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Allosteric regulation and radical transfer in ribonucleotide reductase /Larsson, Karl-Magnus, January 2004 (has links)
Diss. (sammanfattning) Stockholm : Univ., 2004. / Härtill 4 uppsatser.
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Repair, consequence, and profile of ribonucleotides in DNAKoh, Kyung Duk 08 June 2015 (has links)
Ribonucleotides, also known as ribonucleoside monophosphates (rNMPs), are the most abundant non-canonical nucleotides incorporated into genomic DNA. Despite the relevance, information about their repair pathways, consequences, and profiles is still lacking. Exploiting the use of oligonucleotides containing rNMPs in a molecular approach to generate various RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in cells, we show that mispaired rNMPs embedded into genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in E. coli and S. cerevisiae cells. In addition, we discovered that paired rNMPs in DNA are targets of both RNase H type 2 and nucleotide excision repair (NER) in yeast. Also, we report atomic force microscopy (AFM)-based single molecule elasticity measurement, molecular dynamics simulation, and nuclear magnetic resonance spectroscopy results, showing that rNMPs in short DNA duplexes can change the elastic and structural properties of DNA. Lastly, we developed ribose-seq, a method for capturing rNMPs embedded in DNA. High-throughput sequencing of rNMP-captured molecules from the yeast S. cerevisiae revealed widespread but non-random rNMP distribution with preferences in base composition of rNMPs and neighboring DNA sequence context in both nuclear and mitochondrial DNA. With ribose-seq, systematic profiling of rNMP incorporation into genomic DNA is achieved, potentially allowing determination of specific signatures of rNMPs in DNA which could help to better understand the nature of rNMP repair mechanisms, effect of rNMPs on DNA mechanical properties and structure, and eventually rNMP impact on genome integrity.
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Ribonucleotide reductase and DNA damage /Håkansson, Pelle, January 2006 (has links)
Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 3 uppsatser.
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Molecular analysis of the anaerobic-inducible operon nrdDG from Salmonella typhimurium.January 1998 (has links)
by Ng Wai-Leung. / Thesis submitted in: August 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 135-144). / Title page --- p.i / Thesis Committee --- p.ii / Abstract --- p.iii / Acknowledgments --- p.v / Abbreviations --- p.vi / Table of contents --- p.vii / List of figures --- p.x / List of tables --- p.xiii / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review / Chapter 2.1 --- Biosynthesis of deoxyribonucleotide triphosphates --- p.3 / Chapter 2.2 --- Ribonucleotide reductase --- p.6 / Chapter 2.2.1 --- Class I ribonucleotide reductase --- p.6 / Chapter 2.2.2 --- Class II ribonucleotide reductase --- p.13 / Chapter 2.2.3 --- Class III ribonucleotide reductase --- p.14 / Chapter 2.3 --- Proposed mechanism for ribonucleotide reduction --- p.17 / Chapter 2.4 --- Allosteric control of ribonucleotide reductase --- p.21 / Chapter 2.4.1 --- Allosteric control of class I ribonucleotide reductase --- p.21 / Chapter 2.4.2 --- Allosteric control of class II and class III ribonucleotide reductases --- p.23 / Chapter 2.5 --- Evolution of ribonucleotide reductase --- p.25 / Chapter 2.6 --- Central metabolism pathways of enteric bacteria --- p.28 / Chapter 2.7 --- Regulation of gene expression by oxygen in enteric bacteria --- p.33 / Chapter 2.7.1 --- Regulation of gene expression by Fnr --- p.33 / Chapter 2.7.2 --- Regulation of gene expression by AcrAB --- p.39 / Chapter 2.7.3 --- Regulation of gene expression by NarXL and NarQP --- p.42 / Chapter 2.7.4 --- Other aspects in anaerobic gene expression --- p.45 / Chapter 2.7.5 --- Relationship between NrdD and anaerobic metabolism --- p.45 / Chapter 2.8 --- Objectives --- p.46 / Chapter Chapter 3. --- Molecular cloning and sequencing of nrdDG operon from Salmonella typhimurium / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Material and methods --- p.48 / Chapter 3.2.1 --- Bacterial strains and bacteriophages --- p.48 / Chapter 3.2.2 --- Culture media --- p.48 / Chapter 3.2.3 --- Preparation of lambda lysate and phage DNA --- p.48 / Chapter 3.2.3.1 --- Plating out pf lambda phage and preparation of plate lysate --- p.48 / Chapter 3.2.3.2 --- Preparation of lambda lysate stock --- p.49 / Chapter 3.2.3.3 --- Preparation of lambda phage DNA --- p.50 / Chapter 3.2.4 --- Long distance polymerase chain reaction (LD-PCR) of nrdDG gene fragment --- p.51 / Chapter 3.2.5 --- Restriction enzyme digestion of LD-PCR products and subcloning of restriction fragments --- p.52 / Chapter 3.2.6 --- Confirmation of recombinants by colony-PCR --- p.53 / Chapter 3.2.7 --- Preparation of plasmid DNA by alkaline lysis using Wizard´ёØ Plus Miniprep DNA Purification System (Promega) --- p.54 / Chapter 3.2.8 --- DNA cycle sequencing by using dye-labeled dideoxy chain terminator and data collection --- p.55 / Chapter 3.2.9 --- Computer software for analyzing and manipulating DNA sequences --- p.57 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Preparation of lambda DNA --- p.59 / Chapter 3.3.2 --- Long distance PCR amplification of nrdDG from lambda DNA --- p.59 / Chapter 3.3.3 --- Restriction digestion of LD-PCR products --- p.61 / Chapter 3.3.4 --- Subcloning of LD-PCR restriction fragments --- p.61 / Chapter 3.3.5 --- Miniprep of plasmid DNA from recombinants and verification of nrdDG identities --- p.64 / Chapter 3.3.6 --- Nucleotide sequence of nrdDG --- p.66 / Chapter 3.4 --- Discussions --- p.72 / Chapter 3.4.1 --- Sequence analysis of S. typhimurium nrdDG --- p.72 / Chapter 3.4.2 --- Experimental design --- p.79 / Chapter Chapter 4. --- Transcriptional regulation of anaerobic ribonucleotide reductase in Salmonella typhimurium in aerobic and anaerobic environments / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Bacteria and bacteriophages strains / Chapter 4.2.2 --- Culture media --- p.86 / Chapter 4.2.3 --- Construction and characterization of oxrA mutant --- p.87 / Chapter 4.2.3.1 --- Preparation of P22 lysate of TN2336 --- p.87 / Chapter 4.2.3.2 --- P22 transduction for construction of oxrA mutant --- p.87 / Chapter 4.2.3.3 --- Characterization of oxrA mutant --- p.87 / Chapter 4.2.4 --- Extraction of bacterial RNA by hot phenol method --- p.88 / Chapter 4.2.5 --- Formaldehyde gel electrophoresis of RNA --- p.88 / Chapter 4.2.6 --- Reverse transcriptase polymerase chain reaction (RT-PCR) of nrdD transcript --- p.89 / Chapter 4.2.7 --- Transfer of DNA/RNA to solid support --- p.90 / Chapter 4.2.7.1 --- Transfer of DNA to solid support by Southern blotting --- p.90 / Chapter 4.2.7.2 --- Transfer of RNA to solid support by Northern blotting --- p.91 / Chapter 4.2.7.3 --- RNA Dot blot --- p.91 / Chapter 4.2.8 --- Preparation of radioactive-labeled probes for hybridization --- p.92 / Chapter 4.2.8.1 --- Synthesis of radioactive-labeled probes by labeling --- p.92 / Chapter 4.2.8.2 --- Preparation of RNA probe by in vitro transcription --- p.93 / Chapter 4.2.9 --- Hybridization and membrane washing conditions --- p.95 / Chapter 4.2.10 --- Normalization of samples by 16S ribosomal RNA (rRNA) --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Preparation of RNA --- p.97 / Chapter 4.3.2 --- RT-PCR of nrdD transcript --- p.97 / Chapter 4.3.3 --- Northern blot analysis of nrdD transcript --- p.103 / Chapter 4.3.4 --- Dot blot hybridization analysis of nrdD expression in an oxrA mutant --- p.103 / Chapter 4.4 --- Discussions --- p.107 / Chapter 4.4.1 --- Expression of nrdD of S. typhimurium in aerobic and anaerobic environments --- p.107 / Chapter 4.4.2 --- Experimental design --- p.110 / Chapter Chapter 5. --- Characterization of nrdD::Tn10 mutant of S. typhimurium / Chapter 5.1 --- Introduction --- p.112 / Chapter 5.2 --- Materials and methods --- p.112 / Chapter 5.2.1 --- Bacteria and bacteriophages strains --- p.113 / Chapter 5.2.2 --- Transduction of zzz-3875::Tn10 to S. typhimurium --- p.113 / Chapter 5.2.3 --- Characterization of zzz-3875::Tn10 by Southern hybridization --- p.113 / Chapter 5.2.3.1 --- Preparation of genomic DNA from S. typhimurium --- p.113 / Chapter 5.2.3.2 --- Restriction enzyme digestion of genomic DNA and Southern hybridization --- p.114 / Chapter 5.2.4 --- Characterization of growth pattern of nrdD::Tn10 mutant --- p.115 / Chapter 5.3 --- Results --- p.116 / Chapter 5.3.1 --- Characterization of zzz-3 875: :Tn7 0 in S. typhimurium --- p.116 / Chapter 5.3.2 --- Characterization of growth pattern of nrdD mutant --- p.120 / Chapter 5.4 --- Discussions --- p.125 / Chapter Chapter 6. --- General Discussions / Chapter 6.1 --- General discussions --- p.131 / Chapter 6.2 --- Further studies --- p.134 / References --- p.135
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Herpes simplex ribonucleotide reductaseIngemarson, Rolf January 1989 (has links)
In all bacterial, plant and animal cells, as well as in many viruses, genetic information resides in DNA (deoxyribonucleic acid). Replication of DNA is essential for proliferation, and DNA-containing viruses (such as herpesviruses) must carry out this process within the mammalian cells they infect. The enzyme ribonucleotide reductase catalyzes the first unique step leading to the production of the four deoxy-ribonucleotides used to make DNA. Each deoxyribonucleotide is produced by reduction of the corresponding ribonucleotide. After infection of a mammalian cell with herpes simplex virus (HSV) a new ribonucleotide reductase activity appears, which is distinct from the mammalian enzyme activity. This is due to induction of a separate, virally-encoded ribonucleotide reductase. Two monoclonal antibodies were raised against HSV (type 1) ribonucleotide reductase, and were found to bind but not neutralize its enzyme activity. One antibody recognized a larger (140 kD) protein and the other a smaller (40 kD) protein, suggesting the HSV 1 ribonucleotide reductase had a heterodimeric composition similar to that found in many other organisms. The 140 kD protein was sequentially degraded to 110 kD, 93 kD and 81 kD proteins by a host (Vero) cell-specific serine protease. Of these different proteolytic products, at least the 93 kD residue was enzymatically active, suggesting that part of the 140 kD protein may have functions unrelated to ribonucleotide reduction. The 140 and 40 kD proteins bound tightly to each other in a complex of the a2ß2 type, as shown by analytical glycerol gradient centrifugation. An assay system for functional small and large subunits of HSV 1 ribonucleotide reductase was developed, using two temperaturesensitive mutant viruses, defective in either the large (tsl207) or small (tsl222) subunits. Active holoenzyme was reconstituted both in vitro, by mixing extracts from cells infected with either mutant, and in vivo by coinfection of cells with both mutants. The gene encoding the small subunit of HSV 1 ribonucleotide reductase was cloned into an expression plasmid under control of a tac promoter. The recombinant protein was purified to homogeneity from extracts of transfected E. coli, and was active when combined with large subunit, as provided by extracts of tsl222- infected hamster (BHK) cells. The protein contained a novel tyrosyl free radical that spectroscopically resembled, but was distinguishable from, the active-site free radical found in either the E. coli or mammalian small subunits of ribonucleotide reductase. The gene encoding the large subunit of HSV 1 ribonucleotide reductase was also expressed in E. coli, using similar techniques. The recombinant large subunit was immunoprecipitated from extracts of transfected bacteria, and showed weak activity when combined with small subunit, provided by extracts of tsl20-infected hamster (BHK) cells. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1989, härtill 4 uppsatser.</p> / digitalisering@umu
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Regulation of ribonucleotide reductase and the role of dNTP pools in genomic stability in yeast Saccharomyces cerevisiaeTsaponina, Olga January 2011 (has links)
Every living organism is programmed to reproduce and to pass genetic information to descendants. The information has to be carefully copied and accurately transferred to the next generation. Therefore organisms have developed the network of conserved mechanisms to survey the protection and precise transfer of the genetic information. Such mechanisms are called checkpoints and they monitor the correct execution of different cell programs. The DNA damage and the replication blocks are surveyed by the conserved Mec1-Rad53 (human ATM/ATR and Chk2, respectively) protein kinase cascade. Mec1 and Rad53 are essential for survival and when activated orchestrate the multiple cellular responses, including the activation of the ribonucleotide reductase (RNR), to the genotoxic stress. RNR is an enzyme producing all four dNTPs - the building blocks of the DNA - and is instrumental for the maintenance both proper concentration and balance of each of dNTPs. The appropriate concentration of the dNTPs should be strictly regulated since inadequate dNTP production can impede many cellular processes and lead to higher mutation rates and genome instability. Hence RNR activity is regulated at many levels, including allosteric and transcriptional regulation and the inhibition at protein level. In our research, we addressed the question of the transcriptional regulation of RNR and the consequences of dNTP malproduction in the terms of the genomic stability. In yeast S. cerevisiae, four genes encode RNR: 2 genes encode a large subunit (RNR1 and RNR3) and 2 genes encode a small subunit (RNR2 and RNR4). All 4 genes are DNA-damage inducible: transcription of RNR2, RNR3 and RNR4 is regulated via Mec1-Rad53-Dun1 pathway by targeting the transcriptional repressor Crt1 (Rfx1) for degradation; on the contrary, RNR1 gene promoter does not contain Crt1-binding sites and is not regulated through the Mec1-Rad53-Dun1 pathway. Instead, we show that intrastrand cross (X)-link recognition protein (Ixr1) is required for the proper transcription of the RNR1 gene and maintenance of the dNTP pools both during unperturbed cell cycle and after the DNA damage. Thus, we identify the novel regulator of the RNR1 transcription. Next, we show that the depletion of dNTP pools negatively affects genome stability in the hypomorphic mec1 mutants: the hyper-recombination phenotype in those mutants correlates with low dNTP levels. By introducing even lower dNTP levels the hyper-recombination increased even further and conversely all the hyper-recombination phenotypes were suppressed by artificial elevation of dNTP levels. In conclusion, we present Ixr1 as a novel regulator of the RNR activity and provide the evidence of role of dNTP concentration in the genome stability.
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Genome annotation and identification of blood invasiveness genetic determinants in Salmonella Typhimurium clinical isolates from Hong Kong. / 香港沙門氏鼠傷寒桿菌臨床分離菌株的基因序列註釋及全身性感染的遺傳因素的識別 / CUHK electronic theses & dissertations collection / Xianggang Shamen shi shu shang han gan jun lin chuang fen li jun zhu de ji yin xu lie zhu shi ji quan shen xing gan ran de yi chuan yin su de shi bieJanuary 2013 (has links)
食物中毒感染是常見但非常重要的全球性公共健康問題。沙門氏鼠傷寒桿菌乃常被分離出來的細菌性病原體之一。隨著實驗室參考菌株LT2的基因組序列於2001年被發表之後,另外9個沙門氏鼠傷寒菌菌株的基因序列均已陸續進行測序。最近,本實驗室亦對十個本地沙門氏鼠傷寒菌臨床分離菌株的基因序列進行了測序。為了為這些基因組序列提供高品質的註釋,我們把預測的基因組提交到質量控制工具GenePRIMP以識別有潛在錯誤或異常的預測基因。本研究針對血液分離菌株78896和糞便分離菌株1047518的GenePRIMP報告進行人工檢查,並對每個菌株超過270個的基因進行了修訂。此外,本研究亦對上述的10個本地菌株進行了功能註釋。註釋項目包括沙門氏菌致病島(SPIs)、致病因子、tRNA和非編碼小分子RNA、噬菌體和CRISPRs結構等基因組及致病元素。 KEGG通路則提供了進一步的功能註釋。 / 本研究同時對本地的血液和糞便分離菌株,連同國外的臨床分離菌株,進行了廣泛的比對,用以識別全身性沙門氏菌感染的潛在遺傳因素。 本研究進行了以下基因分析:(1)多位點序列分型(MLST);(2)在小鼠全身性感染中涉及的主調控因子和關鍵元素; 及(3)人類腸胃道感染中涉及的基因。然而,這些分析產生只能對全身性沙門氏菌感染提供有限的見解。然而,透過使用RAST註釋系統,我們於其中三個血液分離菌株中發現了一個的額外的螯鐵蛋白aerobactin鐵採集系統。儘管在體外實驗中,這些血液分離菌株並沒有明顯的生長優勢,但實驗結果表明,在缺乏鐵的培養液中,aerobactin基因的表達水平是比較高的。此外,我們亦於其中四個血液分離菌株中,發現負責細胞色素c熟成(ccm)的基因座均被中斷。這可能改變了這些血液分離菌株中細胞色素c的生物合成途徑。這些鐵採集和同化機制的觀察均為未來全身性沙門氏菌感染的研究提供了可能的發展方向。 / 本研究同時識別了用以分別本地及海外的沙門氏鼠傷寒菌菌株的分子標記,並在鮭魚和生菜的接種實驗中,展現了它們分辨本地及海外菌株的能力。然而,在投入實際應用之前,這些標記尚需要進一步的驗證和測試,以便確定快速檢測方法的有效性。 / Foodborne infection is a common but important public health issue worldwide. Salmonella enterica serovar Typhimurium is frequently isolated from outbreaks as one of the common bacterial causative agents. Following the availability of the genome sequence of the reference lab strain LT2 in 2001, nine genomes of S. Typhimurium had been sequenced since then. Recently, genomes of ten local S. Typhimurium clinical isolates have been assembled in our laboratory. In order to provide high quality annotation of these genome sequences, the predicted gene sets were submitted to the quality control tool GenePRIMP (Gene PRediction IMprovement Pipeline) to identify potentially erroneous and abnormal gene calls. The GenePRIMP reports for the local blood isolate 78896 and stool isolate 1047518 were manually inspected and more than 270 genes were amended individually for each isolate. Functional annotation had also been performed for the 10 local isolates. Genomic and virulent elements including Salmonella Pathogenicity Islands (SPIs), virulence factors, tRNAs and small non-coding RNAs, prophage elements and CRISPRs structures had been annotated. The KEGG pathways provided a further means of functional annotation. / The local blood and stool isolates, together with the sequenced foreign clinical isolates, had also been extensively compared to identify potential genetic determinants of Salmonella systemic infection. (1) Multilocus sequence typing (MLST); (2) Alignment of master regulators and key players of systemic infection in mice; and (3) Analyses of the genes responsible for human gastrointestinal tract infection had been performed. However, these analyses yielded limited insights on systemic infection. Alternatively, using subsystems annotation by RAST, an additional aerobactin siderophore iron acquisition system was shown to be prevalent among three of the blood isolates. Despite no obvious growth advantage was offered to the blood isolates in an in vitro experiment, it was demonstrated that expression of the aerobactin genes was higher in iron-depleted culturing medium. In addition, a disrupted cytochrome c maturation (ccm) locus that may alter the cytochrome c biogenesis pathway was also identified in four of the blood isolates. These observations in iron acquisition and assimilation mechanisms suggest their potential in future direction of Salmonella systemic infection studies. / Molecular markers specific to local and foreign S. Typhimurium isolates were also identified and their utility in differentiating local and foreign isolates was demonstrated in a pilot spiking experiment using raw salmon and lettuce. These markers will require further verification and testing prior to actual application in real-world settings in order to examine the validity of the rapid detection method. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Chi Keung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis entitled --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction and Taxonomy --- p.1 / Chapter 1.2 --- Epidemiology of Salmonella Typhimurium infections --- p.2 / Chapter 1.3 --- Pathogenesis of Salmonella Typhimurium infection --- p.4 / Chapter 1.3.1 --- Infection mechanisms --- p.4 / Chapter 1.3.2 --- Salmonella Pathogenicity Islands --- p.6 / Chapter 1.3.3 --- Regulation of virulence --- p.9 / Chapter 1.4 --- Non-typhoid Salmonella (NTS) systemic infection --- p.11 / Chapter 1.4.1 --- Epidemiology of NTS systemic infection --- p.11 / Chapter 1.4.2 --- Salmonella Typhimurium multidrug resistance --- p.12 / Chapter 1.5 --- Salmonella Typhimurium genomics --- p.15 / Chapter 1.5.1 --- Salmonella Typhimurium genome sequencing --- p.15 / Chapter 1.5.2 --- Comparative studies on Salmonella genomes --- p.17 / Chapter 1.6 --- Aims of project --- p.19 / Chapter Chapter 2 --- Curation and detailed annotation of genomes of local Salmonella Typhimurium clinical isolates --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Manual curation of GenePRIMP results --- p.27 / Chapter 2.2.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.29 / Chapter 2.2.3 --- Small RNA and t-RNA annotation --- p.29 / Chapter 2.2.4 --- Phage elements annotation --- p.30 / Chapter 2.2.5 --- CRISPRs annotation --- p.30 / Chapter 2.2.6 --- KEGG annotation --- p.30 / Chapter 2.3 --- Results --- p.32 / Chapter 2.3.1 --- Manual curation of GenePRIMP results --- p.32 / Chapter 2.3.1.1 --- Short genes --- p.35 / Chapter 2.3.1.2 --- Long genes --- p.35 / Chapter 2.3.1.3 --- Unique genes --- p.36 / Chapter 2.3.1.4 --- Overlapped genes --- p.36 / Chapter 2.3.1.5 --- Broken genes --- p.37 / Chapter 2.3.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.37 / Chapter 2.3.2.1 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.37 / Chapter 2.3.2.2 --- Virulence factors annotation --- p.44 / Chapter 2.3.3 --- Small RNA and t-RNA annotation --- p.44 / Chapter 2.3.4 --- Phage elements annotation --- p.44 / Chapter 2.3.5 --- CRISPRs annotation --- p.50 / Chapter 2.3.6 --- KEGG annotation --- p.51 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- Manual curation of GenePRIMP results --- p.53 / Chapter 2.4.1.1 --- Gene amendment not required --- p.54 / Chapter 2.4.1.2 --- Genes with boundaries relocated --- p.54 / Chapter 2.4.1.3 --- Genes to be discarded --- p.55 / Chapter 2.4.1.4 --- Gene pairs to be fused --- p.55 / Chapter 2.4.1.5 --- Potential pseudogenes formation --- p.56 / Chapter 2.4.2 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.57 / Chapter 2.4.3 --- Virulence factors annotation --- p.57 / Chapter 2.4.4 --- Small RNA and t-RNA annotation --- p.58 / Chapter 2.4.5 --- Phage elements annotation --- p.59 / Chapter Chapter 3 --- Identification of genetic determinants of blood invasiveness in local S. Typhimurium clinical isolates --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods --- p.66 / Chapter 3.2.1 --- Multilocus Sequence Typing (MLST) --- p.66 / Chapter 3.2.2 --- Phage elements annotation for foreign isolates --- p.67 / Chapter 3.2.3 --- Alignment of genes inferred to play important roles in NTS systemic --- p.infection67 / Chapter 3.2.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.68 / Chapter 3.2.5 --- Subsystems assignment using Rapid Annotation using Subsystem Technology (RAST) server --- p.68 / Chapter 3.2.6 --- Growth analysis of local S. Typhimurium clinical isolates in iron-limiting environment --- p.69 / Chapter 3.2.7 --- Reverse transcription and real-time PCR --- p.70 / Chapter 3.2.7.1 --- Primer design and verification --- p.70 / Chapter 3.2.7.2 --- cDNA synthesis and real-time PCR --- p.70 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Multilocus Sequence Typing (MLST) --- p.73 / Chapter 3.3.2 --- Phage elements annotation for foreign isolates --- p.73 / Chapter 3.3.3 --- Alignment of genes inferred to play important roles in NTS systemic infection --- p.74 / Chapter 3.3.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.79 / Chapter 3.3.4.1 --- Acid tolerance response --- p.79 / Chapter 3.3.4.2 --- Epithelial cells attachment --- p.80 / Chapter 3.3.4.3 --- Epithelial cells invasion --- p.83 / Chapter 3.3.4.4 --- Survival within macrophages --- p.83 / Chapter 3.3.5 --- RAST subsystem analysis --- p.86 / Chapter 3.3.6 --- Growth analysis and aerobactin genes expression --- p.87 / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter 4 --- Molecular markers identification and testing on selected foodstuff for local S. Typhimurium isolates --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Molecular markers identification --- p.101 / Chapter 4.2.2 --- Primer design and verification --- p.101 / Chapter 4.2.3 --- Spiking experiments on selected food samples --- p.103 / Chapter 4.2.4 --- Quantitative TaqMan real-time PCR --- p.103 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- Molecular markers identification --- p.105 / Chapter 4.3.2 --- Spiking experiments and TaqMan real-time PCR --- p.109 / Chapter 4.4 --- Discussion --- p.113 / Chapter 4.4.1 --- Molecular markers identification --- p.113 / Chapter 4.4.2 --- Spiking experiments and TaqMan real-time PCR --- p.114 / Chapter Chapter 5 --- General discussion --- p.116 / Chapter 5.1 --- Manual curation of GenePRIMP results --- p.116 / Chapter 5.2 --- Functional annotation of local S. Typhimurium genomes --- p.118 / Chapter 5.3 --- Systemic infection studies --- p.120 / Chapter 5.4 --- Molecular markers identification and spiking experiments --- p.121 / Chapter 5.5 --- Conclusion and future perspectives --- p.122 / References --- p.124
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Vaccinia virus DNA polymerase and ribonucleotide reductase: their role in replication, recombination and drug resistanceGammon, Donald Brad Unknown Date
No description available.
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Mechanisms controlling DNA damage survival and mutation rates in budding yeastWiberg, Jörgen January 2012 (has links)
All living organisms are made of cells, within which genetic information is stored on long strands of deoxyribonucleic acid (DNA). The DNA encodes thousands of different genes and provides the blueprint for all of the structures and activities occurring within the cell. The building blocks of DNA are the four deoxyribonucleotides, dATP, dGTP, dTTP, and dCTP, which are collectively referred to as dNTPs. The key enzyme in the production of dNTPs is ribonucleotide reductase (RNR). In the budding yeast Saccharomyces cerevisiae, the concentrations of the individual dNTPs are not equal and it is primarily RNR that maintains this balance. Maintenance of the dNTP pool balance is critical for accurate DNA replication and DNA repair since elevated and/or imbalanced dNTP concentrations increase the mutation rate and can ultimately lead to genomic instability and cancer. In response to DNA damage, the overall dNTP concentration in S. cerevisiae increases. Cell survival rates increase as a result of the elevated concentration of dNTPs, but the cells also suffer from a concomitant increase in mutation rates. When the replication machinery encounters DNA damage that it cannot bypass, the replication fork stalls and recruits specialized translesion synthesis (TLS) polymerases that bypass the damage so that replication can continue. We hypothesized that elevated dNTP levels in response to DNA damage may allow the TLS polymerases to more efficiently bypass DNA damage. To explore this possibility, we deleted all known TLS polymerases in a yeast strain in which we could artificially increase the dNTP concentrations. Surprisingly, even though all TLS polymerases had been deleted, elevated dNTP concentrations led to increased cell survival after DNA damage. These results suggest that replicative DNA polymerases may be involved in the bypass of certain DNA lesions under conditions of elevated dNTPs. We confirmed this hypothesis in vitro by demonstrating that high dNTP concentrations result in an increased efficiency in the bypass of certain DNA lesions by DNA polymerase epsilon, a replicative DNA polymerase not normally associated with TLS activity. We asked ourselves if it would be possible to create yeast strains with imbalanced dNTP concentrations in vivo, and, if so, would these imbalances be recognized by the checkpoint control mechanisms in the cell. To address these questions, we focused on the highly conserved loop2 of the allosteric specificity site of yeast Rnr1p. We introduced several mutations into RNR1-loop2 that resulted in changes in the amino acid sequence of the protein. Each of the rnr1-loop2 mutation strains obtained had different levels of individual dNTPs relative to the others. Interestingly, all of the imbalanced dNTP concentrations led to increased mutation rates, but these mutagenic imbalances did not activate the S-phase checkpoint unless one or several dNTPs were present at concentrations that were too low to sustain DNA replication. We were able to use these mutant yeast strains to successfully correlate amino acid substitutions within loop2 of Rnr1p to specific ratios of dNTP concentrations in the cells. We also demonstrated that specific imbalances between the individual dNTP levels result in unique mutation spectra. These mutation spectra suggest that the mutagenesis that results from imbalanced dNTP pools is due to a decrease in fidelity of the replicative DNA polymerases at specific DNA sequences where they are more likely to make a mistake. The mutant rnr1-loop2 strains that we have created with defined dNTP pool imbalances will be of great value for in vivo studies of polymerase fidelity, translesion synthesis by specialized DNA polymerases, and lesion recognition by the DNA repair machinery.
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Vaccinia virus DNA polymerase and ribonucleotide reductase: their role in replication, recombination and drug resistanceGammon, Donald Brad 06 1900 (has links)
Despite the eradication of smallpox, poxviruses continue to cause human disease around the world. At the core of poxvirus replication is the efficient and accurate synthesis and repair of the viral genome. The viral DNA polymerase is critical for these processes. Acyclic nucleoside phosphonate (ANP) compounds that target the viral polymerase are effective inhibitors of poxvirus replication and pathogenesis. Cidofovir (CDV) is an ANP that inhibits vaccinia virus (VAC) DNA polymerase (E9) DNA synthesis and 3-to-5 exonuclease (proofreading) activities. We determined that point mutations in the DNA polymerase genes of ANP-resistant (ANPR) VAC strains were responsible for CDV resistance and resistance to the related compound, HPMPDAP. Although these resistant strains replicated as well as wild-type VAC in culture, they were highly attenuated in mice. The generation of ANPR VAC strains, in combination with our knowledge of how CDV inhibits E9 activities, allowed us to study the hypothesized role of E9 in catalyzing double-strand break repair through homologous recombination. We provide evidence that VAC uses E9 proofreading activity to catalyze genetic recombination through single-strand annealing reactions (SSA) in infected cells. Both the polarity of end resection of recombinant intermediates and the involvement of polymerase proofreading activity establish these poxviral SSA reactions as unique among homologous recombination schemes. Furthermore, we identified roles for the VAC single-stranded DNA-binding (SSB) protein and nucleotide pools in regulating these reactions. During these later studies we uncovered a differential requirement for the large and small subunits of the VAC ribonucleotide reductase (RR) in viral replication and pathogenesis. Our studies suggest that poxviral RR small subunits form functional complexes with host large RR subunits to provide sufficient nucleotide pools to support DNA replication. We present a model whereby interaction of VAC SSB and RR proteins at replication forks allows for modulation of E9 activity through local nucleotide pool changes, which serves to maximize replication rates while still allowing for recombinational repair. / Virology
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