

MicroRNA-155 Suppresses Activation-Induced Cytidine Deaminase-Mediated Myc-Igh Translocation  

PubMed Central

SUMMARY MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA-mRNA interaction in vivo, we generated mice that carry a mutation in the putative microRNA-155 (miR-155) binding site in the 3?-untranslated region of activation-induced cytidine deaminase (AID), designated Aicda155 mice. AID is required for immunoglobulin gene diversification in B lymphocytes, but it also promotes chromosomal translocations. Aicda155 caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID. PMID:18455451

Dorsett, Yair; McBride, Kevin M.; Jankovic, Mila; Gazumyan, Anna; Thai, To-Ha; Robbiani, Davide F.; Di Virgilio, Michela; San-Martin, Bernardo Reina; Heidkamp, Gordon; Schwickert, Tanja A.; Eisenreich, Thomas; Rajewsky, Klaus; Nussenzweig, Michel C.

2009-01-01

326

Cerebrospinal fluid adenosine deaminase activity for the diagnosis of tuberculous meningitis in adults.  

PubMed

We studied adenosine deaminase (ADA) activity in cerebrospinal fluid (CSF) of 16 cases of tuberculous meningitis, 4 cases of cryptococcal meningitis, 5 cases of bacterial meningitis, 12 cases of eosinophilic meningitis, 26 cases of aseptic meningitis, 6 cases of carcinomatous meningitis and 108 cases with normal CSF. The mean CSF ADA values for the different groups were: 39.44 +/- 41.46, 13.00 +/- 7.43, 34.20 +/- 40.81, 3.17 +/- 4.82, 10.03 +/- 9.23, 8.67 +/- 13.60, and 2.58 +/- 2.90 U/I, respectively. Comparing the ADA activity between patients with tuberculous meningitis and non-tuberculous meningitis, the receiver-operating characteristic (ROC) curve identified a CSF ADA level of 15.5 U/I as the best cut-off value to differentiate between the two, with a sensitivity of 75% and a specificity of 93%, with an area under the curve of 0.92. When tuberculous meningitis was compared with aseptic and carcinomatous meningitis, the ROC curve identified a CSF ADA level of 19.0 U/I as the best cut-off value for differentiation, with a sensitivity of 69% and a specificity of 94%, with an area under the curve of 0.83. The level of CSF ADA may be useful as a complementary tool in the early diagnosis of tuberculous meningitis. PMID:17333738

Chotmongkol, Verajit; Teerajetgul, Yaovalak; Yodwut, Chattanong

2006-09-01

327

AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle  

NASA Technical Reports Server (NTRS)

The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

1992-01-01

328

Reinforcement of antitumor effect of Cordyceps sinensis by 2'-deoxycoformycin, an adenosine deaminase inhibitor.  

PubMed

In this study, an attempt was made to elucidate the combined effect of 2'-deoxycoformycin (DCF), an adenosine deaminase inhibitor, with a water extract of Cordyceps sinensis (WECS), on the growth curves of mouse melanoma and lung carcinoma cells. Sub-confluent cells were harvested with an EDTA trypsin solution, and resuspended to appropriate concentrations in DMEM containing 10% fetal bovine serum. Using 1x10(5) cells/2 ml in each well of a 12-well culture plate, cells were incubated for 24, 48 and 72 h in the presence of WECS alone, or WECS plus DCF in a CO2 incubator at 37 degrees C. Duplicate samples of viable cells were enumerated with a Coulter counter. The antitumor effect of WECS on the growth curves of tumor cell lines increased over 3-fold in combination with DCF. These results suggest that DCF has a remarkable reinforcement effect on the antitumor activity of WECS. DCF is a potent adjuvant for WECS. PMID:17436579

Yoshikawa, Noriko; Nakamura, Kazuki; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

2007-01-01

329

The Adenosine Deaminase Gene Polymorphism Is Associated with Chronic Heart Failure Risk in Chinese  

PubMed Central

Adenosine (Ado) is an important cardioprotective agent. Since endogenous Ado levels are affected by the enzyme Ado deaminase (ADA), polymorphisms within the ADA gene may exert some effect on chronic heart failure (CHF). This study applied a case-control investigation to 300 northern Chinese Han CHF patients and 400 ethnicity-matched healthy controls in which nine single-nucleotide polymorphisms (SNPs) of ADA were genotyped and association analyses were performed. Odds ratios (ORs) with 95% confidence intervals (CI) were used to assess the association. Overall, rs452159 polymorphism in ADA gene was significantly associated with susceptibility to CHF under the dominant model (p = 0.013, OR = 1.537, 95% CI = 1.10–2.16), after adjustment for age, sex, and traditional cardiovascular risk factors. No difference in genotype distribution and allele frequency for the rs452159 according to the functional New York Heart Association class was found. Furthermore, the values of left ventricular ejection fraction, left-ventricle end-diastolic diameter or left-ventricle end-systolic diameter did not differ significantly among the different rs452159 genotype CHF patients. Although further studies with larger cohorts and other ethnicities are required to validate the conclusions, the findings of this study potentially provide novel insight into the pathogenesis of CHF. PMID:25170811

He, Hai-Rong; Li, Yuan-Jie; He, Gong-Hao; Wang, Ya-Jun; Zhai, Ya-Jing; Xie, Jiao; Zhang, Wei-Peng; Dong, Ya-Lin; Lu, Jun

2014-01-01

330

The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase  

PubMed Central

Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (?-aqua)(?-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated. PMID:24970226

Ranieri-Raggi, Maria; Moir, Arthur J. G.; Raggi, Antonio

2014-01-01

331

Target DNA sequence directly regulates the frequency of activation-induced deaminase-dependent mutations.  

PubMed

Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core S?1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted S?1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely. PMID:22962683

Chen, Zhangguo; Viboolsittiseri, Sawanee S; O'Connor, Brian P; Wang, Jing H

2012-10-15

332

Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis  

PubMed Central

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE. PMID:23167594

2013-01-01

333

Yeast Metabolism Lab Mrs. Zimmerman  

E-print Network

Energy from sunlight #12;Respiration #12;Cellular Respiration C6H12O6 + 6 O2 6 CO2 + 6 H2O + energyYeast Metabolism Lab Mrs. Zimmerman 10/22/10 #12;Photosynthesis 6 CO2 + 6 H2O C6H12O6 + 6 O2 Oxygen Glucose Carbon Dioxide Water Energy #12;Yeast · Unicellular · Eukaryotic (like us) · Kingdom Fungi

Rose, Michael R.

334

Study of amyloids using yeast  

PubMed Central

Summary Saccharomyces cerevisiae has been a useful model organism in such fields as the cell cycle, regulation of transcription, protein trafficking and cell biology, primarily because of its ease of genetic manipulation. This is no less so in the area of amyloid studies. The endogenous yeast amyloids described to date include prions, infectious proteins (Table 1), and some cell wall proteins (1). and amyloids of humans and a fungal prion have also been studied using the yeast system. Accordingly, the emphasis of this chapter will be on genetic, biochemical, cell biological and physical methods particularly useful in the study of yeast prions and other amyloids studied in yeast. We limit our description of these methods to those aspects which have been most useful in studying yeast prions, citing more detailed expositions in the literature. Volumes on yeast genetics methods (2–4), and on amyloids and prions (5, 6) are useful, and Masison has edited a volume of Methods on “Identification, analysis and characterization of fungal prions” which covers some of this territory (7). We also outline some useful physical methods, pointing the reader to more extensive and authoratative descriptions. PMID:22528100

Wickner, Reed B.; Kryndushkin, Dmitry; Shewmaker, Frank; McGlinchey, Ryan; Edskes, Herman K.

2012-01-01

335

Yeast models for amyloid disease.  

PubMed

Saccharomyces cerevisiae (baker's yeast) is a well-established eukaryotic model organism, which has significantly contributed to our understanding of mechanisms that drive numerous core cellular processes in higher eukaryotes. Moreover, this has led to a greater understanding of the underlying pathobiology associated with disease in humans. This tractable model offers an abundance of analytical capabilities, including a vast array of global genetics and molecular resources that allow genome-wide screening to be carried out relatively simply and cheaply. A prime example of the versatility and potential for applying yeast technologies to explore a mammalian disease is in the development of yeast models for amyloid diseases such as Alzheimer's, Parkinson's and Huntington's. The present chapter provides a broad overview of high profile human neurodegenerative diseases that have been modelled in yeast. We focus on some of the most recent findings that have been developed through genetic and drug screening studies using yeast genomic resources. Although this relatively simple unicellular eukaryote seems far removed from relatively complex multicellular organisms such as mammals, the conserved mechanisms for how amyloid exhibits toxicity clearly underscore the value of carrying out such studies in yeast. PMID:25131588

Panaretou, Barry; Jones, Gary W

2014-01-01

336

Yeasts preservation: alternatives for lyophilisation.  

PubMed

The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6 months storage at 4 and 25 °C. None of the yeast cultures showed a significant loss in viable cell count during 6 months of storage at 4 °C upon lyophilisation and preservation in dry rice cakes. During storage at 25 °C in the dark, yeast cultures preserved in dry rice cakes, and lyophilised cultures of Saccharomyces cerevisiae and Issatchenkia orientalis showed no significant loss of viable cells up to 4 months of storage. Yeast cultures preserved in dry plant fibre strands had the greatest loss of viable count during the 6 months of storage at 25 °C. Preservation of yeasts cultures in dry rice cakes provided better survival during storage at 4 °C than lyophilisation. The current study demonstrated that traditional methods can be useful and effective for starter culture preservation in small-scale, low-tech applications. PMID:22806747

Nyanga, Loveness K; Nout, Martinus J R; Smid, Eddy J; Boekhout, Teun; Zwietering, Marcel H

2012-11-01

337

The effect of CH3, F and NO2 substituents on the individual hydrogen bond energies in the adenine-thymine and guanine-cytosine base pairs  

Microsoft Academic Search

The substituent effects on the geometrical parameters and the individual hydrogen bond (HB) energies of base pairs such as\\u000a X–adenine–thymine (X–A–T), X–thymine–adenine (X–T–A), X–guanine–cytosine (X–G–C), and X–cytosine–guanine (X–C–G) have been studied by the quantum mechanical calculations at the B3LYP and MP2 levels with the 6–311++G(d,p) basis set. The\\u000a electron withdrawing (EW) substituents (F and NO2) increase the total binding energy

A. Ebrahimi; S. M. Habibi Khorassani; H. Delarami; H. Esmaeeli

2010-01-01

338

The cytidine deaminase signature HxE(x)n CxxC of DYW1 binds zinc and is necessary for RNA editing of ndhD-1.  

PubMed

In flowering plants, RNA editing involves deamination of specific cytidines to uridines in both mitochondrial and chloroplast transcripts. Pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factor (MORF) proteins have been shown to be involved in RNA editing but none have been shown to possess cytidine deaminase activity. The DYW domain of some PPR proteins contains a highly conserved signature resembling the zinc-binding active site motif of known nucleotide deaminases. We modified these highly conserved amino acids in the DYW motif of DYW1, an editing factor required for editing of the ndhD-1 site in Arabidopsis chloroplasts. We demonstrate that several amino acids of this signature motif are required for RNA editing in vivo and for zinc binding in vitro. We conclude that the DYW domain of DYW1 has features in common with cytidine deaminases, reinforcing the hypothesis that this domain forms part of the active enzyme that carries out RNA editing in plants. PMID:25041347

Boussardon, Clément; Avon, Alexandra; Kindgren, Peter; Bond, Charles S; Challenor, Michael; Lurin, Claire; Small, Ian

2014-09-01

339

ACC (1-Aminocyclopropane-1-Carboxylate) Deaminase Activity, a Widespread Trait in Burkholderia Species, and Its Growth-Promoting Effect on Tomato Plants?  

PubMed Central

The genus Burkholderia includes pathogens of plants and animals and some human opportunistic pathogens, such as the Burkholderia cepacia complex (Bcc), but most species are nonpathogenic, plant associated, and rhizospheric or endophytic. Since rhizobacteria expressing ACC (1-aminocyclopropane-1-carboxylate) deaminase may enhance plant growth by lowering plant ethylene levels, in this work we investigated the presence of ACC deaminase activity and the acdS gene in 45 strains, most of which are plant associated, representing 20 well-known Burkholderia species. The results demonstrated that ACC deaminase activity is a widespread feature in the genus Burkholderia, since 18 species exhibited ACC deaminase activities in the range from 2 to 15 ?mol of ?-ketobutyrate/h/mg protein, which suggests that these species may be able to modulate ethylene levels and enhance plant growth. In these 18 Burkholderia species the acdS gene sequences were highly conserved (76 to 99% identity). Phylogenetic analysis of acdS gene sequences in Burkholderia showed tight clustering of the Bcc species, which were clearly distinct from diazotrophic plant-associated Burkholderia species. In addition, an acdS knockout mutant of the N2-fixing bacterium Burkholderia unamae MTl-641T and a transcriptional acdSp-gusA fusion constructed in this strain showed that ACC deaminase could play an important role in promotion of the growth of tomato plants. The widespread ACC deaminase activity in Burkholderia species and the common association of these species with plants suggest that this genus could be a major contributor to plant growth under natural conditions. PMID:19700546

Onofre-Lemus, Janette; Hernandez-Lucas, Ismael; Girard, Lourdes; Caballero-Mellado, Jesus

2009-01-01

340

RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase.  

PubMed

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV. PMID:23341589

Liang, Guoxin; Kitamura, Kouichi; Wang, Zhe; Liu, Guangyan; Chowdhury, Sajeda; Fu, Weixin; Koura, Miki; Wakae, Kousho; Honjo, Tasuku; Muramatsu, Masamichi

2013-02-01

341

Tumour necrosis factor, interleukin-1 and adenosine deaminase in tuberculous pleural effusion.  

PubMed

Tumour necrosis factor (TNF) and interleukin-1 (IL-1) are powerful mediators with a key role in inflammation. This study was undertaken to study the presence of TNF and IL-1 in tuberculous effusion where there is marked inflammation and where examination of the pleural fluid may give information about the local inflammatory reaction. Adenosine deaminase activity (ADA, a marker of TB pleurisy) was also tested. Tumour necrosis factor, IL-1 and ADA levels were measured in the pleural fluid and serum of 97 patients; 33 with tuberculous effusion, 33 with malignant effusion, and 31 patients with benign non-tuberculous effusion. Pleural fluid TNF and ADA levels were higher in tuberculous (TB) patients than in patients with benign disorders or cancer (P < 0.01). Serum TNF levels were also higher in TB patients than other benign (P < 0.01) or malignant (P < 0.05) effusions. There was a positive correlation between serum and pleural fluid values (r = 0.998-0.999, P < 0.001) although pleural fluid concentration was higher (P < 0.001), possibly suggesting local production in the pleural cavity. Pleural fluid IL-1 levels were not raised in any patient group but there was a positive correlation between TNF and IL-1. In addition, a positive correlation was found between TNF and ADA levels, probably indicating some common production mechanism. Furthermore, ADA sensitivity in the diagnosis of tuberculous effusion was augmented by the combined use of TNF and ADA. The use of both these markers may prove useful in the differential diagnosis of TBC pleurisy. PMID:8730328

Orphanidou, D; Gaga, M; Rasidakis, A; Dimakou, K; Toumbis, M; Latsi, P; Pandalos, J; Christacopoulou, J; Jordanoglou, J

1996-02-01

342

Activation-Induced Cytidine Deaminase Does Not Impact Murine Meiotic Recombination  

PubMed Central

Activation-induced cytidine deaminase (AID) was first described as the triggering enzyme of the B-cell?specific reactions that edit the immunoglobulin genes, namely somatic hypermutation, gene conversion, and class switch recombination. Over the years, AID was also detected in cells other than lymphocytes, and it has been assigned additional roles in the innate defense against transforming retroviruses, in retrotransposition restriction and in DNA demethylation. Notably, AID expression was found in germline tissues, and in heterologous systems it can induce the double-strand breaks required for the initiation of meiotic recombination and proper gamete formation. However, because AID-deficient mice are fully fertile, the molecule is not essential for meiosis. Thus, the remaining question that we addressed here is whether AID influences the frequency of meiotic recombination in mice. We measured the recombination events in the meiosis of male and female mice F1 hybrids of C57BL/6J and BALB/c, in Aicda+/+ and Aicda?/? background by using a panel of single-nucleotide polymorphisms that distinguishes C57BL/6J from BALB/c genome across the 19 autosomes. In agreement with the literature, we found that the frequency of recombination in the female germline was greater than in male germline, both in the Aicda+/+ and Aicda?/? backgrounds. No statistical difference was found in the average recombination events between Aicda+/+ and Aidca?/? animals, either in females or males. In addition, the recombination frequencies between single-nucleotide polymorphisms flanking the immunoglobulin heavy and immunoglobulin kappa loci was also not different. We conclude that AID has a minor impact, if any, on the overall frequency of meiotic recombination. PMID:23550130

Cortesao, Catarina S.; Freitas, Raquel F.; Barreto, Vasco M.

2013-01-01

343

Activities of ectonucleotidases and adenosine deaminase in platelets of dogs experimentally infected with Rangelia vitalii.  

PubMed

Rangeliosis is a disease which affects dogs in Brazil, caused by a piroplasm known as Rangelia vitalii. This disease causes a lot of clinico-pathological features, including the coagulation disorders associated with bleeding. The cause of these changes has not yet been determined. Considering the association of purinergic system and hemostasis this study aimed to evaluate the activity of enzymes that hydrolyze ATP, ADP and AMP; and deamination of adenosine in platelets from dogs experimentally infected with R. vitalii. For this study, 12 healthy young dogs (females) were used, separated in two groups. Group A (n=5) were uninfected controls, and group B were experimentally infected with R. vitalii (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite after five days post-inoculation (PI). Blood samples were collected to quantitate and separate platelets (Day 0, 12 and 21 PI) and to measure the enzymatic activities (Day 12 and 21 PI). The activity of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) was measured in platelets. A reduction (P<0.01) in the number of platelets was observed in R. vitalii-infected blood at Days 12 and 21 PI. At Day 12 PI, a reduction (P<0.01) in the hydrolysis of ATP, ADP and AMP, and deamination of adenosine was observed in dogs infected with R. vitalii. At Day 21 PI the ADA activity remained decreased, unlike the activity of NTPDase which increased (P<0.05). Based on these results we can conclude that ATP, ADP and AMP hydrolysis and adenosine deamination were altered in platelets of R. vitalii-infected dogs. Considering the importance of the purinergic system in hemostasis, it is believed that those changes contribute to the coagulation disorders and bleeding observed in R. vitalii-infected dogs and discussed in this manuscript. PMID:22475775

Paim, Carlos Breno V; Da Silva, Aleksandro S; Paim, Francine C; França, Raqueli T; Costa, Marcio M; Souza, Viviane C G; Pimentel, Victor C; Jaques, Jeandre A; Mazzanti, Cinthia M; Leal, Daniela B R; Monteiro, Silvia G; Schetinger, Maria Rosa C; Lopes, Sonia T A

2012-06-01

344

Diagnosis of tuberculosis pleurisy with adenosine deaminase (ADA): a systematic review and meta-analysis  

PubMed Central

This systematic review and meta-analysis was performed to determine accuracy and usefulness of adenosine deaminase (ADA) in diagnosis of tuberculosis pleurisy. Medline, Google scholar and Web of Science databases were searched to identify related studies until 2014. Two reviewers independently assessed quality of studies included according to standard Quality Assessment of Diagnosis Accuracy Studies (QUADAS) criteria. The sensitivity, specificity, diagnostic odds ratio and other parameters of ADA in diagnosis of tuberculosis pleurisy were analyzed with Meta-DiSC1.4 software, and pooled using the random effects model. Twelve studies including 865 tuberculosis pleurisy patients and 1379 non-tuberculosis pleurisy subjects were identified from 110 studies for this meta-analysis. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnosis odds ratio (DOR) of ADA in the diagnosis of tuberculosis pleurisy were 45.25 (95% CI 27.63-74.08), 0.86 (95% CI 0.84-0.88), 0.88 (95% CI 0.86-0.90), 6.32 (95% CI 4.83-8.26) and 0.15 (95% 0.11-0.22), respectively. The area under the summary receiver operating characteristic curve (SROC) was 0.9340. Our results demonstrate that the sensitivity and specificity of ADA are high in the diagnosis of tuberculosis pleurisy especially when ADA?50 (U/L). Thus, ADA is a relatively sensitive and specific marker for tuberculosis pleurisy diagnosis. However, it is cautious to apply these results due to the heterogeneity in study design of these studies. Further studies are required to confirm the optimal cut-off value of ADA.

Gui, Xuwei; Xiao, Heping

2014-01-01

345

Adenosine deaminase deficiency: genotype-phenotype correlations based on expressed activity of 29 mutant alleles.  

PubMed Central

Adenosine deaminase (ADA) deficiency causes lymphopenia and immunodeficiency due to toxic effects of its substrates. Most patients are infants with severe combined immunodeficiency disease (SCID), but others are diagnosed later in childhood (delayed onset) or as adults (late onset); healthy individuals with "partial" ADA deficiency have been identified. More than 50 ADA mutations are known; most patients are heteroallelic, and most alleles are rare. To analyze the relationship of genotype to phenotype, we quantitated the expression of 29 amino acid sequence-altering alleles in the ADA-deleted Escherichia coli strain SO3834. Expressed ADA activity of wild-type and mutant alleles ranged over five orders of magnitude. The 26 disease-associated alleles expressed 0.001%-0.6% of wild-type activity, versus 5%-28% for 3 alleles from "partials." We related these data to the clinical phenotypes and erythrocyte deoxyadenosine nucleotide (dAXP) levels of 52 patients (49 immunodeficient and 3 with partial deficiency) who had 43 genotypes derived from 42 different mutations, including 28 of the expressed alleles. We reduced this complexity to 13 "genotype categories," ranked according to the potential of their constituent alleles to provide ADA activity. Of 31 SCID patients, 28 fell into 3 genotype categories that could express <=0.05% of wild-type ADA activity. Only 2 of 21 patients with delayed, late-onset, or partial phenotypes had one of these "severe" genotypes. Among 37 patients for whom pretreatment metabolic data were available, we found a strong inverse correlation between red-cell dAXP level and total ADA activity expressed by each patient's alleles in SO3834. Our system provides a quantitative framework and ranking system for relating genotype to phenotype. PMID:9758612

Arredondo-Vega, F X; Santisteban, I; Daniels, S; Toutain, S; Hershfield, M S

1998-01-01

346

Activation-Induced Deaminase, AID, is catalytically active as a monomer on single-stranded DNA  

PubMed Central

Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate non-homologous recombination in the case of class-switch recombination. In vitro studies have demonstrated that AID deaminates single-stranded but not double-stranded substrates unless AID is in a complex with RPA and the substrate is actively undergoing transcription. However, it is not clear whether AID deaminates its substrates primarily as a monomer or as a higher order oligomer. To examine the oligomerization state of AID alone and in the presence of single stranded DNA substrates of various structures, including loops embedded in double-stranded DNA, we used atomic force microscopy (AFM) to visualize AID protein alone or in complex with DNA. Surprisingly, AFM results indicate that most AID molecules exist as a monomer and that it binds single-stranded DNA substrates as a monomer at concentrations where efficient deamination of single-stranded DNA substrates occur. The rate of deamination, under conditions of excess and limiting protein, also imply that AID can deaminate single-stranded substrates as a monomer. These results imply that non-phosphorylated AID is catalytically active as a monomer on single stranded DNA in vitro, including single-stranded DNA found in loops similar to those transiently formed in the immunoglobulin switch regions during transcription. PMID:17889624

Brar, Sukhdev S.; Sacho, Elizabeth J.; Tessmer, Ingrid; Croteau, Deborah L.; Erie, Dorothy A.; Diaz, Marilyn

2009-01-01

347

Expression of Drosophila Adenosine Deaminase in Immune Cells during Inflammatory Response  

PubMed Central

Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. Herein we present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. We show that the reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. Our vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. Our results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, we show that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented here, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role. PMID:21412432

Novakova, Milena; Dolezal, Tomas

2011-01-01

348

Crystallization and preliminary X-ray analysis of the hypothetical deaminase RPB_0146 from Rhodopseudomonas palustris HaA2.  

PubMed

RPB_0146, a putative deaminase from Rhodopseudomonas palustris HaA2, was expressed in Escherichia coli BL21 (DE3) cells and purified using a His6 tag by Ni(2+)-chelating affinity chromatography for X-ray crystallographic analysis. Diffraction-quality crystals were grown by the hanging-drop vapour-diffusion method at 289?K and diffracted to a resolution of 2.44?Å using a wavelength of 1.000?Å at the Photon Factory (KEK), Japan. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 66.26, b = 123.94, c = 155.95?Å. PMID:25372831

Zhang, Guofang; Yu, Dan; Yang, Guodong; Dong, Hui; Zhang, Tongcun; Liu, Xiang

2014-11-01

349

Adenosine Deaminase Acts as a Natural Antagonist for Dipeptidyl Peptidase 4-Mediated Entry of the Middle East Respiratory Syndrome Coronavirus  

PubMed Central

Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection. PMID:24257613

Raj, V. Stalin; Smits, Saskia L.; Provacia, Lisette B.; van den Brand, Judith M. A.; Wiersma, Lidewij; Ouwendijk, Werner J. D.; Bestebroer, Theo M.; Spronken, Monique I.; van Amerongen, Geert; Rottier, Peter J. M.; Fouchier, Ron A. M.; Bosch, Berend Jan; Osterhaus, Albert D.M.E.

2014-01-01

350

Evidence from ¹⁵N and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase  

Microsoft Academic Search

They have determined ¹⁵N and solvent deuterium isotope effects (SDIE) for adenosine deaminase using both adenosine and the slow alternate substrate, 8-oxoadenosine. With adenosine, the isotope effects were small: ¹⁵(V\\/K) was 1.0040 in HâO and 1.0023 in DâO, and the SDIE was 0.9. With 8-oxoadenosine, however, larger values were observed: ¹⁵(V\\/K) was 1.015 in HâO and 1.013 in DâO and

Paul M. Weiss; P. F. Cook; J. D. Hermes; W. W. Cleland

1987-01-01

351

Nuclear transport of yeast proteasomes.  

PubMed

Proteasomes are conserved protease complexes enriched in the nuclei of dividing yeast cells, a major site for protein degradation. If yeast cells do not proliferate and transit to quiescence, metabolic changes result in the dissociation of proteasomes into proteolytic core and regulatory complexes and their sequestration into motile cytosolic proteasome storage granuli. These granuli rapidly clear with the resumption of growth, releasing the stored proteasomes, which relocalize back to the nucleus to promote cell cycle progression. Here, I report on three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells. The first model applies for dividing yeast and is based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin ??. The second model applies for quiescent yeast cells, which resume growth and use Blm10, a HEAT-like repeat protein structurally related to karyopherin ?, for nuclear import of proteasome core particles. In the third model, the fully-assembled proteasome is imported into the nucleus. Our still marginal knowledge about proteasome dynamics will inspire the discussion on how protein degradation by proteasomes may be regulated in different cellular compartments of dividing and quiescent eukaryotic cells. PMID:25333764

Enenkel, Cordula

2014-01-01

352

Microhydration of the guanine-cytosine (GC) base pair in the neutral and anionic radical states: a density functional study.  

PubMed

A density functional study of the effects of microhydration on the guanine-cytosine (GC) base pair and its anion radical is presented. Geometries of the GC base pair in the presence of 6 and 11 water molecules were fully optimized in the neutral (GC-nH2O) and anion radical [(GC-nH2O)*-] (n = 6 and 11) states using the B3LYP method and the 6-31+G** basis set. Further, vibrational frequency analysis at the same level of theory (B3LYP/6-31+G**) was also performed to ensure the existence of local minima in these hydrated structures. It was found that water molecules surrounding the GC base pair have significant effects on the geometry of the GC base pair and promote nonplanarity in the GC base pair. The calculated structures were found to be in good agreement with those observed experimentally and obtained in molecular dynamics (MD) simulation studies. The water molecules in neutral GC-nH2O complexes lie near the ring plane of the GC base pair where they undergo hydrogen bonding with both GC and each other. However, in the GC anion radical complexes (GC-nH2O, n = 6, 11), the water molecules are displaced substantially from the GC ring plane. For GC-11H2O*-, a water molecule is hydrogen-bonded with the C6 atom of the cytosine base. We found that the hydration shell initially destabilizes the GC base pair toward electron capture as a transient anion. Energetically unstable diffuse states in the hydration shell are suggested to provide an intermediate state for the excess electron before molecular reorganization of the water molecules and the base pair results in a stable anion formation. The singly occupied molecular orbital (SOMO) in the anion radical complexes clearly shows that an excess electron localizes into a pi orbital of cytosine. The zero-point-energy (ZPE-) corrected adiabatic electron affinities (AEAs) of the GC-6H2O and GC-11H2O complexes, at the B3LYP/6-31+G** level of theory, were found to be 0.74 and 0.95 eV, respectively. However, the incorporation of bulk water as a solvent using the polarized continuum model (PCM) increases the EAs of these complexes to 1.77 eV. PMID:18380501

Kumar, Anil; Sevilla, Michael D; Suhai, Sandor

2008-04-24

353

Cdc42 Oscillations in Yeasts  

NSDL National Science Digital Library

A fundamental problem in cell biology is how cells define one or several discrete sites of polarity. Through mechanisms involving positive and negative feedback, the small Rho-family guanosine triphosphatase Cdc42 breaks symmetry in round budding yeast cells to define a single site of polarized cell growth. However, it is not clear how cells can define multiple sites of polarization concurrently. We discuss a study in which rod-shaped fission yeast cells, which naturally polarize growth at their two cell ends, exhibited oscillations of Cdc42 activity between these sites. We compare these findings with similar oscillatory behavior of Cdc42 detected in budding yeast cells and discuss the possible mechanism and functional outputs of these oscillations.

Felipe O. Bendezu (Switzerland;University of Lausanne REV); Sophie G. Martin (Switzerland;University of Lausanne REV)

2012-12-04

354

The yeasts of cheese brines.  

PubMed

A total of 365 yeasts were isolated from the brines of soft, semihard and hard cheeses from different manufacturers. Identification was based on 131 characteristics, primarily employing a method with microtitration plates. Most brines exhibited a characteristic yeast flora. The predominant strains proved to be mainly Debaryomyces hansenii and Candida versatilis. In a few brines Trichosporon beigelii, C. rugosa, C. intermedia, Kluyveromyces marxianus, Saccharomyces sp. and C. tenuis/polymorpha were predominant. Also of importance were C. tropicalis, C. parapsilosis, C. zeylanoides, Issatchenkia orientalis and Geotrichum klebahnii. Not all strains could be clearly identified. Lists of characters are provided for subdividing D. hansenii and T. beigelii. The specificity of the yeast flora of brines is assumed to contribute to the sensory variety of cheeses. PMID:2282287

Seiler, H; Busse, M

1990-12-01

355

280 EXPRESSION IN YEAST [23] [23] Manipulating Yeast Genome Using Plasmid Vectors  

E-print Network

280 EXPRESSION IN YEAST [23] [23] Manipulating Yeast Genome Using Plasmid Vectors By TIM STEARNS, HONG MA, and DAVID BOTSTEIN The yeast Saccharomyces cerevisiae has proved to be a popular high status of yeast as an experimental system is in large part due to the work of the many geneticists

Botstein, David

356

APPENDIX 4LGrowth and Manipulation of Yeast PREPARATION OF SELECTED YEAST MEDIA  

E-print Network

APPENDIX 4LGrowth and Manipulation of Yeast PREPARATION OF SELECTED YEAST MEDIA Like Escherichia coli, yeast can be grown in either liquid media or on the surface of (or embedded in) solid agar plates. Yeast cells grow well on a minimal medium containing dextrose (glucose) as a carbon source and salts

Winston, Fred

357

Pre-Absorbing Antibody with Yeast Cells Preparation of Fixed Yeast  

E-print Network

106 Pre-Absorbing Antibody with Yeast Cells Preparation of Fixed Yeast 1. Plan to do steps 1-10 in the yeast immunofluorescence method. But, start with 100 mls of cells at OD600=0.2. Then, do all steps in quadruplicate. Do pretreatment, and digest cells for 10 minutes. 2. Pool all yeast in SPC + Pics in one

Aris, John P.

358

A 5? cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs  

SciTech Connect

A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the 'core' PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

Zhu, Deyu; Stumpf, Craig R.; Krahn, Joseph M.; Wickens, Marvin; Tanaka Hall, Traci M.; (NIEHS); (UW)

2010-11-03

359

The Yeast Nuclear Pore Complex  

PubMed Central

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport. PMID:10684247

Rout, Michael P.; Aitchison, John D.; Suprapto, Adisetyantari; Hjertaas, Kelly; Zhao, Yingming; Chait, Brian T.

2000-01-01

360

Chromatin and Transcription in Yeast  

PubMed Central

Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

Rando, Oliver J.; Winston, Fred

2012-01-01

361

Modelling the Yeast Interactome Vuk Janjic1  

E-print Network

Modelling the Yeast Interactome Vuk Janjic´1 , Roded Sharan2 & Natasa Przulj1 1 Department of any empirical observation regarding those networks. Here, we perform a comprehensive analysis of yeast complexity (human and yeast); (3) clear topological difference is present between PPI networks

Shamir, Ron

362

Yeast communities associated with stingless bees  

Microsoft Academic Search

The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied. The bees T. angustula and F. varia showed a strong association with the yeast Starmerella meliponinorum. M. quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S. meliponinorum. Some of the yeasts isolated from adult bees

Carlos A Rosa; Marc-André Lachance; Jana??na O. C Silva; Ana Carolina P Teixeira; Marjorie M Marini; Yasmine Antonini; Rogerio P Martins

2003-01-01

363

Red yeast rice: a new hypolipidemic drug  

Microsoft Academic Search

Red yeast rice is a source of fermented pigment with possible bioactive effect. Evidence shows that fermented red yeast rice lowers cholesterol levels moderately compared to other statin drugs, but with the added advantage of causing less adverse effects. A review of the body of evidence surrounding the properties of red yeast rice underscores its potential as a new alternative

Mélanie Journoud; Peter J. H Jones

2004-01-01

364

Yeast community survey in the Tagus estuary  

Microsoft Academic Search

The yeast community in the waters of the Tagus estuary, Portugal, was followed for over a year in order to assess its dynamics. Yeast occurrence and incidence were measured and this information was related to relevant environmental data. Yeast occurrence did not seem to depend upon tides, but river discharge had a dramatic impact both on the density and diversity

João M. G. C. F. de Almeida

2005-01-01

365

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression  

PubMed Central

Background DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. Results We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. Conclusions We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation. PMID:22251412

2012-01-01

366

Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum.  

PubMed

A series of overlapping clones complementary to the Arabidopsis cytosine-5 DNA methyltransferase (C-5 MTase) has been isolated from pea cDNA libraries. The assembled nucleic acid sequence contains an open reading frame of 4761 bp encoding a protein of 1554 amino acids. Like other eukaryotic C-5 MTases, the inferred protein has a presumed regulatory N-terminal region linked to a catalytic C-terminal domain, which has eight of the ten conserved motifs found in prokaryotic C-5 MTases. The pea C-5 MTase has 65% identity at the nucleotide level and 61% identity at the protein level, with the Arabidopsis C-5 MTase. The catalytic domain of the pea enzyme shares 78% identity with Arabidopsis and approximately 52% identity with murine and human C-5 MTases, including the relative position of the proline-cysteine dipeptides of the catalytic centre. Using the conserved region of the cDNA as a probe, we have identified a transcript of 5 kb. Southern blot analysis of pea genomic DNA with the above probe indicates the presence of a single gene. Using poly(A)+ RNA from different developmental stages and different tissues, we have observed that expression is confined mostly to the rapidly dividing tissues of the plant. Expression of this assembled cDNA in a baculovirus system gives a protein of approximately 174 kDa. The expressed protein can be cross-linked, in an AdoMet-dependent manner, to duplex oligonucleotide substrates containing FdC in place of target cytosines in either CG or CAG/CTG sequences. PMID:9469828

Pradhan, S; Cummings, M; Roberts, R J; Adams, R L

1998-03-01

367

Evidence for a dynamic role for proline376 in the purine-cytosine permease of Saccharomyces cerevisiae.  

PubMed

The purine-cytosine permease (PCP), a carrier located in the plasma membrane of Saccharomyces cerevisiae, mediates the active transport of purine (adenine, guanine and hypoxanthine) and cytosine into the cell. Previous studies [Ferreira, T, Brèthes, D., Pinson, B., Napias, C. & Chevallier, J. et al. (1997) J. Biol. Chem. 272, 9697-9702] suggest that the hydrophilic segment 371-377 (-I-A-N-N-I-P-N-) of the polypeptide chain may play a key role in the correct three-dimensional structure of the active carrier. This paper describes the effects of mutations in this particular segment: a four-residue deletion, Delta374-377, and two substitutions, P376G and P376R. The Delta374-377 PCP was expressed in tiny amounts and was totally inactive. When compared with the wild-type, the P376G PCP showed slightly decreased amounts and was able to transport the bases with significantly increased affinity and decreased turnover. The P376R PCP was normally expressed and targeted to the plasma membrane; however, despite a normal number of base-binding sites [1000-1200 pmol.(mg protein)-1], this mutated carrier was completely unable to transport any of its ligands. In addition, the Kd(app) for hypoxanthine binding was completely independent of the pH (within the range 3.5-6.0), showing that the conformational change induced by ligand binding was no longer present. Our results show that the 374-377 segment is essential for the expression and activity of this carrier. They also show that the P376 residue is part of an unusual secondary structure, probably a beta-turn motif, which must play a crucial dynamic role in the translocation process. PMID:10429187

Ferreira, T; Napias, C; Chevallier, J; Brèthes, D

1999-07-01

368

Piracetam prevents scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities.  

PubMed

Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 ?mol/5 ?L, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5'-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5'-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5'-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam. PMID:23677777

Marisco, Patricia C; Carvalho, Fabiano B; Rosa, Michelle M; Girardi, Bruna A; Gutierres, Jessié M; Jaques, Jeandre A S; Salla, Ana P S; Pimentel, Víctor C; Schetinger, Maria Rosa C; Leal, Daniela B R; Mello, Carlos F; Rubin, Maribel A

2013-08-01

369

A novel activation-induced cytidine deaminase (AID) mutation in Brazilian patients with hyper-IgM type 2 syndrome.  

PubMed

Activation-induced cytidine deaminase (AID) is a DNA editing protein that plays an essential role in three major events of immunoglobulin (Ig) diversification: somatic hypermutation, class switch recombination and Ig gene conversion. Mutations in the AID gene (AICDA) have been found in patients with autosomal recessive Hyper-IgM (HIGM) syndrome type 2. Here, two 9- and 14-year-old Brazilian sisters, from a consanguineous family, were diagnosed with HIGM2 syndrome. Sequencing analysis of the exons from AICDA revealed that both patients are homozygous for a single C to G transversion in the third position of codon 15, which replaces a conserved Phenylalanine with a Leucine. To our knowledge, this is a new AICDA mutation found in HIGM2 patients. Functional studies confirm that the homologous murine mutation leads to a dysfunctional protein with diminished intrinsic cytidine deaminase activity and is unable to rescue CSR when introduced in Aicda(-/-)stimulated murine B cells. We briefly discuss the relevance of AICDA mutations found in patients for the biology of this molecule. PMID:23803409

Caratão, Nadine; Cortesão, Catarina S; Reis, Pedro H; Freitas, Raquel F; Jacob, Cristina M A; Pastorino, Antonio C; Carneiro-Sampaio, Magda; Barreto, Vasco M

2013-08-01

370

Structural and Kinetic Characterization of Escherichia coli TadA, the Wobble-Specific tRNA Deaminase  

SciTech Connect

The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k{sub cat} = 13 {+-} 1 min{sup -1} and K{sub M} = 0.83 {+-} 0.22 {micro}M). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 {micro}M). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.

Kim,J.; Malashkevich, V.; Roday, S.; Lisbin, M.; Schramm, V.; Almo, S.

2006-01-01

371

Stability of the valence anion of cytosine is governed by nucleobases sequence in the double stranded DNA ?-stack: A computational study  

NASA Astrophysics Data System (ADS)

The stabilities of the valence anion of cytosine (C-) in model trimers of complementary base pairs that possess the B-DNA geometry but differ in base sequence are reported. In order to estimate the energetics of electron attachment to the middle cytosine incorporated in the trimer, a thermodynamic cycle employing all possible two-body interaction energies in the neutral and anionic duplex as well as the adiabatic electron affinity of isolated cytosine were developed. All calculations were carried out at the MP2 level of theory with the aug-cc-pVDZ basis set. We have demonstrated that contrary to the literature reports, concerning single stranded DNA, the sequence of nucleic bases has a profound effect on the stability of the cytosine valence anion. The anionic 3'-CCC-5' complex is the most stable configuration (EA=0.399 eV) and the 3'-GCG-5' trimer anion is the most unstable species (EA=-0.193 eV). Moreover, with the energetic correction for the presence of sugar-phosphate backbone all possible double stranded DNA sequences lead to the stable C-. The predicted electron affinities of the cytosine anion have been compared to the results of analogous studies on the thymine anion published recently [M. Koby?ecka et al., J. Am. Chem. Soc. 130, 15683 (2008)]. The consequences of low-energy barrier proton transfer in the GC anion have been discussed in the context of induced by electrons DNA single strand breaks. The DNA sequences that should dramatically differ in their vulnerability to be damaged by low energy electrons have been proposed.

Koby?ecka, Monika; Leszczynski, Jerzy; Rak, Janusz

2009-08-01

372

Stability of the valence anion of cytosine is governed by nucleobases sequence in the double stranded DNA pi-stack: A computational study.  

PubMed

The stabilities of the valence anion of cytosine (C(-)) in model trimers of complementary base pairs that possess the B-DNA geometry but differ in base sequence are reported. In order to estimate the energetics of electron attachment to the middle cytosine incorporated in the trimer, a thermodynamic cycle employing all possible two-body interaction energies in the neutral and anionic duplex as well as the adiabatic electron affinity of isolated cytosine were developed. All calculations were carried out at the MP2 level of theory with the aug-cc-pVDZ basis set. We have demonstrated that contrary to the literature reports, concerning single stranded DNA, the sequence of nucleic bases has a profound effect on the stability of the cytosine valence anion. The anionic 3(')-CCC-5(') complex is the most stable configuration (EA=0.399 eV) and the 3(')-GCG-5(') trimer anion is the most unstable species (EA=-0.193 eV). Moreover, with the energetic correction for the presence of sugar-phosphate backbone all possible double stranded DNA sequences lead to the stable C(-). The predicted electron affinities of the cytosine anion have been compared to the results of analogous studies on the thymine anion published recently [M. Koby?ecka et al., J. Am. Chem. Soc. 130, 15683 (2008)]. The consequences of low-energy barrier proton transfer in the GC anion have been discussed in the context of induced by electrons DNA single strand breaks. The DNA sequences that should dramatically differ in their vulnerability to be damaged by low energy electrons have been proposed. PMID:19725636

Koby?ecka, Monika; Leszczynski, Jerzy; Rak, Janusz

2009-08-28

373

Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.  

PubMed Central

Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w. PMID:10445884

Motorin, Y; Grosjean, H

1999-01-01

374

Observations on the Yeast Lipomyces  

Microsoft Academic Search

IN 1946, Starkey1 isolated and described a soil yeast characterized by a peculiar method of spore formation after a relatively long period of growth on solid medium. Large, round vegetative cells containing fat globules gave rise to irregularly shaped protuberances in which were afterwards formed 4-16 or more lightly pigmented spores. Lodder and Kregervan Rij2 considered these spores to be

Catherine Roberts

1957-01-01

375

Genetically modified industrial yeast ready for application.  

PubMed

Tremendous progress in the genetic engineering of yeast had been achieved at the end of 20th century, including the complete genome sequence, genome-wide gene expression profiling, and whole gene disruption strains. Nevertheless, genetically modified (GM) baking, brewing, wine, and sake yeasts have not, as yet, been used commercially, although numerous industrial recombinant yeasts have been constructed. The recent progress of genetic engineering for the construction of GM yeast is reviewed and possible requirements for their application are discussed. 'Self-cloning' yeast will be the most likely candidate for the first commercial application of GM microorganisms in food and beverage industries. PMID:16233347

Akada, Rinji

2002-01-01

376

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation.  

PubMed

Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion. PMID:22846334

Barnawal, Deepti; Bharti, Nidhi; Maji, Deepamala; Chanotiya, Chandan Singh; Kalra, Alok

2012-09-01

377

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala.  

PubMed

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala. PMID:18406559

Tittabutr, Panlada; Awaya, Jonathan D; Li, Qing X; Borthakur, Dulal

2008-06-01

378

Effects of Retroviral Vector Design on Expression of Human Adenosine Deaminase in Murine Bone Marrow Transplant Recipients Engrafted with Genetically Modified Cells  

Microsoft Academic Search

To determine which features of retroviral vector design most critically affect gene expression in hematopoietic cells in vivo, we have constructed a variety of different retroviral vectors which encode the same gene product, human adenosine deaminase (EC 3.5.4.4), and possess the same vector backbone yet differ specifically in transcriptional control sequences suggested by others to be important for gene expression

Isabelle Riviere; Katja Brose; Richard C. Mulligan

1995-01-01

379

Substrate- and product-affinity resins for adenosine deaminase obtained by immobilisation of adenosine and inosine via 2',3'-cyclic acetal derivatives.  

PubMed

Immobilised inosine (6a) and adenosine (6c) and their 5'-phosphates have been synthesized. Reaction of the nucleosides with ethyl levulinate, followed by saponification or phosphorylation and then saponification, gave the 2',3'-O-[1-(2-carboxyethyl)ethylidene] derivatives 3 and 4 and the corresponding 5'-phosphates 2b and 2d. 6-Aminohexylagarose (5) was severally coupled to 2b, 2d, 3, and 4 through the carboxyl groups to give the polymers 6a-d. Adenosine deaminase converts 3 into 4, and 6c into 6a. The polymers can be used as affinity resins for adenosine deaminase, which is bound more strongly to 6c than to 6a. The operational capacity of 6a for adenosine deaminase is constant at 15--25 degrees, but decreases by approximately 16% from 25 degrees to 35 degrees. The resin 6a has been used to separate adenosine deaminase from mixtures containing other enzymes, for example, guanase or alcohol dehydrogenase. PMID:647708

Rosemeyer, H; Seela, F

1978-04-01

380

EFFECT OF REPEATED PESTICIDE APPLICATIONS ON SOIL PROPERTIES IN COTTON FIELDS. II. INSECTICIDE RESIDUES AND IMPACT ON DEHYDROGENASE AND ARGININE DEAMINASE ACTVITIES  

Microsoft Academic Search

Insecticides were applied sequentially at recommended dosages post crop emergence in cotton fields and soil was sampled at regular intervals after each treatment. Soil was analysed for insecticide residues and activity of the enzymes dehydrogenase and arginine deaminase. Insecticide residues detected in the soil were in small quantities and they did not persist for long. Only endosulfan leached below 15

KOMAL VIG; DILEEP K. SINGH; H. C AGARWAL; A. K. DHAWAN; PREM DUREJA

381

Guanine repeat-containing sequences confer transcription-dependent instability in an orientation-specific manner in yeast  

PubMed Central

Non-B DNA structures are a major contributor to the genomic instability associated with repetitive sequences. Immunoglobulin switch Mu (S?) region sequence is comprised of guanine-rich repeats and has high potential for forming G4 DNA, in which one strand of DNA folds into an array of guanine quartets. Taking advantage of the genetic tractability of Saccharomyces cerevisiae, we developed a recombination assay to investigate mechanisms involved in maintaining stability of G-rich repetitive sequence. By embedding S? sequence within recombination substrates under the control of a tetracycline-regulatable promoter, we demonstrate that the rate and orientation of transcription both affect the stability of S? sequence. In particular, the greatest instability was observed under high-transcription conditions when the S? sequence was oriented with the C-rich strand as the transcription template. The effect of transcription orientation was enhanced in the absence of the Type IB topoiosmerase Top1, possibly due to enhanced R-loop formation. Loss of Sgs1 helicase and RNase H activity also increased instability, suggesting they may cooperatively function to reduce the formation of non-B DNA structures in highly transcribed regions. Finally, the S? sequence was unstable when transcription elongation was perturbed due to a defective THO complex. In a THO-deficient background, there was further exacerbation of orientation-dependent instability associated with the ectopically expressed, single-strand cytosine deaminase AID. The implications of our findings to understanding instability associated with potential G4 DNA forming sequences are discussed. PMID:21813340

Kim, Nayun; Jinks-Robertson, Sue

2011-01-01

382

Occurrence and Growth of Yeasts in Yogurts  

PubMed Central

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 103 cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 ?g of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar. PMID:16345853

Suriyarachchi, V. R.; Fleet, G. H.

1981-01-01

383

Control of ATP homeostasis during the respiro-fermentative transition in yeast  

PubMed Central

Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content ([ATP]+[ADP]+[AMP]=[AXP]). The unknown fate of ‘lost' AXP nucleotides represented a long-standing problem for the understanding of the yeast's physiological response to changing growth conditions. Transient accumulation of the purine salvage pathway intermediate, inosine, accounted for the apparent loss of adenine nucleotides. Conversion of AXPs into inosine was facilitated by AMP deaminase, Amd1, and IMP-specific 5?-nucleotidase, Isn1. Inosine recycling into the AXP pool was facilitated by purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1. Analysis of changes in 24 intracellular metabolite pools during the respiro-fermentative growth transition in wild-type, amd1, isn1, and pnp1 strains revealed that only the amd1 mutant exhibited significant deviations from the wild-type behavior. Moreover, mutants that were blocked in inosine production exhibited delayed growth acceleration after glucose addition. It is proposed that interconversion of adenine nucleotides and inosine facilitates rapid and energy-cost efficient adaptation of the AXP pool size to changing environmental conditions. PMID:20087341

Walther, Thomas; Novo, Maite; Rossger, Katrin; Letisse, Fabien; Loret, Marie-Odile; Portais, Jean-Charles; Francois, Jean-Marie

2010-01-01

384

Bacterial biosynthesis of 1-aminocyclopropane-1-caboxylate (ACC) deaminase, a useful trait to elongation and endophytic colonization of the roots of rice under constant flooded conditions.  

PubMed

This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Pseudomonas fluorescens strain REN1 and its ability to reduce ethylene levels produced during stress, endophytically colonize and promote the elongation of the roots of rice seedlings under gnotobiotic conditions. We isolated 80 bacteria from inside roots of rice plants grown in the farmers' fields in Guilan, Iran. All of the isolates were characterized for plant growth promoting (PGP) traits. In addition, the colonization assay of these isolates on rice seedlings was carried out to screen for competent endophytes. The best bacterial isolate, based on ACC deaminase production, was identified and used for further study. 16S rDNA sequence analysis revealed that the endophyte was closely related to Pseudomonas fluorescens. The results of this study showed ACC deaminase containing P. fluorescens REN1 increased in vitro root elongation and endophytically colonized the root of rice seedlings significantly, as compared to control under constant flooded conditions. The trait of low amount of indole-3-acetic acid (IAA) production (<15 ?g mL(-1)) and the high production of ACC deaminase by bacteria may be main factors in colonizing rice seedling roots compared to other PGP traits (siderophore production and phosphate solubilization) in this study. Endophytic IAA and ACC deaminase-producing bacteria may be preferential selections by rice seedlings. Therefore, it may be suggested that the utilization of ACC as a nutrient gives the isolates advantages in more endophytic colonization and increase of root length of rice seedlings. PMID:25320466

Etesami, Hassan; Mirseyed Hosseini, Hossein; Alikhani, Hossein Ali

2014-10-01

385

Preparation of extracts from yeast.  

PubMed

Because yeast is exceptionally well suited to genetic analysis, both classical and molecular, it is an attractive system for expressing recombinant animal proteins for purification purposes. Methods available for lysing yeast cells include autolysis, pressure cells (e.g., French press), abrasives (glass bead vortexing), and enzymatic lysis (e.g., zymolase). One of the simplest methods, discussed in this protocol, involves the abrasive action of well-agitated glass beads. This is a very effective method for both low volumes (e.g., <1 mL using a microcentrifuge tube) and many liters using a specialized DynoMill apparatus. Cell breakage is typically >95%, as assessed by phase-contrast microscopy. PMID:21205845

Simpson, Richard J

2011-01-01

386

Oxidative stress responses in yeast  

Microsoft Academic Search

Yeast, and especially S. cerevisiae, is a unique eukaryotic model organism for studying oxidative stress and its cellular responses. S. cerevisiae has become a very powerful tool to decipher the complexity of these biologically important responses, because it offers the\\u000a relative simplicity of a single celled eukaryotic organism that enables the combination and integration of genetic, biochemical,\\u000a physico-chemical, cell biological,

Michel B. Toledano; Agnes Delaunay; Benoit Biteau; Daniel Spector; Dulce Azevedo

387

Yeast adaptation on softwood prehydrolysate  

Microsoft Academic Search

Several strains and genera of yeast, includingSaccharomyces cerevisiae D5A,Pachysolen tannophilus, S. cerevisiae K-l,Brettanomyces custersii, Candida shehatae, andCandida acidothermophilum, are screened for growth on dilute acid-pretreated softwood prehydrolysate. Selected softwood species found in forest underbrush\\u000a of the western United States, which contain predominantly hexosan hemicellulose, were studied. This phase of the work emphasized\\u000a debarked Douglas fir. The two best initial isolates

Fcred A. Keller; Delicia Bates; Ray Ruiz; Quang Nguyen

1998-01-01

388

Gas-Phase Interactions between Lead(II) Ions and Cytosine: Tandem Mass Spectrometry and Infrared Multiple-Photon Dissociation Spectroscopy Study.  

PubMed

Gas-phase interactions between Pb(2+) ions and cytosine (C) were studied by combining tandem mass spectrometry, infrared multiple photon dissociation spectroscopy, and density functional theory (DFT) calculations. Both singly and doubly charged complexes were generated by electrospray. The [Pb(C)-H](+) complex was extensively studied, and this study shows that two structures, involving the interaction of the metal with the deprotonated canonical keto-amino tautomer of cytosine, are generated in the gas phase; the prominent structure is the bidentate form involving both the N1 and O2 electronegative centers. The DFT study also points out a significant charge transfer from the nucleobase to the low-lying p orbitals of the metal and a strong polarization of the base upon complexation. The various potential energy surfaces explored to account for the fragmentation observed are consistent with the high abundance of the [PbNH2 ](+) fragment ion. PMID:25044836

Salpin, Jean-Yves; Haldys, Violette; Guillaumont, Sébastien; Tortajada, Jeanine; Hurtado, Marcela; Lamsabhi, Al Mokhtar

2014-10-01

389

Structure and the energy of base pairing in non-natural bases of nucleic acids: the azaguanine-cytosine and azaadenine-thymine base pairs.  

PubMed

Watson-Crick optimized geometries and the energies of base pairing for the natural pairs of nucleic bases: adenine-thymine (AT) and guanine-cytosine (GC) have been recalculated by ab initio methods in order to compare results to those found for the non-natural azaadenine-thymine (AAT) and azaguanine-cytosine (AGC) pairs. Geometry optimizations carried out at the HF/6-31G** level and energies obtained at MP2/6-31G**, show that AAT and AGC have hydrogen bonding patterns similar to the natural AT and GC and that the interaction energies (DeltaH0int) for the former are ca. 7 kcal/mol more stable than the latter. Accordingly, the pairs based on azapurines would be favored with respect to the natural pairs. Some possible explanations why nature does not use extensively the azabases in base pairing are given. PMID:12941289

Contreras, J Guillermo; Madariaga, Sandra T

2003-10-01

390

Biodegradation of Oil Pollutants by Yeasts and Yeast-Like Fungi.  

National Technical Information Service (NTIS)

In exploring the feasibility of the use of microbial systems for the facilitated biodegradation of waste oils, yeasts and yeast-like fungi from marine, freshwater and terrestrial sources were screened for their ability to utilize hydrocarbons. Mixed cultu...

D. G. Ahearn, N. H. Berner

1978-01-01

391

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine  

Microsoft Academic Search

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C\\/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J\\/m2). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks\\/108 daltons following cell culture exposure to ara-C or ara-C\\/HU in a series

William C. Dunn; James D. Regan; Ronald D. Snyder

1985-01-01

392

Calculation and analysis of vibrational spectra of adenine–thymine, guanine–cytosine, and adenine–uracil complementary pairs in the condensed state  

Microsoft Academic Search

We have calculated the frequencies of the normal vibrations of the complementary nucleic acid base pairs adenine–thymine,\\u000a guanine–cytosine, adenine–uracil, corresponding to the Watson–Crick structure, and the adenine–uracil pair, corresponding\\u000a to the Hoogsteen structure, in condensed states and we interpret the spectra. We determine the contributions of hydrogen bonds\\u000a to the vibrational modes of the complementary pairs. We have analyzed the

G. N. Ten; T. G. Burova; V. I. Baranov

2009-01-01

393

The Electron Affinities of the Radicals Formed by the Loss of an Aromatic Hydrogen Atom from Adenine, Guanine, Cytosine, Uracil, and Thymine  

Microsoft Academic Search

The major ion formed in Negative Ion Chemical Ionization Mass Spectrometry of Adenine, Guanine, Cytosine, Uracil and Thymine is the dehydrogenated anion. The CURES EC procedure for optimizing Austin Model-1 Multiconfigurational Configuration Interaction semi-empirical calculations is applied to the electron affinities of the corresponding dehydrogenated bases and N–H bond dissociation energies. These calculated values will be compared with literature values

E. S. D. Chen; E. C. M. Chen; Neeta Sane

1998-01-01

394

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)

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.

Shanak, Siba; Helms, Volkhard

2014-12-01

395

Assembly of eukaryotic algal chromosomes in yeast  

PubMed Central

Background Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (>?~?150 kb), high G?+?C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G?+?C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G?+?C content. The model diatom Phaeodactylum tricornutum has an average G?+?C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast. Results We present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency. Conclusions Entire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G?+?C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly. PMID:24325901

2013-01-01

396

Yeasts in floral nectar: a quantitative survey  

PubMed Central

Background and Aims One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored. Methods The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37–63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated. Key Results Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 × 105 cells mm?3). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 104 cells mm?3 were commonplace, and densities >105 cells mm?3 were not rare. About one-fifth of species at each site had mean yeast cell densities >104 cells mm?3. Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees. Conclusions Incorporating nectar yeasts into the scenario of plant–pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence of yeasts in nectar samples. PMID:19208669

Herrera, Carlos M.; de Vega, Clara; Canto, Azucena; Pozo, Maria I.

2009-01-01

397

The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants  

PubMed Central

Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005). The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes) compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes) (p = 0.002). Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine deaminase locus 1 phenotype 1 carriers (p = 0.04). The two systems show a cooperative effect on the correlation between birth weight and placental weight: the highest value is observed in newborns carrying adenosine deaminase locus 1 allele*2 and acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (p = 0.005). Conclusion These data suggest that zygotes with low adenosine deaminase locus 1 activity and low F activity may experience the most favourable intrauterine conditions for a balanced development of the feto-placental unit. PMID:18768081

Gloria-Bottini, Fulvia; Pietropolli, Adalgisa; Coppeta, Luca; Magrini, Andrea; Bergamaschi, Antonio; Bottini, Egidio

2008-01-01

398

NH⋯Br, Br⋯? and ?⋯? interactions toward self-assembly of the cytosine hydrobromide: Crystal structure, infrared spectroscopy and thermal behavior  

NASA Astrophysics Data System (ADS)

The cytosine hydrobromide (CytHBr) was obtained and studied by single-crystal X-ray diffraction, FT-IR spectroscopy and thermal analysis. This salt crystallizes in the P21/n space group with one ionic-pair CytH+Br- in the asymmetric unit. The CytHBr compound forms centrosymmetric R22(8) dimers, linking the cationic units (CytH+) in infinite strands of ionic pairs arranged in zigzag layers, connected by ?⋯? and ?⋯Br- interactions. Also, the Hirshfeld Surface (HS) and fingerprint plots were used to explore the H⋯Br, C⋯H, C⋯C, C⋯N, C⋯O and C⋯Br intermolecular contacts. The molecular assembly of CytHBr is similar compared with the cytosine hydrochloride salt (CytHCl). As a result, the FT-IR spectrum of the CytHBr is similar to that one reported for the CytHCl. On the other hand, small shifts are observed comparing with the pure cytosine (Cyt) spectrum. The CytHBr was also studied by thermal analysis (DSC and TGA), supported by Hot Stage and Optical Microscopy (HSOM) technique, confirming the high-quality of the crystalline modification.

Tenorio, Juan C.; Corrêa, Rodrigo S.; Batista, Alzir A.; Ellena, Javier

2013-09-01

399

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)

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 ?* shape resonance in energy region <1 eV. This is mainly due to the high activation energy barrier associated with the electron transfer from the ?* orbital of the base to the ?* 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.

Bhaskaran, Renjith; Sarma, Manabendra

2014-09-01

400

Distinguishing Between Relaxation Pathways by Combining Dissociative Ionization Pump Probe Spectroscopy and Ab Initio Calculations: A Case Study of Cytosine  

SciTech Connect

We present a general method for tracking molecular relaxation along different pathways from an excited state down to the ground state. We follow the excited state dynamics of cytosine pumped near the S0-S1 resonance using ultrafast laser pulses in the deep ultraviolet and probed with strong field near infrared pulses which ionize and dissociate the molecules. The fragment ions are detected via time of flight mass spectroscopy as a function of pump probe delay and probe pulse intensity. Our measurements reveal that different molecular fragments show different timescales, indicating that there are multiple relaxation pathways down to the ground state. We interpret our measurements with the help of ab initio electronic structure calculations of both the neutral molecule and the molecular cation for different conformations en route to relaxation back down to the ground state. Our measurements and calculations show passage through two seams of conical intersections between ground and excited states and demonstrate the ability of dissociative ionization pump probe measurements in conjunction with ab initio electronic structure calculations to track molecular relaxation through multiple pathways.

Kotur, Marija; Weinacht, Thomas C.; Zhou, Congyi; Kistler, Kurt A.; Matsika, Spiridoula

2011-05-10

401

Role of DNA methylation in growth and differentiation in Physcomitrella patens and characterization of cytosine DNA methyltransferases.  

PubMed

Epigenetic mechanisms such as DNA methylation are known to regulate important developmental processes in higher eukaryotes. However, little is known about the necessity and role of this process in early land plants. Using the methyltransferase (MTase) inhibitor zebularine (1-(?-d-ribofuranosyl)-1,2-dihydropyrimidine-2-one), the impact of loss of genome-wide methylation on the overall development in Physcomitrella patens was analyzed. It is observed that various aspects of growth and differentiation during gametophyte development become aberrant. A search for the core molecular components of methylation machinery, cytosine DNA MTases, revealed the presence of seven loci in the P. patens genome. Five of the loci code for MTases that are similar to corresponding proteins in higher plants, while two MTases appear specific to P. patens and are closely related to human DNMT3a and DNMT3b, respectively. These proteins possess all the conserved catalytic motifs characteristic of MTases and a domain of unknown function, DUF3444. Association of these highly conserved motifs with a DUF has not been reported in any of the MTases known so far. All the seven genes are differentially but ubiquitously expressed in gametophytes at low levels. Subcellular localization of GFP-fused proteins shows patterns of distribution that can be correlated with their putative cellular functions. This work bridges the knowledge of MTases in P. patens and makes this simple model plant accessible for studies on epigenetic aspects that remain intractable in higher plants. PMID:22943564

Malik, Garima; Dangwal, Meenakshi; Kapoor, Sanjay; Kapoor, Meenu

2012-11-01

402

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

PubMed Central

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

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

2013-01-01

403

High Preservation of CpG Cytosine Methylation Patterns at Imprinted Gene Loci in Liver and Brain of Aged Mice  

PubMed Central

A gradual loss of the correct patterning of 5-methyl cytosine marks in gene promoter regions has been implicated in aging and age-related diseases, most notably cancer. While a number of studies have examined DNA methylation in aging, there is no consensus on the magnitude of the effects, particularly at imprinted loci. Imprinted genes are likely candidate to undergo age-related changes because of their demonstrated plasticity in utero, for example, in response to environmental cues. Here we quantitatively analyzed a total of 100 individual CpG sites in promoter regions of 11 imprinted and non-imprinted genes in liver and cerebral cortex of young and old mice using mass spectrometry. The results indicate a remarkably high preservation of methylation marks during the aging process in both organs. To test if increased genotoxic stress associated with premature aging would destabilize DNA methylation we analyzed two DNA repair defective mouse models showing a host of premature aging symptoms in liver and brain. However, also in these animals, at the end of their life span, we found a similarly high preservation of DNA methylation marks. We conclude that patterns of DNA methylation in gene promoters of imprinted genes are surprisingly stable over time in normal, postmitotic tissues and that the multiple documented changes with age are likely to involve exceptions to this pattern, possibly associated with specific cellular responses to age-related changes other than genotoxic stress. PMID:24039963

Gravina, Silvia; Dolle, Martijn E. T.; Wang, Tao; van Steeg, Harry; Hasty, Paul; Hoeijmakers, Jan; Vijg, Jan

2013-01-01

404

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

PubMed Central

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

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

405

Nanoswitches based on DNA base pairs: why adenine-thymine is less suitable than guanine-cytosine.  

PubMed

Substituted Watson-Crick guanine-cytosine (GC) base pairs were recently shown to yield robust three-state nanoswitches. Here, we address the question: Can such supramolecular switches also be based on Watson-Crick adenine-thymine (AT) base pairs? We have theoretically analyzed AT pairs in which purine-C8 and/or pyrimidine-C6 positions carry a substituent X=NH(-), NH(2), NH(3) (+) (N series), O(-), OH or OH(2) (+) (O series), using the generalized gradient approximation (GGA) of density functional theory at the BP86/TZ2P level. Thus, we explore the trend in geometrical shape and hydrogen bond strengths in AT pairs along a series of stepwise protonations of the substituents. Introducing a charge on the substituents leads to substantial and characteristic changes in the individual hydrogen bond lengths when compared to the neutral AT pair. However, the trends along the series of negative, neutral, and positive substituents are less systematic and less pronounced than for GC. In certain instances, internal proton transfer from thymine to adenine occurs. Our results suggest that AT is a less suitable candidate than GC in the quest for chemically controlled nanoswitches. PMID:16888742

Fonseca Guerra, Célia; van der Wijst, Tushar; Bickelhaupt, F Matthias

2006-09-11

406

Intermolecular proton transfer in microhydrated guanine-cytosine base pairs: a new mechanism for spontaneous mutation in DNA.  

PubMed

Accurate calculations of the double proton transfer (DPT) in the adenine-thymine base pair (AT) were presented in a previous work [J. Phys. Chem. A 2009, 113, 7892.] where we demonstrated that the mechanism of the reaction in solution is strongly affected by surrounding water. Here we extend our methodology to the guanine-cytosine base pair (GC), for which it turns out that the proton transfer in the gas phase is a synchronous concerted mechanism. The O(G)-H-N(C) hydrogen bond strength emerges as the key parameter in this process, to the extent that complete transfer takes place by means of this hydrogen bond. Since the main effect of the molecular environment is precisely to weaken this bond, the direct proton transfer is not possible in solution, and thus the tautomeric equilibrium must be assisted by surrounding water molecules in an asynchronous concerted mechanism. This result demonstrates that water plays a crucial role in proton reactions. It does not act as a passive element but actually catalyzes the DPT. PMID:19736955

Cerón-Carrasco, J P; Requena, A; Zúñiga, J; Michaux, C; Perpète, E A; Jacquemin, D

2009-10-01

407

Methylation by a unique ?-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725.  

PubMed

Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique ?-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation. PMID:22928042

Chung, Daehwan; Farkas, Joel; Huddleston, Jennifer R; Olivar, Estefania; Westpheling, Janet

2012-01-01

408

The Drosophila Cytosine-5 Methyltransferase Dnmt2 Is Associated with the Nuclear Matrix and Can Access DNA during Mitosis  

PubMed Central

Cytosine-5 methyltransferases of the Dnmt2 family are highly conserved in evolution and their biological function is being studied in several organisms. Although all structural DNA methyltransferase motifs are present in Dnmt2, these enzymes show a strong tRNA methyltransferase activity. In line with an enzymatic activity towards substrates other than DNA, Dnmt2 has been described to localize to the cytoplasm. Using molecular and biochemical approaches we show here that Dnmt2 is both a cytoplasmic and a nuclear protein. Sub-cellular fractionation shows that a significant amount of Dnmt2 is bound to the nuclear matrix. Sub-cellular localization analysis reveals that Dnmt2 proteins are enriched in actively dividing cells. Dnmt2 localization is highly dynamic during the cell cycle. Using live imaging we observed that Dnmt2-EGFP enters prophase nuclei and shows a spindle-like localization pattern during mitotic divisions. Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions. Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2. PMID:18183295

Schaefer, Matthias; Steringer, Julia P.; Lyko, Frank

2008-01-01

409

Distinguishing between relaxation pathways by combining dissociative ionization pump probe spectroscopy and ab initio calculations: A case study of cytosine  

NASA Astrophysics Data System (ADS)

We present a general method for tracking molecular relaxation along different pathways from an excited state down to the ground state. We follow the excited state dynamics of cytosine pumped near the S0-S1 resonance using ultrafast laser pulses in the deep ultraviolet and probed with strong field near infrared pulses which ionize and dissociate the molecules. The fragment ions are detected via time of flight mass spectroscopy as a function of pump probe delay and probe pulse intensity. Our measurements reveal that different molecular fragments show different timescales, indicating that there are multiple relaxation pathways down to the ground state. We interpret our measurements with the help of ab initio electronic structure calculations of both the neutral molecule and the molecular cation for different conformations en route to relaxation back down to the ground state. Our measurements and calculations show passage through two seams of conical intersections between ground and excited states and demonstrate the ability of dissociative ionization pump probe measurements in conjunction with ab initio electronic structure calculations to track molecular relaxation through multiple pathways.

Kotur, Marija; Weinacht, Thomas C.; Zhou, Congyi; Kistler, Kurt A.; Matsika, Spiridoula

2011-05-01

410

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

SciTech Connect

CCA-adding enzymes [ATP(CTP):tRNA nucleotidyltransferases] add CCA onto the 3{prime} 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{prime}-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{prime}-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{prime} hydroxyl group of cytidine75.

Pan, Baocheng; Xiong, Yong; Steitz, Thomas A. (Yale)

2010-11-22

411

Construction of an efficient amylolytic industrial yeast strain containing DNA exclusively derived from yeast  

Microsoft Academic Search

An amylolytic industrial yeast strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis