Dissertations / Theses on the topic 'Genetic code'
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Nicholls, Felicity K. M. "Genetic analysis of the gene Additional sex combs and interacting loci." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/29644.
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In order to recover new mutant alleles of the Polycomb group gene Additional sex combs (Asx), mutagenized chromosomes were screened over the putative Asx allele XT129. Thirteen new mutant strains that fail to complement XT129 were recovered. Unexpectedly, the thirteen strains sorted into four complementation groups. Recombination mapping suggests that each complementation group represents a separate locus. The largest group fails to complement a deletion of Asx and maps in the vicinity of 2-72, the published location of Asx. All new mutant strains enhance the phenotype of Polycomb mutant flies and are not allelic to any previously discovered second chromosome Polycomb group genes. Therefore, the new mutants may be considered putative new members of the Polycomb group. This study suggests that Asx belongs to a sub-group of genes displaying intergenic non-complementation.Science, Faculty of
Zoology, Department of
Graduate
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Mat, Wai Kin. "Genetic code mutants of bacillus subtilis /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BICH%202007%20MAT.
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Freeland, Stephen J. "Natural selection and the genetic code." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313922.
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Gutfraind, Alexander. "Error-Tolerant Coding and the Genetic Code." Thesis, University of Waterloo, 2006. http://hdl.handle.net/10012/2913.
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The following thesis is a project in mathematical biology building upon the so-called "error minimization hypothesis" of the genetic code. After introducing the biological context of this hypothesis, I proceed to develop some relevant information-theoretic ideas, with the overall goal of studying the structure of the genetic code. I then apply the newfound understanding to an important question in the debate about the origin of life, namely, the question of the temperatures in which the genetic code, and life in general, underwent their early evolution. The main advance in this thesis is a set of methods for calculating the primordial evolutionary pressures that shaped the genetic code. These pressures are due to genetic errors, and hence the statistical properties of the errors and of the genome are imprinted in the statistical properties of the code. Thus, by studying the code it is possible to reconstruct, to some extent, the primordial error rates and the composition of the primordial genome. In this way, I find evidence that the fixation of the genetic code occurred in organisms which were not thermophiles.
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Mateus, Denisa Daud. "Molecular reconstruction of a genetic code alteration." Doctoral thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7501.
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Doutoramento em BioquímicaThe genetic code establishes the rules that govern gene translation into proteins. It was established more than 3.5 billion years ago and it is one of the most conserved features of life. Despite this, several alterations to the standard genetic code have been discovered in both prokaryotes and eukaryotes, namely in the fungal CTG clade where a unique seryl transfer RNA (tRNACAG Ser) decodes leucine CUG codons as serine. This tRNACAG Ser appeared 272±25 million years ago through insertion of an adenosine in the middle position of the anticodon of a tRNACGA Ser gene, which changed its anticodon from 5´-CGA-3´ to 5´-CAG-3´. This most dramatic genetic event restructured the proteome of the CTG clade species, but it is not yet clear how and why such deleterious genetic event was selected and became fixed in those fungal genomes. In this study we have attempted to shed new light on the evolution of this fungal genetic code alteration by reconstructing its evolutionary pathway in vivo in the yeast Saccharomyces cerevisiae. For this, we have expressed wild type and mutant versions of the C. albicans tRNACGA Ser gene into S. cerevisiae and evaluated the impact of the mutant tRNACGA Ser on fitness, tRNA stability, translation efficiency and aminoacylation kinetics. Our data demonstrate that these mutants are expressed and misincorporate Ser at CUGs, but their expression is repressed through an unknown molecular mechanism. We further demonstrate, using in vivo forced evolution methodologies, that the tRNACAG Ser can be easily inactivated through natural mutations that prevent its recognition by the seryl-tRNA synthetase. The overall data show that repression of expression of the mistranslating tRNACAG Ser played a critical role on the evolution of CUG reassignment from Leu to Ser. In order to better understand the evolution of natural genetic code alterations, we have also engineered partial reassignment of various codons in yeast. The data confirmed that genetic code ambiguity affects fitness, induces protein aggregation, interferes with the cell cycle and results in nuclear and morphologic alterations, genome instability and gene expression deregulation. Interestingly, it also generates phenotypic variability and phenotypes that confer growth advantages in certain environmental conditions. This study provides strong evidence for direct and critical roles of the environment on the evolution of genetic code alterations.
O código genético regula a correcta descodificação da informação contida nos genes durante a síntese de proteínas. Apresenta um elevado grau de conservação e estima-se que tenha sido originado há mais de 3.5 biliões de anos. Contudo, várias alterações ao código genético foram identificadas em procariotas e eucariotas, nomeadamente nos fungos denominados de “CTG clade”, nos quais um tRNA de serina atípico (tRNACAG Ser) descodifica o codão de leucina CUG como serina. Este tRNACAG Ser foi originado há 272±25 milhões de anos, pela inserção de uma adenosina no centro do anticodão do gene do tRNACGA Ser que alterou a sequência original do anticodão de 5´-CGA-3´ para 5´-CAG-3´. Esta alteração ao código genético promoveu a restruturação do proteoma das espécies denominadas de “CTG clade”. Contudo, permanece por esclarecer o motivo que permitiu que esta alteração atípica fosse preservada no genome destes fungos. Numa tentativa de clarificar os aspectos evolutivos desta alteração ao código genético, procedemos à reconstrução da via evolutiva, proposta para esta alteração, na levedura Saccharomyces cerevisiae. Para tal, induzimos a expressão do gene do tRNACGA Ser de C. albicans, nas versões mutantes e original, em S. cerevisiae e determinámos o impacto das mesmas no crescimento celular, bem como na estabilidade, eficiência na tradução e aminoacilação do tRNA. Os nossos dados, demonstram que as versões mutantes do tRNA, apesar de sua reduzida expressão, induzem a incorporação de serina nos codões CUG de leucina. Observámos ainda, através de uma estratégia de evolução forçada, que o tRNACAG Ser é facilmente inactivado por mutações naturais que impedem o seu reconhecimento pela seryl-tRNA synthetase. O nosso estudo demonstra que a repressão da expressão do tRNACAG Ser, terá desempenhado um papel fundamental na evolução da redefinição do codão CUG de leucina para serina. Com o intuito de compreender a evolução das alterações ao código genético, induzimos redefinições parciais em vários codões de levedura. Os nossos resultados confirmam que a ambiguidade no código genético afecta o crescimento, induz a produção de agregados proteicos, interfere no ciclo celular e promove alterações nucleares, morfológicas, instabilidade genómica e desregulação da expressão genética. Contudo, origina também variedade fenotípica e fenótipos vantajosos em determinadas condições ambientais. Este estudo demonstra o impacto do ambiente na evolução das alterações ao código genético.
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Gomes, Ana Catarina Batista. "Molecular evolution of a genetic code alteration." Doctoral thesis, Universidade de Aveiro, 2008. http://hdl.handle.net/10773/939.
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Doutoramento em BiologiaDurante os últimos anos, foram descritas alterações ao código genético, quer em procariotas, quer em eucariotas, quebrando o dogma de que o código genético é universal e imutável. Estudos recentes sugerem que a evolução de tais alterações requerem modificações ao nível da estrutura da maquinaria da tradução e são promovidas por mecanismos de descodificação ambígua. Em C. albicans, um organismo que é patogénico para o Homem, a alteração ao código genético é mediada por uma alteração na estrutura de um novo tRNACAG de serina que descodifica o codão CUG de leucina como serina. De forma a determinar se este tRNA, que é aminoacilado pelas Seryl- e Leucyl- tRNA sintetases, promove a descodificação ambígua do codão CUG, foi desenvolvido um sistema para a quantificar in vivo, por espectrometria de massa, os níveis de incorporação de serina e de leucina em codões CUG. Os resultados mostraram que em condições normais de crescimento leucina é incorporada a uma taxa de 3% e que serina é incorporada a uma taxa de 97%. No entanto, o nível de ambiguidade na descodificação de codões CUG aumentou para 5% em células crescidas em condições de stress, indicando que a incorporação de leucina em codões CUG é sensível a factores ambientais e é manipulada durante a tradução do mRNA. Tal, levanta a hipótese de que a incorporação de leucina poderá atingir níveis superiores aos determinados neste estudo. Para testar esta hipótese e determinar os níveis máximos de ambiguidade na descodificação do codão CUG tolerados pelas células, aumentou-se artificialmente a ambiguidade do codão CUG em C. albicans. Surpreendentemente, a incorporação de leucina subiu de 5% para 28%, o que representa um aumento na taxa de erro da tradução de 3500 vezes, relativamente ao descrito para o mecanismo de tradução. Dado existirem 13.000 codões CUG no genoma de C. albicans, a sua descodificação ambígua expande de uma forma exponencial o proteoma deste fungo, criando assim um proteoma estatístico, resultante da síntese de um conjunto de moléculas diferentes para cada proteína a partir de um único RNA mensageiro (mRNA) que contenha codões CUG. Os resultados obtidos demonstraram que o proteoma de C. albicans tem uma dimensão muito superior à prevista pelo seu genoma e demonstram um papel central da descodificação ambígua na evolução do código genético.
Alterations to the standard genetic code have been found in both prokaryotes and eukaryotes, demolishing the dogma of an immutable and universal genetic code. Recent studies suggest that evolution of such alterations require structural change of the translation machinery and are driven through mechanisms that require codon decoding ambiguity. In the human pathogen C. albicans, a structural change in a novel sertRNACAG allows for its recognition by both the LeuRS and SerRS in vitro and in vivo, providing such molecular device. In order to determine whether this tRNA charging ambiguity results in ambiguous CUG decoding, we have developed a system for quantification of the level of serine and leucine at the CUG codon by Mass-Spectrometry. The data showed that 3.0% of leucine and 97.0% of serine are incorporated at CUG codons in vivo under standard growth conditions. Moreover, this ambiguity increases up to 5.0% under stress, indicating that it is sensitive to environmental change and raising the hypothesis that leucine incorporation may be higher than determine experimentally. In order to determine the scope of C. albicans tolerance to CUG ambiguity, we have created highly ambiguous C. albicans cell lines through tRNA engineering. These cell lines tolerated up to 28% leucine incorporation at CUGs, which represents an increase of 3500 fold in decoding error rate. Since there are 13,000 CUG codons in C. albicans such ambiguity expands the proteome exponentially and creates a statistical proteome due to synthesis of arrays of protein molecules from mRNAs containing CUG codons. The overall data showed that the dimension of the C. albicans proteome is far higher than that predicted from its genome and provides important new evidence for a pivotal role for codon ambiguity in the evolution of the genetic code.
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Silva, Raquel Monteiro Marques da. "Molecular reconstruction of a genetic code alteration." Doctoral thesis, Universidade de Aveiro, 2005. http://hdl.handle.net/10773/948.
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Doutoramento em BiologiaVárias espécies de Candida traduzem o codão CUG de leucina como serina. Esta alteração ao código genético é mediada por um novo tRNA (sertRNACAG), que pode ser carregado com serina (97 %) e leucina (3 %) in vivo. Por esta razão o codão CUG é ambíguo, pois pode ser descodificado como serina ou leucina. Para elucidar o impacto da ambiguidade do código genético na expressão génica e na fisiologia da célula, o ser-tRNACAG de C. albicans foi expresso em Saccharomyces cerevisiae. Isto induz a descodificação ambígua do codão CUG, devido à competição entre o tRNA endógeno que traduz o codão CUG como leucina e o C. albicans ser-tRNACAG, que o traduz maioritariamente como serina. A caracterização do transcriptoma e do proteoma das linhas celulares manipuladas de S. cerevisiae mostra que a ambiguidade do código genético induz alterações globais na expressão de genes e proteínas, com alterações na resposta ao stress, metabolismo dos hidratos de carbono e dos aminoácidos, estrutura e função da parede celular, síntese e degradação de proteínas. Adicionalmente, os resultados indicam que a tradução errada do codão CUG regula a expressão génica ao nível da tradução. A ambiguidade do codão CUG gera instabilidade do proteoma e genoma, contudo, estas células não perdem viabilidade. Pelo contrário, os dados sugerem que a resposta ao stress despoletada pela ambiguidade do codão CUG aumenta o potencial de adaptação, como é demonstrado pela tolerância que as células ambíguas têm a várias condições de stress. Por estas razões, a reconstrução da alteração na descodificação do codão CUG providenciou dados importantes sobre o impacto que alterações ao código genético têm na adaptação e evolução das células. Este estudo também trouxe novas ideias acerca dos mecanismos que permitem a tolerância das células eucarióticas a elevados níveis de erro na tradução do mRNA.
Several Candida species translate the standard leucine CUG codon as serine. This genetic code alteration is mediated by a novel tRNA (ser-tRNACAG), which can be charged both with serine (97 %) and leucine (3%) in vivo. Therefore, the CUG codon is ambiguous, since it can be decoded either as serine or leucine. To elucidate the impact of genetic code ambiguity on gene expression and cell physiology, the C. albicans ser-tRNACAG was expressed in Saccharomyces cerevisiae. This induces ambiguous decoding of the CUG codon, due to competition between the endogenous tRNA that decodes the CUG codon as leucine and the C. albicans ser-tRNACAG, which decodes it mainly as serine. Transcriptome and proteome characterization of the engineered S. cerevisiae cell lines show that genetic code ambiguity induces global gene and protein expression changes, with alterations in the stress response, carbohydrate and amino acid metabolism, cell wall structure and function, protein synthesis and protein degradation. Additionally, the results indicate that CUG mistranslation regulates gene expression at the translational level. CUG ambiguity generates proteome and genome instability, however, these cells do not lose viability. Instead, the data suggests that the stress response triggered by CUG ambiguity increases adaptation potential, as shown by the tolerance of ambiguous cells to several stress conditions. Therefore, the reconstruction of the CUG reassignment pathway provided important insight on the impact that genetic code alterations have on cell adaptation and evolution. This study also sheds new light on the mechanisms that allow eukaryotic cells to tolerate high levels of mRNA mistranslation.
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Bezerra, Ana Rita Macedo. "Molecular genomics of a genetic code alteration." Doctoral thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12499.
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Doutoramento em BiologiaThe genetic code is not universal. Alterations to its standard form have been discovered in both prokaryotes and eukaryotes and demolished the dogma of an immutable code. For instance, several Candida species translate the standard leucine CUG codon as serine. In the case of the human pathogen Candida albicans, a serine tRNA (tRNACAGSer) incorporates in vivo 97% of serine and 3% of leucine in proteins at CUG sites. Such ambiguity is flexible and the level of leucine incorporation increases significantly in response to environmental stress. To elucidate the function of such ambiguity and clarify whether the identity of the CUG codon could be reverted from serine back to leucine, we have developed a forced evolution strategy to increase leucine incorporation at CUGs and a fluorescent reporter system to monitor such incorporation in vivo. Leucine misincorporation increased from 3% up to nearly 100%, reverting CUG identity from serine back to leucine. Growth assays showed that increasing leucine incorporation produced impressive arrays of phenotypes of high adaptive potential. In particular, strains with high levels of leucine misincorporation exhibited novel phenotypes and high level of tolerance to antifungals. Whole genome re-sequencing revealed that increasing levels of leucine incorporation were associated with accumulation of single nucleotide polymorphisms (SNPs) and loss of heterozygozity (LOH) in the higher misincorporating strains. SNPs accumulated preferentially in genes involved in cell adhesion, filamentous growth and biofilm formation, indicating that C. albicans uses its natural CUG ambiguity to increase genetic diversity in pathogenesis and drug resistance related processes. The overall data provided evidence for unantecipated flexibility of the C. albicans genetic code and highlighted new roles of codon ambiguity on the evolution of genetic and phenotypic diversity.
O código genético não é universal. Alterações à identidade de vários codões descobertas em procariotas e eucariotas invalidam a hipótese dum código genético universal e imutável. Por exemplo, várias espécies do género Candida traduzem o codão CUG de leucina como serina. Em Candida albicans, um único tRNA de serina (tRNACAGSer) incorpora in vivo 97% de serina e 3% de leucina nas proteínas em resposta a codões CUG presentes nos mRNAs deste fungo patogénico. Esta ambiguidade é flexível e a incorporação de leucina aumenta em condições de stress. De forma a elucidar a função desta ambiguidade e determinar se a identidade dos codões CUG podia ser revertida de serina para leucina, desenvolvemos uma estratégia de evolução forçada e uma proteína recombinante fluorescente cuja actividade depende da incorporação de leucina num codão CUG. Construímos estirpes que incorporam leucina nas proteínas em resposta a codões CUGs em níveis que variam entre 0,64% e 98,46%. Esta reversão de uma alteração ao código genético demostrou de modo inequívoco que o código é flexível e pode evoluir. Testes de crescimento em diferentes meios de cultivo revelaram uma série impressionante de fenótipos com elevado potencial adaptativo nas estirpes mais ambíguas, nomeadamente tolerância a antifúngicos. A sequenciação dos genomas das estirpes que construímos revelou que a ambiguidade do codão CUG resulta na acumulação de polimorfismos de nucleótido únicos (SNP) no genoma. Verificámos também perda de heterozigozidade (LOH) nos cromossomas 5 e R das estirpes que incorporam 80,84% e 98,46% de leucina em locais proteicos codificados por codões CUG. Os SNPs acumularam-se preferencialmente em genes envolvidos na adesão celular, no crescimento filamentoso e na formação de biofilmes, sugerindo que C. albicans utiliza a sua ambiguidade natural para aumentar a diversidade genética dos processos relacionados com a patogénese e resistência a drogas. Estes resultados evidenciam uma notável flexibilidade do código genético de C. albicans e revelam funções inesperadas da ambiguidade do código genético na evolução da diversidade genética e fenotípica.
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Lajoie, Marc Joseph. "Genome Engineering Technologies to Change the Genetic Code." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11265.
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New technologies are making it possible to engineer organisms with fundamentally new and useful properties. In vivo genome engineering technologies capable of manipulating genomes from the nucleotide to the megabase scale were developed and applied to reassign the genetic code of Escherichia coli. Such genomically recoded organisms show promise for thwarting horizontal gene transfer with natural organisms, resisting viral infection, and expanding the chemical properties of proteins.
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Jääskelä, E. (Esa). "Genetic algorithm in code coverage guided fuzz testing." Master's thesis, University of Oulu, 2016. http://urn.fi/URN:NBN:fi:oulu-201601151058.
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The security of computers is a growing concern when the amount of devices increases. New and more comprehensive testing methods need to be done to avoid damages to the users and their computers. Fuzzing is a testing method that inserts semi-valid input to the tested system and has before been considered as a good method for the security testing. However, it usually either does not get high code coverage or it requires a long set-up process or a source code analysis to achieve better code coverage. This work presents a genetic algorithm that automatically balances the probabilities of multiple mutators in a fuzzing program. This balancing aims to maximize the code coverage fuzz testing. After fuzzing two different open source libraries it was found that the grey-box approach in fuzzing gives better results than pure black-box fuzzingTietokoneiden tietoturva on kasvava huolenaihe, kun laitteiden määrä lisääntyy. Uusia ja kattavampia testauksia täytyy suorittaa, jotta voidaan estää käyttäjille ja heidän laitteilleen tapahtuvat vahingot. Fuzzausta on pidetty hyvänä testausmetodina, mutta yleensä se ei saavuta hyvää koodikattavuutta tai vaatii joko monimutkaisen asennuksen tai lähdekoodianalyysin. Tämä työ esittelee geneettisen algoritmin, joka automaattisesti tasapainottaa fuzzerin eri mutaatiofunktioiden todennäköisyydet. Tämä tasapainotus pyrkii maksimoimaan saavutetun koodikattavuuden ja parantamaan fuzzaamisen tehokkuutta. Kahden avoimen lähdekoodin kirjaston testaamisen perusteella mutatorit koodikattavuuden perusteella tasapainottava työkalu pärjäsi paremmin kuin perinteinen, lisätietoa hyödyntämätön black-box fuzzaus
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Grasso, Katherine Taylor. "Resolving the Limitations of Genetic Code Expansion Platforms:." Thesis, Boston College, 2021. http://hdl.handle.net/2345/bc-ir:109076.
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Thesis advisor: Abhishek ChatterjeeThesis advisor: Eranthie Weerapana
Over the past twenty years, the site-specific incorporation of unnatural amino acids (UAAs) into a target protein through genetic code expansion (GCE) has emerged as one of the foremost technologies to selectively modify proteins in their native cellular context. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs. Traditionally, scientists look towards evolutionarily distant domains of life to identify orthogonal aaRS/tRNA pairs that can be further engineered for GCE applications in the host system. For example, bacterial aaRS/tRNA pairs are used for GCE in eukaryotes. The directed evolution of orthogonal aaRS/tRNA pairs for eukaryotic GCE has been less fortuitous due to the cumbersome nature of established yeast-based selection platforms. Recently, our lab circumvented this platform-based limitation by developing “altered translational machinery” (ATM) Escherichia coli strains that enabled the directed evolution of bacterial aaRS/tRNA pairs for eukaryotic GCE applications. In the ATM-tyrosyl (ATMY) E. coli strain, reintroduction of the E. coli tyrosyl-tRNA (tRNAEcTyrCUA) as a nonsense suppressor led to cross-reactivity with the endogenous E. coli glutaminyl-tRNA synthetase (EcGlnRS), restricting the activity range of aaRSs that could be selected, ultimately diminishing the scope of incorporable UAAs. To recover the dynamic range of this platform, cross-reactivity of the tRNAEcTyrCUA was eliminated through directed evolution of the tRNA acceptor stem. This new, orthogonal tRNA revealed weak mutant aaRSs whose suppression efficiencies were boosted through additional rounds of directed evolution. Improved aaRS mutants exhibited higher solubility, thermal stability, and suppression efficiency than their predecessor. While the newly engineered, orthogonal tRNAEcTyrCUA gave access to novel aaRS/tRNA pairs for eukaryotic GCE, some notable UAAs were still missing that could be incorporated with the archaeal Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair in bacteria. Following a systematic investigation into the discrepancy between the E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA and MjTyrRS/tRNA pairs, we found that it can be partially attributed to the low structural robustness of the EcTyrRS. This limitation was overcome by rationally designing chimeric TyrRSs composed of EcTyrRS and a structural homologue from the thermophilic bacterium Geobacillus stearothermophilus. The chimeric scaffolds demonstrated enhanced stability, activity, and resilience to destabilizing active site mutations, offering a potentially more attractive scaffold for GCE
Thesis (PhD) — Boston College, 2021
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Iqbal, Emil S. "In vitro genetic code expansion and selected applications." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5580.
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The ability of incorporation non-canonical amino acids (ncAAs) using translation offers researchers the ability of extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the three projects where the addition of ncAAs to in vitro translation systems creates useful chemical biology techniques. In the first, a fluorinated histidine derivative is used to create a novel affinity tag that allows for the selective purification of peptides from a complex mixture of proteins. In the second, the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P) is used to demonstrate ribosomal translation of 13 ncAAs including those with novel side chains, α,α disubstitutions, and cyclic β amino acids. Lastly, a couple of these amino acids are integrated into the powerful ligand discovery tool of mRNA display for the discovery of helical peptide ligands.
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Ma, Natalie Jing. "Altering the Genetic Code to Probe and Control the Flow of Genetic Information." Thesis, Yale University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10584955.
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The genetic code is highly conserved across all domains of life, enabling horizontal gene transfer (HGT) between organisms and across ecosystems via horizontally-transferred genetic elements such as viruses and plasmids. While HGT increases genetic diversity, it poses a risk to engineered biological systems by introducing new genes that destabilize engineered functions or allowing the expression of engineered genes in wild organisms with unknown effects. A model organism engineered with an alternative genetic code may provide new insight into the origins of the genetic code while also providing a stable chassis for engineered biological systems.
The Isaacs Lab recently developed an Escherichia coli strain lacking both UAG stop codons and Release Factor 1, resulting in the first genomically recoded organism (GRO) with an unassigned codon in its genetic code. Here, we demonstrate that this alternative genetic code lacking UAG codon assignment confers resistance to multiple viruses (λ, M13, PI, MS2) at titers up to 1011 PFU/mL and impairs conjugative plasmid function (F and RK2) up to 105-fold. Propagating viruses on a mixed microbial community containing standard and alternative genetic codes also reduced viral population fitness and prompted viral adaptation to the alternative genetic code. In investigating the molecular mechanism underlying the resistance to viruses and conjugative plasmids, we found that UAG-ending genes elicit ribosomal stalling and the tmRNAmediated ribosomal rescue response, resulting in degradation of UAG-ending proteins and suggesting that genomic recoding may be a broadly applicable strategy to impair horizontal gene transfer into other organisms.
To prevent the expression of engineered genes in wild organisms, we reassigned the UAG codon in the GRO to a sense codon incorporating the non-standard amino acid 4-acetylphenylalanine (pAcF) through the introduction of an orthogonal translation system (OTS). We then created a library of UAG-containing variants and assessed escape of UAG-containing genes from the GRO into wild-type organisms for both a non-selective green fluorescent protein (GFP) and selective chloramphenicol acetyltransferase (CAT) gene. While 1 UAG codon impaired the expression of GFP in wild-type organisms, at least 2 UAG codons were required in CAT to consistently prevent escaped expression in wild-type organisms with a standard genetic code. Additionally, sequencing revealed that wild-type organisms enabled expression of CAT by mutating UAG codons to UGG coding for tryptophan or CAG coding for glutamine. By placing UAG at sites in proteins that cannot tolerate a tryptophan or glutamine substitution, we can create UAG-containing genes further isolated from expression in wild organisms.
As biotechnology increasingly targets open-environment applications such as bioremediation or disease treatment in humans, we require methods to stabilize and control the genetic information that we encode in engineered biological systems. Because alternative genetic codes can both confer resistance to horizontal gene transfer into an engineered system and restrict expression of engineered genes in wild-type organisms, genomic recoding of organisms to contain alternative genetic codes is a promising path towards increasing the stability and safety of engineered biological systems. However, open-environment applications will expose engineered biological systems to new stresses not represented in the laboratory environment, and further work is required to validate these methods will be robust in conditions of limiting nutrients or other cellular stresses. Additionally, while we have demonstrated genetic isolation of the GRO with respect to genes both entering and leaving the cell, we cannot currently have both properties simultaneously because UAG is the sole open codon. We envision that current research into further codon reassignments, including the reassignment of sense codons, will pave the way for alternate genetic codes with multiple codon reassignments. By expanding recoding efforts to multiple species, we envision the development of synthetic microbial communities with alternate genetic codes that are genetically isolated and robust to perturbation by HGT.
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Ording, Marcus. "Context-Sensitive Code Completion : Improving Predictions with Genetic Algorithms." Thesis, KTH, Skolan för informations- och kommunikationsteknik (ICT), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-205334.
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Within the area of context-sensitive code completion there is a need for accurate predictive models in order to provide useful code completion predictions. The traditional method for optimizing the performance of code completion systems is to empirically evaluate the effect of each system parameter individually and fine-tune the parameters. This thesis presents a genetic algorithm that can optimize the system parameters with a degree-of-freedom equal to the number of parameters to optimize. The study evaluates the effect of the optimized parameters on the prediction quality of the studied code completion system. Previous evaluation of the reference code completion system is also extended to include model size and inference speed. The results of the study shows that the genetic algorithm is able to improve the prediction quality of the studied code completion system. Compared with the reference system, the enhanced system is able to recognize 1 in 10 additional previously unseen code patterns. This increase in prediction quality does not significantly impact the system performance, as the inference speed remains less than 1 ms for both systems.Inom området kontextkänslig kodkomplettering finns det ett behov av precisa förutsägande modeller för att kunna föreslå användbara kodkompletteringar. Den traditionella metoden för att optimera prestanda hos kodkompletteringssystem är att empiriskt utvärdera effekten av varje systemparameter individuellt och finjustera parametrarna. Det här arbetet presenterar en genetisk algoritm som kan optimera systemparametrarna med en frihetsgrad som är lika stor som antalet parametrar att optimera. Studien utvärderar effekten av de optimerade parametrarna på det studerade kodkompletteringssystemets pre- diktiva kvalitet. Tidigare utvärdering av referenssystemet utökades genom att även inkludera modellstorlek och slutledningstid. Resultaten av studien visar att den genetiska algoritmen kan förbättra den prediktiva kvali- teten för det studerade kodkompletteringssystemet. Jämfört med referenssystemet så lyckas det förbättrade systemet korrekt känna igen 1 av 10 ytterligare kodmönster som tidigare varit osedda. Förbättringen av prediktiv kvalietet har inte en signifikant inverkan på systemet, då slutledningstiden förblir mindre än 1 ms för båda systemen.
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Massey, Steven Edward. "Codon reassignment and the evolution of the genetic code." Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399606.
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Italia, James Sebastian. "Development and Applications of Universal Genetic Code Expansion Platforms:." Thesis, Boston College, 2019. http://hdl.handle.net/2345/bc-ir:108354.
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Thesis advisor: Abhishek ChatterjeeThe emergence of genetic code expansion (GCE) technology, which enables sitespecific incorporation of unnatural amino acids (UAAs) into proteins, has facilitated powerful new ways to probe and engineer protein structure and function. Using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that suppress repurposed nonsense codons, a variety of structurally diverse UAAs have been incorporated into proteins in living cells. This technology offers tremendous potential for deciphering the complex biology of eukaryotes, but its scope in eukaryotic systems remains restricted due to several technical limitations. For example, development of the engineered tRNA/aaRS pairs for eukaryotic GCE traditionally relied on a eukaryotic cell-based directed evolution system, which are significantly less efficient relative to bacteria-based engineering platforms. The work described in this thesis establishes a new paradigm in GCE through the development of a novel class of universal tRNA/aaRS pairs, which can be used for ncAA incorporation in both E. coli and eukaryotes. We achieve this by developing engineered strains of E. coli, where one of its endogenous tRNA/aaRS pair is functionally replaced with an evolutionarily distant counterpart. The liberated pair can then be used for GCE in the resulting altered translational machinery (ATM) strain, as well as any eukaryote. Using this strategy, we have been able to genetically encode new bioconjugation chemistries, post-translational modifications, and facilitate the incorporation of multiple, distinct ncAAs into a single protein. The ATM technology holds enormous promise for significantly expanding the scope of the GCE technology in both bacteria and eukaryotes
Thesis (PhD) — Boston College, 2019
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Mukre, Prakash. "Hardware accelerator for DNA code word searching." Diss., Online access via UMI:, 2008.
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Thesis (M.S.)--State University of New York at Binghamton, Thomas J. Watson School of Engineering and Applied Science, Department of Electrical and Computer Engineering, 2008.Includes bibliographical references.
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Abedi, Saied. "Genetic multi-user detection for code division multiple access systems." Thesis, University of Surrey, 2000. http://epubs.surrey.ac.uk/843016/.
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The origins of spread spectrum are in navigation and military systems. Techniques originally developed to reduce the effects of the intentional jamming also proved suitable for communications through dispersive channels in cellular applications. In 1949 the first time hoping spread spectrum multiple access system has been introduced by John Pierce. A direct-sequence spread spectrum system has been proposed by De Rosa-Rogoff later in 1950. He has also introduced the concept of processing gain. However the commercialisation of cellular direct-sequence code division multiple access (DS-CDMA) systems became possible only during the 1980's and 1990's. In 2000 and beyond we will be witness to the standardisation and commercialisation of wide band CDMA system with a bandwidth of 5 MHz or more. While CDMA presents a number of advantages for cellular mobile communications, it has its own drawbacks. Good air interface designs provide efficient solutions for the terrestrial cellular system. We first take a look at the problem of narrow-band and partial band interference suppression in a CDMA system. By using Poor's model for partial-band interference, some interference suppression techniques are analysed. Then the concept of hybrid genetic prediction is introduced which outperforms the nonlinear techniques in terms of SNR improvements. The performance of a CDMA system can be degraded by Multiple Access Interference (MAI) due to the presence of many users in the same bandwidth. After analysing the structure and performance of existing multi user detection techniques, a low complexity bit level detector is proposed to reduce MAI in synchronous CDMA (S-CDMA) system for an AWGN channel. It is shown how it is possible to reach almost the single user bound by combining detection theory and the fundamentals laws of evolution found in nature. Nonlinear mappings are added to the proposed detector to increase detector's performance by reducing the misleading effect of noise on the detection process. Then for Asynchronous CDMA (A-CDMA), a novel packet level genetic detector is proposed. The near-far resistance feature of the proposed detector is studied. The effect of different parameters of genetic engine i.e. chromosome length, gene's value or mutation and crossover probabilities on its performance are characterised. Different initialisation methods are introduced. For high bit rate CDMA system in multimedia applications in a dispersive CDMA channel, Inter Symbol Interference (ISI) becomes another highly important factor that degrades system performance. It is shown how it is possible to use a signal sub-space based detector as a core detector for a hybrid genetic Multi user detector.
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Grewal, Gary William. "Enhanced genetic algorithms and their application in retargetable code generation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0008/NQ33302.pdf.
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Miranda, Isabel Alexandra Marcos. "Molecular study of a genetic code alteration in C. albicans." Doctoral thesis, Universidade de Aveiro, 2006. http://hdl.handle.net/10773/8980.
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Doutoramento em BiologiaA maioria dos organismos utiliza o mesmo código genético, no entanto alterações a este código padrão foram descobertas em procariotas e eucariotas. A maior parte das alterações ao código genético ocorre em mitocôndrias. No citoplasma eucariótico, o único exemplo conhecido de alteração ao código genético envolvendo a substituição de um aminoácido por outro aminoácido, ocorre em várias espécies do género Candida. Em Candida albicans o codão CUG é ambíguo, ou seja, pode ser traduzido como serina ou leucina, com predominância para o primeiro aminoácido. Na origem desta ambiguidade está um tRNACAG Ser de C. albicans que possui elementos de identidade para duas aminoacil-tRNA sintetases, nomeadamente a seril e leucil-tRNA sintetases, podendo, por isso, ser aminoacilado com serina e leucina. Este tRNA surgiu há cerca de 272 milhões de anos no antepassado das leveduras e introduziu dupla identidade (ambiguidade) no codão CUG que começou a ser descodificado como leucina e serina. As consequências biológicas desta ambiguidade e da alteração de identidade do codão CUG de leucina para serina são desconhecidas. O objectivo deste estudo foi elucidar a função biológica da ambiguidade do codão CUG que foi preservada em C. albicans. Pretendeu-se compreender porque é que a ambiguidade do codão CUG foi preservada e conhecer melhor os mecanismos de evolução ao código genético. Para tal, aumentou-se a ambiguidade do codão CUG, usando engenharia de tRNAs e estudaram-se as consequências de tal ambiguidade ao nível fenotípico. Os resultados demonstram de forma inequívoca que a ambiguidade do codão CUG é um gerador de diversidade fenotípica e sugerem que uma das funções da alteração ao código genético é potenciarem a evolução rápida de novos fenótipos. A ambiguidade do codão CUG induz a expressão de vários factores de virulência de C. albicans, nomeadamente variabilidade morfológica, alteração fenotípica, produção de hidrolases extracelulares e adesinas. Assim, a ambiguidade do código genético é fundamental para a biologia de C. albicans.
Most organisms use the same genetic code, however several alterations to the standard code have been found in prokaryotes and eukaryotes. Most alterations occur in mitochondria and the only known case of a cytoplasmatic sense-to-sense codon identity change occurs in several species of the genus Candida. In Candida albicans, standard leucine-CUG codon is decoded mainly as serine but to a lesser extent as leucine. This is due the existence of a novel tRNACAG Ser that has identity elements for both the seryl- and the leucyl-tRNA aminoacyl synthetases and hence can be aminoacylated with serine and leucine. The tRNACAG Ser appeared 272 million years ago in the yeast ancestor, and created a CUG codon with double identity due to its decoding as both serine and leucine. The biological function of such ambiguity, which was preserved to the present day, is still unknown. The objective of this study was to elucidate the role of CUG ambiguity in C. albicans biology. An attempt was made to shed new light i) on the biological role of genetic code ambiguity, ii) on why CUG ambiguity was preserved and iii) on why genetic code alterations evolve. For this, highly ambiguous C. albicans strains were created through tRNA engineering techniques and the effects of such ambiguity were studied at phenotypic level. The data presented herein shows for the first time that genetic code ambiguity is a generator of phenotypic diversity and strongly suggests that genetic code alterations speed up evolution of new phenotypes. Ambiguous decoding of the CUG codon triggers expression of C. albicans virulence factors, namely morphogenesis, phenotypic switching, extracellular hydrolases production and adhesion, indicating that it plays a critical role on C. albicans biology.
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Madrzak, Julia. "Site-specific ubiquitination of recombinant proteins via genetic code expansion." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708739.
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Shi, Aishan. "Decoding the Genetic Code: Unraveling the Language of Scientific Paradigms." Thesis, The University of Arizona, 2013. http://hdl.handle.net/10150/297762.
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Scientific revolutions have not only significantly broadened our knowledge underlying physical laws and natural patterns, but also shifted the cultural paradigm through which science is understood and practiced. These paradigm shifts, as Thomas Kuhn denoted them, are facilitated through changes in language, because language is the only method of articulating - and thereby establishing - truth, according to Friedrich Nietzsche and Michel Foucalt. Steven Shapin analyzed the progression of these linguistic changes in global scientific revolutions and Bruno Latour categorized them in the local laboratory setting. One of the most recent revolutions in science, the discovery of the double helix by James Watson and Francis Crick, altered the understanding and application of genetics. James Watson’s personal account of this discovery, the Double Helix, makes the point to change the general perception of science as intellectual labor to innovative play. However, he does not portray his discovery in the scientific elegance that it deserves as the culmination of nearly a century of research and the marriage of quantum physics and biology. This thesis explores the paradigm shifts that developed with each scientific revolution, how they led to the double helix, and finally, the paradigm shift of the "Structure-Function Relationship" that accompanied this discovery.
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Willis, Julian C. W. "Developing new orthogonal tRNA/synthetase pairs for genetic code expansion." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274057.
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Pinto, Desterro Maria Joana. "Role of SUMO-1 modification in transcriptional activation." Thesis, University of St Andrews, 1999. http://hdl.handle.net/10023/2724.
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In unstimulated cells, the transcription factor NF-κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Activation of NF--KB is mediated by signal induced degradation of IκBα via the ubiquitin proteasome-dependent pathway. Targeting the proteins for ubiquitin-mediated proteolysis is an irrevocable decision, and as such, the process needs to be highly specific and tightly regulated. This task is achieved by conjugation and deconjugation enzymes that act in a dynamic and coordinated mechanism. In a yeast two hybrid screen designed to identify proteins involved in IκBα signalling Ubch9 was found to interact with the N-terminal regulatory region of IκBα. Although Ubch9 is an enzyme homologous to E2 ubiquitin conjugating enzymes we have shown that is unable to form a thioester with ubiquitin but it is capable to form a thioester with the small ubiquitin-like protein SUMO- 1. To fully characterise the SUMO-1 modification reaction we have purified the proteins and cloned the genes encoding the SUMO-1 activating enzyme (SAEl/SAE2) and shown that it is homologous to enzymes involved in the activation of ubiquitin, Smt3p, the yeast SUMO-1 homologue, and Rublp/Nedd8, another ubiquitin-like protein. SUMO-1 is conjugated to target proteins by a pathway that is distinct from, but analogous to, ubiquitin conjugation. SUMO-1 was efficiently conjugated, both in vivo and in vitro, to IκBα on lysine 21, which is also utilised for ubiquitin modification. Thus, by blocking ubiquitination SUMO-1 modification acts antagonistically to generate a pool of IκBα resistant to proteasome-mediated degradation which consequently inhibits NF-κB dependent transcription activation. In view of several lines of similarity between NF-kB and p53, the involvement of SUMO-1 modification in the metabolism of the tumour supressor p53 was investigated. We have shown that p53 is modified by SUMO-1 at a single site, lysine 386 in the C-terminus of p53. Although p53 is regulated by ubiquitination, SUMO-1 and ubiquitin modification do not compete for the same lysine in p53. However, overexpression of SUMO-1 activates the transcriptional activity of wild type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. A consensus sequence was obtained by comparison of the sequences surrounding the SUMO-1 acceptor lysine in proteins that have been shown to be modified by SUMO-1 and revealed a possible recognition site for SUMO-1 conjugation machinery. Tagging of proteins with SUMO-1 regulates transcriptional activation, either by interfering with subcellular location or with the ubiquitination pathway. The pathway may represent a novel target for drug development.
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Tsai, Ya-Lin. "Development of parallel processing algorithms to provide automatic image analysis for medical application." Thesis, University of Sunderland, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336914.
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This thesis describes the development of: (i) an automatic chromosome analysis system capable of producing to a high degree of accuracy and consistency a correct classification for damaged chromosomes at a low cost and (ii) a parallel computer system to enable more rapid chromosome analysis. Chromosomes can be examined in a cytogenetics laboratory for a variety of purposes including an assessment of the affects of ionisation exposure on the genetic code of the cell. Scoring of chromosome aberrations caused by ionisation of radiation exposure, is possible by detecting dicentric chromosomes. In addition this approach provides a good biological radiation measure (dosimeter). However, currently manual methods are extremely time consuming and expensive with respect to labour costs. For the low radiation doses it is necessary to analyse a large number of chromosomes to identify a small number of damaged ones to score the number of aberrations. Consequently, the main objective of this research programme is to develop a rapid, low cost, and accurate automated chromosome analysis system. This research has concentrated solely on scoring dicentric chromosome since their characteristic shape is relatively easy to recognise in most cases and they most commonly created by exposure to radiation. The methods and theories considered in this thesis concerns chromosome image selection by automatic segment extraction using of the following: grey levels; image extraction by seed aggregation, a two dimensional function, a moment algorithm, for chromosome orientation; chromosome centreline determination; rapid detection of the chromosome centromere of the candidate. The new methods developed by the author and presented herein concern three steps or processes in automatic chromosome analysis. These include (i) a new segmentation scheme (ii) automatic selection the cell threshold grey scale level and (iii) the design a new methods capable of detecting bent chromosome with rapid determination the chromosome centromere. Parallel processing using the processor farm technique has been successfully developed to enable a more rapid chromosome classification system. The techniques described have been carefully tested and evaluated and have clearly demonstrated the potential application of the analysis methods by the author.
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Han, Tony. "SWASAD Smith & Waterman-algorithm-specific ASIC design /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16391.pdf.
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Henderson, Daryl Stewart. "A genetic analysis of mutagen-sensitive mutations on the second chromosome of Drosophila melanogaster." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26418.
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Mutagen-sensitive (mus) mutations in Drosophila melanogaster render developing flies hypersensitive to the lethal effects of DNA-damaging agents. In general, mus mutations identify DNA repair-related genes. In this study, 5 new second chromosome mus mutations (mus205B¹, mus208B¹, mus209B¹, mus210B¹ and mus211B¹), selected on the basis of sensitivity to methyl methanesulfonate (MMS), were characterized using a variety of genetic tests. One test measured the MMS-sensitivity of double mutant mus strains compared to their component single mutants. Mutant interactions were examined in 8 double mus and in 2 triple mus strains containing combinations of mus201D¹, mus205B¹, mus208B¹, mus210B¹ and mus211B¹ (or mus211B²). These analyses have revealed predominantly synergistic and epistatic responses to MMS. Taken together with the findings of previous genetic and biochemical studies of Drosophila mus strains, these results suggest that 3 major repair pathways may operate in flies to correct damage caused by MMS.
Mutagen cross-sensitivity data and the results of the interaction studies suggest that mus mutations might serve as rapid and sensitive bioassays of somatic genotoxicity caused by mutagens and carcinogens. To explore this possibility, a simple mutagen test system was devised employing triple mutant mus strains. One strain (mus208B¹ mus210B¹ mus211B²) was tested for sensitivity to 14 mutagens/carcinogens and 2 non-carcinogens. Eleven of the mutagens/carcinogens were readily detected as genotoxic. Both non-carcinogens were non-genotoxic. These preliminary results demonstrate the feasibility (and some limitations) of the proposed somatic genotoxicity assay and emphasize the need for further test validation using a larger chemical data base.
The temperature-sensitive lethal mutation mus209B¹ was subjected to extensive genetic analyses and to temperature shift experiments during development. This locus was found to encode a product(s) that (1) is essential for viability at virtually all pre-imaginal developmental stages (the latter half of pupation appears to be an exception), (2) is necessary for wildtype levels of resistance to the genotoxic effects of MMS and ionizing radiation, and (3) is required for female fertility. Confirmation of the pleiotropic nature of this mutation was obtained by meiotic and cytogenetic mapping studies and by complementation tests with a series of allelic mutations. The mus209B¹ phenotypes are similar to ones conferred by mutations in Drosophila and yeast that disrupt various aspects of chromosome metabolism. In this context, some possible roles for mus209B¹ are discussed.Science, Faculty of
Zoology, Department of
Graduate
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Cai, Zesi. "Genetic Algorithm for Integrated SoftwarePipelining." Thesis, Linköpings universitet, Institutionen för datavetenskap, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-76088.
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The purpose of the thesis was to study the feasibility of using geneticalgorithm (GA) to do the integrated software pipelining (ISP). Different from phasedcode generation, ISP is a technique which integrates instruction selection, instructionscheduling, and register allocation together when doing code generation. ISP is able toprovide a lager solution space than phased way does, which means that ISP haspotential to generate more optimized code than phased code generation. However,integrated compiling costs more than phased compiling. GA is stochastic beam searchalgorithm which can accelerate the solution searching and find an optimized result.An experiment was designed for verifying feasibility of implementing GA for ISP(GASP). The implemented algorithm analyzed data dependency graphs of loop bodies,created genes for the graphs and evolved, generated schedules, calculated andevaluated fitness, and obtained optimized codes. The fitness calculation wasimplemented by calculating the maximum value between the smallest possibleresource initiation interval and the smallest possible recurrence initiation interval. Theexperiment was conducted by generating codes from data dependency graphsprovided in FFMPEG and comparing the performance between GASP and integerlinear programming (ILP). The results showed that out of eleven cases that ILP hadgenerated code, GASP performed close to ILP in seven cases. In all twelve cases thatILP did not have result, GASP did generate optimized code. To conclude, the studyindicated that GA was feasible of being implemented for ISP. The generated codesfrom GASP performed similar with the codes from ILP. And for the dependencygraphs that ILP could not solve in a limited time, GASP could also generate optimizedresults.
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Brodyk, Andr?? Art College of Fine Arts UNSW. "Genetic art and recombinants: introns non-code and the proto-animate condition." Awarded By:University of New South Wales. Art, 2009. http://handle.unsw.edu.au/1959.4/44087.
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This thesis examines how a contemporary biological model namely genetic recombinant DNA and technologies can be interpreted to develop new creative art-based recombinant processes and media. This question is investigated through a focus on practice-led research, on materiality and novel methodologies. The materiality centers on the development of new biologically based Genetic art media. These new methodologies entail practice-based interpretations and experimentation of literal and invented molecular recombinant DNA processes. These procedures necessitated working inside molecular biology laboratories as art studios. The idiosyncratic and problematical dimensions of working within a science domain as part of this practical paradigm are presented to contextualize the work relative to more conventional approaches. The conceptual basis of the project is shown to emanate out of the practice. This experiential strategy was part of the overall approach adopted for this project. Accordingly that underpins the practice as research and also forms the general structure for the written chapters. Conceptually, the broader results were the development of two main concepts for Genetic art. These are shown to be a direct consequence of a shift in the material nature of the projects mature phases of practice. These concepts are the Genetic Portmanteaux and the concept of the Protoanimate Condition. They entail reinterpretations of recombinant semi ready-mades and the notion of the inanimate applied to non-coding DNA. This is important in relation to extant and novel DNA code scripts, especially regarding a particular form of biological inanimate material, the noncoding DNA known as introns. In the Proto-animate Condition argument, introns represent a recondite and temporal state in a pending or protoanimated condition within a recombinant environment. The major results and conclusions expressed in creative terms are the development of several novel DNA scripts and the practical materialization of these recombinants via the agency of installation. Seven exhibitions including five installations were created and published within curated forums. Overall they imbue qualities such as the temporal, the recondite, the anticipatory and the creative recombinant complexion of DNA material. This is proffered as a fecund area for new art-based explorations and critiques of developments in both contemporary art and science.
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Moghal, Adil Baig. "Context-dependent threats to the fidelity of translation of the genetic code." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1465925323.
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Cardno, Tony Stuart, and n/a. "Development of a high throughput fluorescent screening assay for genetic recoding." University of Otago. Department of Biochemistry, 2007. http://adt.otago.ac.nz./public/adt-NZDU20071218.145806.
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The development of new drug therapies traditionally requires mass screening of thousands if not millions of substances to identify lead compounds. They are then further optimised to increase potency. The screening of the large pharmaceutical compound libraries can be incredibly expensive, with the industry responding by miniaturising the assays to smaller formats, enabling the compound screening to be automated and, importantly, eliminating assay reagents that are a major contributing cost for running large screens.
A potential target for such an approach is the genetic recoding site of viruses like HIV-1 and SARS. They use programmed recoding of the genetic code to regulate the translation of necessary proteins required for viable virus production. For example HIV-1 uses a -1 frameshift mechanism to regulate the ratio of the Gag to the Pol proteins, crucial for viable virus formation.
The study of recoding, including readthrough of premature termination codons have most recently used bicistronic reporters with different combinations of enzymes. The most widely used plasmid bicistronic reporter utilises a dual luciferase arrangement comprised of firefly luciferase and Renilla luciferase reporters flanking the DNA being studied. Both of the luciferase enzymatic reporters emit light in response to their respective substrates. The cost of these substrates is the major issue to using luciferase reporters for high throughput screening.
My study aimed at designing and developing a bicistronic assay suitable for genetic recoding that was amenable to high throughput screening. The luciferase reporters were replaced with Green Fluorescent Protein (GFP) reporters that do not require the addition of substrates. The development of a dual GFP assay required the appropriate selection of GFP fluorophores, the best arrangement of the GFPs to maximise the ratio of relative fluorescence intensity signal to background, the optimisation of the cells and growth conditions, DNA transfection, plate reader selection, and optical filter sets. Cassettes encoding protein linkers were also incorporated into the design of the constructs to separate the fluorescent proteins spatially to facilitate unimpaired folding into their functional units within the fusion protein. The assay was further improved by moving from transient transfection to stably expressing cell lines. A viable assay was almost achieved for 96 (and 384) well plates with a Z� factor compatible with the assay being suitable for high throughput screening.
The assay was used to test a small collection of compounds known to interact with the ribosome and compounds known in the literature to affect frameshifting. This proof of concept was important, since it showed that the assay, with the various modifications, optimisations and miniaturisation steps, still retained the capability of correctly measuring the -1 frameshifting efficiency at the HIV-1 recoding site, and recording compound-induced modulations to the frameshifting efficiency. The compounds cycloheximide and anisomycin, for example, were shown to decrease -1 frameshifting albeit at some expense to overall protein synthesis.
The dual GFP assay was also shown to be able to measure accurately changes in the frameshift efficiency brought about by mutations to the frameshift element, and additionally, it would be suitable for the detection and study of compounds, like the recently reported PTC-124 (currently undergoing phase II clinical trial for Duchenne Muscular Dystrophy and cystic fibrosis) that increases readthrough of a UGA premature stop codon mutation.
The dual GFP assay developed in this study is at most only 1/10th of the cost of a comparable dual luciferase assay, largely due to removal of assay substrates and transfection reagents. The assay has a robust Z� factor comparable to that of the dual luciferase assay, and would substantially decrease the costs of high throughput screening in situations where a bicistronic reporter is required. The HIV-1 frameshift element is such a site.
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Butarbutar, Nunut. "Analysis of yeast codon usage patterns using the movable ORF collection /." Online version of thesis, 2007. http://hdl.handle.net/1850/5700.
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Forrest, Megan E. "Regulation of Mammalian Messenger RNA Stability via the Open Reading Frame." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1579862741902687.
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Wang, Shu-Zhen. "Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm." Diss., Virginia Polytechnic Institute and State University, 1985. http://hdl.handle.net/10919/49959.
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Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A)⁺ mRNA were found to direct in vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A)⁺ mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with ³²P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not.
Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single U gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons. There are eight essentially identical tandem repeats of the hexapeptide Pro-Pro-Pro-Val-His-Leu and two of the octapeptide Gln-Pro-His-Pro-Cys-Pro-Cys-Gln in the N-terminal one-half of the polypeptide. The codon specifying the third proline in the hexapeptide repeating unit is identical, CCG, in all eight repeats. It is likely that these highly conserved tandem repeats are of critical importance to the function of gamma-zein which is presently unknown. Alternatively, it is conceivable that selective pressures responsible for conserving these tandem repeats may be operating at the nucleic acid level.Ph. D.
incomplete_metadata
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Johansen-Leete, Jason Paul. "Discovery of Bioactive macrocyclic peptides using mRNA display with genetic reprogramming." Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/29467.
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Macrocyclic peptides are attractive for their favourable drug-like properties, bridging the gap between small molecules and biologics. Whilst nature is a rich source of bioactive macrocyclic peptides, their de novo discovery would access the full potential of this chemotype for therapeutic use. Using its ultra-large libraries of >1012 unique peptides, mRNA display is a powerful method of de novo ligand discovery against protein target. Genetic reprogramming can be exploited to display peptides with modified structures to improve their drug-like properties, such as affinity and cell-permeability.
Chapter 2 describes the first reprogramming of sulfotyrosine into mRNA display for the discovery of chemokine-binding cyclic peptides, discovering several high-affinity sulfated ligands which inhibited chemotactic signalling. Chapter 3 provides an overview of COVID-19 proteases. Replication of SARS-CoV-2 uses two viral proteases, the papain-like protease (PLpro) and the main protease (Mpro), and are of extreme interest as antiviral drug targets for COVID-19. Chapter 4 describes the discovery macrocyclic inhibitors of SARS-CoV-2 PLpro. Initially discovering peptide binders to PLpro with modest inhibitory activity against the protease, we hypothesised leveraging genetic code reprogramming to discover covalent inhibitors would generate higher affinity PLpro ligands. After panning several covalent warhead-containing amino acids, we identified a cysteine-reactive methylacrylamide-moeity compatible with ribosomal incorporation. This lays the foundation for the future discovery of covalent cyclic peptide inhibitors of PLpro and other cysteine proteases. Chapter 5 describes genetically reprogrammed mRNA display to discover several potent inhibitors of the SARS-CoV-2 Mpro which also exhibited in vitro antiviral activity. The novel antiviral molecules discovered in this work exemplify the power of the cyclic peptide chemotype and the need for its further development.
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Silva, Ana Rita Guimarães Rodrigues da. "Codon ambiguities as a mechanism to alter the genetic code in Saccharomyces cerevisiae." Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/15394.
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Mestrado em Biologia Molecular e CelularAlthough the genetic code is generally viewed as immutable, alterations to its standard form occur in the three domains of life. A remarkable alteration to the standard genetic code occurs in many fungi of the Saccharomycotina CTG clade where the Leucine CUG codon has been reassigned to Serine by a novel transfer RNA (Ser-tRNACAG). The host laboratory made a major breakthrough by reversing this atypical genetic code alteration in the human pathogen Candida albicans using a combination of tRNA engineering, gene recombination and forced evolution. These results raised the hypothesis that synthetic codon ambiguities combined with experimental evolution may release codons from their frozen state. In this thesis we tested this hypothesis using S. cerevisiae as a model system. We generated ambiguity at specific codons in a two-step approach, involving deletion of tRNA genes followed by expression of non-cognate tRNAs that are able to compensate the deleted tRNA. Driven by the notion that rare codons are more susceptible to reassignment than those that are frequently used, we used two deletion strains where there is no cognate tRNA to decode the rare CUC-Leu codon and AGG-Arg codon. We exploited the vulnerability of the latter by engineering mutant tRNAs that misincorporate Ser at these sites. These recombinant strains were evolved over time using experimental evolution. Although there was a strong negative impact on the growth rate of strains expressing mutant tRNAs at high level, such expression at low level had little effect on cell fitness. We found that not only codon ambiguity, but also destabilization of the endogenous tRNA pool has a strong negative impact in growth rate. After evolution, strains expressing the mutant tRNA at high level recovered significantly in several growth parameters, showing that these strains adapt and exhibit higher tolerance to codon ambiguity. A fluorescent reporter system allowing the monitoring of Ser misincorporation showed that serine was indeed incorporated and possibly codon reassignment was achieved. Beside the overall negative consequences of codon ambiguity, we demonstrated that codons that tolerate the loss of their cognate tRNA can also tolerate high Ser misincorporation. This raises the hypothesis that these codons can be reassigned to standard and eventually to new amino acids for the production of proteins with novel properties, contributing to the field of synthetic biology and biotechnology.
O código genético é geralmente visto como imutável, no entanto várias alterações à sua forma padrão são conhecidas. Uma das mais notáveis acontece em várias espécies do género Candida, onde o codão Leu-CUG é descodificado como serina por um novo RNA transferência (Ser-tRNACAG). O laboratório de acolhimento fez um grande progresso ao reverter a alteração atípica do código genético do fungo patogénico humano C. albicans, usando uma combinação de tRNAs mutantes, recombinação genética e evolução forçada. Estes resultados levantaram a hipótese que as ambiguidades sintéticas do codão, combinadas com evolução experimental, poderem libertar os codões do seu estado fixo. Nesta tese testamos esta hipótese usando S. cerevisiae como modelo biológico. Geramos ambiguidade em codões específicos, de forma bifásica, envolvendo a deleção de genes de tRNA, seguida pela expressão de tRNAs não-cognatos capazes de compensar o tRNA eliminado. Tendo como base a ideia que os codões raros são mais suscetíveis a alterações que aqueles usados frequentemente, usamos duas estirpes knock-out, nas quais não existem os tRNAs cognatos capazes de descodificar os codões raros CUC-Leu e AGG-Arg. Exploramos então a vulnerabilidade destes codões pela construção de tRNAs mutantes que incorporam erradamente Ser nestes locais. Estas estirpes recombinantes foram evoluídas ao longo do tempo, usando evolução experimental. Apesar de ter havido um forte impacto negativo na taxa de crescimento de estirpes que expressam o tRNA mutante a altos níveis, esta expressão a baixos níveis teve pouco impacto no fitness celular. Descobrimos que não só a ambiguidade do codão, mas também destabilizações da pool de tRNAs endógenos têm um impacto negativo na taxa de crescimento. Após a evolução, as estirpes com elevada expressão do tRNA mutante recuperaram significativamente em vários parâmetros de crescimento, o que mostra que estas adaptam-se e exibem maior tolerância à ambiguidade do codão. Através do sistema repórter fluorescente desenvolvido monitorizamos a incorporação errónea de Ser, o que nos indica que a Ser está de facto a ser incorporada e que, possivelmente, a alteração da identidade do codão foi atingida. Apesar das consequências negativas gerais da ambiguidade do codão, demonstramos que os codões capazes de tolerar a perda do seu tRNA cognato, conseguem também tolerar a incorporação elevada de Ser. Isto levanta a hipótese que estes codões podem ser recodificados para outros aminoácidos naturais e/ou artificiais para a produção de proteínas com novas propriedades, contribuindo assim para o campo da Biologia Sintética e Biotecnologia.
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Qi, Xin Dervan Peter B. "Unnatural amino acid incorporation to rewrite the genetic code and RNA-peptide interactions /." Diss., Pasadena, Calif. : California Institute of Technology, 2005. http://resolver.caltech.edu/CaltechETD:etd-05272005-133323.
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Kinney, William D. "Expansion of the Genetic Code to Include Acylated Lysine Derivatives and Photocaged Histidine." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5903.
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The genetic code of all known organisms is comprised of the 20 proteinogenic amino acids that serve as building blocks on a peptide chain to form a vast array of proteins. Proteins are responsible for virtually every biological process in all organisms; however, the 20 amino acids contain a limited number of functional groups that often leaves much to be desired. The lack of diversity addresses the need to increase the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids. In order to expand the chemical scope of the genetic code beyond the functionalities that can be directly genetically encoded, unnatural amino acids must be added to the proteome. The ability to incorporate unnatural amino acids (UAAs) into proteins at defined sites has a direct impact on the ability of scientists to study biological processes that are difficult or impossible to address by more classical methods.
The UUAs of interest are acylated lysine derivatives (isovaleryl, isobutyryl, and β-hydroxybutyryl) and photocaged histidine. Acylation of histone lysine has been linked to epigenetic regulation of metabolism.1 A means to site-specifically incorporate each acylated lysine derivative would help study the effect of acylated lysine in epigenetic regulation. Likewise, in order to elucidate the role of histidine in specific protein functions, one can replace a critical histidine with a photocaged histidine. Photocaged amino acids are those that possess a photo-cleavable, aromatic caged group. Light-induced protein activation allows for the biological activity of the protein to be spatiotemporally regulated under non-invasive external control.2
The site-specific in vivo incorporation of unnatural amino acids is made possible by amber codon suppression by an orthogonal suppressor aminoacyl-tRNA synthetase (aaRS)/tRNA pair.3 In amber codon suppression the amber stop codon is decoded for an UAA by a suppressor aaRS/tRNA pair. To accept the UAA, the aaRS must be evolved to achieve orthogonal activity with specific UUAs. The pyrrolysyl aaRS/tRNA (PylRS/PylT) pair from M. barkeri and M. mazei was used to construct multiple, large-scale aaRS mutant libraries where critical residues within the active site of PylRS are mutated via site-saturated mutagenesis.4 The libraries were subjected to directed evolution through a series of positive and negative selections to enrich aaRS variants that exclusively bind to acylated lysine derivatives and photocaged histidine as substrates.5 The PylRS selection survivors were screened for UAA activity and identified successful clones underwent a fluorescent activity assay. The active aaRS were used for amber codon suppression to express the respective UAA in ubiquitin and green fluorescent protein constructs.
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Mathew, Suneeth Fiona, and n/a. "Understanding genetic recoding in HIV-1 : the mechanism of -1 frameshifting." University of Otago. Department of Biochemistry, 2008. http://adt.otago.ac.nz./public/adt-NZDU20081006.115352.
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The human immunodeficiency virus type 1 (HIV-1) uses a mechanism of genetic recoding known as programmed ribosomal frameshifting to translate the proteins encoded by the pol gene. The pol gene overlaps the preceding gag gene in the -1 reading frame relative to gag. It contains neither a start codon nor an internal ribosome entry site (IRES) to initiate translation of its proteins. Rather the host ribosomes are forced to pause due to tension placed on the mRNA when they encounter a specific secondary structural element in the mRNA. This tension is relieved by disruption of the contacts between the mRNA codons and tRNA anticodons at a �slippery� sequence within the ribosomal decoding centre. Re-pairing of the tRNAs occurs in the new -1 frame after movement of the mRNA backwards by one nucleotide, allowing the ribosome to translate the pol gene as a Gag-Pol polyprotein. A change in ratio of Gag to Gag-Pol proteins affects viral assembly, and most significantly dramatically reduces viral infectivity.
The prevailing model for the mechanism of -1 frameshifting has focussed on a pre-translocational event, where slippage occurs when the slippery sequence is within the ribosomal A and P sites. This model precludes a contribution from the codon immediately downstream of the slippery sequence leading into the secondary structural element. I have termed this the �intercodon�. Often at frameshifting sites it is a termination codon, whereas in HIV-1 it is a glycine codon, GGG. When the intercodon within the frameshift element was changed from the wild-type GGG to a termination codon UGA, the efficiency of frameshifting decreased 3-4-fold in an in vivo assay in cultured human cells. This result mimicked previous data in the group within bacterial cells and cultured monkey COS-7 cells. Changing the first nucleotide of the intercodon to each of the three other bases altered frameshifting to varying degrees, but not following expected patterns for base stacking effects. Such a result would support a post-translocational model for -1 frameshifting. It suggested that the intercodon might be within the ribosomal A site before frameshifting, and that the slippery sequence was therefore within the P and E sites.
This was investigated by modulating the expression of decoding factors for the intercodon - the release factor eRF1 and cognate suppressor tRNAs when it was either of the UGA or UAG termination codons, and tRNA[Gly] for the native GGG glycine codon. These were predicted to affect frameshifting only if slippage were occurring when the ribosomal elongation cycle was in the post-translocational state. Overexpression of tRNA[Gly] gave inconsistent effects on frameshifting in vivo, implying that its concentration may not be limiting within the cell. When eRF1 was overexpressed or depleted by RNAi, significant functional effects of decreased or increased stop codon readthrough respectively were documented. Expression of suppressor tRNAs increased readthrough markedly in a stop codon-specific manner. These altered levels of eRF1 expression were able to modulate the +1 frameshifting efficiency of the human antizyme gene. Overexpression of eRF1 caused significant reduction of frameshifting of the HIV-1 element with the UAG or UGA intercodon. Depletion of the protein by contrast had unexplained global effects on HIV-1 frameshifting. Suppressor tRNAs increased frameshifting efficiency at the UAG or UGA specifically in a cognate manner. These results strongly indicate that a post-translocational mechanism of frameshifting is used to translate the HIV-1 Gag-Pol protein.
A new model (�almost� post-translocational) has been proposed with -1 frameshifting occurring for 1 in 10 or 20 ribosomal passages during the end stages of translocation, because of opposing forces generated by translocation and by resistance to unwinding of the secondary structural element. With translocation still incomplete the slippery sequence is partially within the E and P sites, and the intercodon partially within the A site. The nature of the intercodon influences frameshifting efficiency because of how effectively the particular decoding factor is able to bind to the partially translocated intercodon and maintain the normal reading frame.
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Hartley, Andrew M. "Using a reprogrammed genetic code to modulate protein activity by novel post-translational control." Thesis, Cardiff University, 2014. http://orca.cf.ac.uk/68901/.
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Despite the diverse structures and functions sampled by the proteome, all proteins comprise 20 canonical amino acids that sample only a small percentage of available chemistry. This limitation is lifted somewhat through the use of post-translational modifications, however the limit imposed by the restricted number of amino acids inherently limits the variety of protein function and control that can be accessed. One powerful route to diversify the chemistry sampled by proteins is through genetically encoded unnatural amino acid (uAA) incorporation. The uAA p-azido-L-phenylalanine (AzPhe) can introduce two novel methods of control, photochemical covalent rearrangement and Click chemistry. AzPhe incorporation combined with these two methods of novel post-translational control were used to modulate the function of two distinct proteins; TEM β-lactamase and sfGFP. This thesis introduces the use of uAAs and the technical modifications required to enable uAA incorporation in vivo. It describes the in silico approach taken to evaluate potential mutations based on the likelihood of them imparting novel changes to protein function. Nine positions in TEM β-lactamase were chosen for uAA incorporation and the effect on activity was then determined using kinetic analyses. AzPhe incorporation alone resulted in a variety of effects on enzyme activity, ranging from small increases to complete loss of activity. Subsequent post-translational modification using UV light resulted in only slight changes in activity. Modification via Click chemistry using dibenzyl cyclo-octyne (DBCO) derivatives resulted in either inhibition or increased catalytic activity, depending on the position of AzPhe incorporation and the type of adduct used. Click chemistry was then used to modify TEM β-lactamase with other chemical modifications that enable the immobilization of proteins onto two different surfaces. The π-π stacking interaction between a DBCO-pyrene moiety and graphene was exploited to attach TEM β-lactamase to graphene in a defined and controlled manner, placing the active site in close proximity to the electron cloud of the sp2-bonded material. TEM β-lactamase was then modified using two DNA oligonucleotides that define assembly of a DNA origami “tile”. DNA origami can be used to immobilize multiple proteins at several defined positions, enabling the re-creation of enzyme pathways or signalling cascades in vitro. Finally, AzPhe was incorporated into sfGFP and the effects of its incorporation and subsequent modification on fluorescence were explored. The incorporation of AzPhe resulted in a blue shifted λmax, a change that was reversed upon UV irradiation. X-ray crystallography suggested that a hydrogen-bonding network involving the chromophore and surrounding residues was disrupted upon AzPhe incorporation, but then reformed upon modification of the uAA. Click chemistry had a variable effect on fluorescence depending on the modification used. Modification of AzPhe with a large fluorescent dye had no effect on the sfGFP fluorescence spectrum, but enabled FRET between the two chromophores. Modification with a DBCO-amine had the same effect as UV irradiation. Overall, this thesis has shown that the use of genetically encoded uAA incorporation coupled with novel post-translational modifications is a powerful approach for modifying protein function, and facilitating defined interfacing with new and useful materials.
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Kelemen, Rachel Elizabeth. "New tools at the intersection of genetic code expansion, virus engineering, and directed evolution:." Thesis, Boston College, 2019. http://hdl.handle.net/2345/bc-ir:108612.
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Thesis advisor: Abhishek ChatterjeeIn the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. We describe the application of this technology to the modification of adeno-associated virus (AAV) for the first time, enabling the generation of vectors with precisely re-engineered cell-targeting for gene therapy. Our UAA-AAV production platform enables the incorporation of UAAs bearing bio-orthogonal reactive handles into multiple specific sites on the virus capsid and their subsequent functionalization with various labeling molecules. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity. Next, we used our unique UAA-AAV vector as a tool for the directed evolution of more active UAA incorporation machinery in mammalian cells. It is well known that the efficiency of unnatural amino acid mutagenesis in mammalian cells is limited by the suboptimal activity of the suppressor tRNAs currently in use. The ability to improve their performance through directed evolution can address this limitation, but no suitable selection system was previously available to achieve this. We have developed a novel platform for virus-assisted directed evolution of enhanced suppressor tRNAs (VADER) in live mammalian cells. Our system applies selective pressure for tRNA activity via the nonsense suppression-dependent production of UAA-AAV, and selectivity for the specific incorporation of interest comes from a novel virus purification strategy based on the unique chemistry of the UAA. We demonstrated > 10,000-fold selectivity for active tRNAs out of mock libraries and used this system to evolve libraries generated from the commonly used archaeal pyrrolysyl suppressor tRNA, ultimately identifying a variant which is three times as active as the original tRNA. Finally, we used next-generation sequencing to analyze the fate of every library member over the course of the selection and found that our VADER selection scheme is indeed selective for the enrichment of more active tRNA variants. This work provides a general blueprint for the evolution of better orthogonal suppressor tRNAs in mammalian cells
Thesis (PhD) — Boston College, 2019
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Schmidt, Moritz Johannes [Verfasser]. "Expanding the Genetic Code to Study the Structure and Interactions of Proteins / Moritz Johannes Schmidt." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1112604634/34.
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Simova, Zuzana. "Importance of single molecular determinant in bacterial tryptophanyl-tRNA synthetase fidelity in expanded genetic code." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/55120/.
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Nonnatural amino acid incorporation is a valuable method for introducing novel chemical functional groups into proteins. For this method, an orthogonal arninoacyl-tRNA synthetase (AARS) and a cognate tRNA that suppress an encoded stop codon are introduced into the cell (these components are required to be orthogonal). Nonnatural amino acids (NAAs) are usually incorporated efficiently by using
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Hart, Jennifer A. "An analysis of the primordial soup hypothesis with respect to DNA structure and the genetic code." Lynchburg, Va. : Liberty University, 1995. http://digitalcommons.liberty.edu.
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Pouyet, Fanny. "Étude bioinformatique de l'évolution de l'usage du code génétique." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1140/document.
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Le code génétique est la table de correspondance entre codons (unité structurelle d'un gène) et acides aminés (brique élémentaire des protéines). Le code génétique est (1) universel, tous les êtres vivants ou presque partagent le même code; (2) univoque, chaque codon spécifie un seul acide aminé et (3) dégénéré, les acides aminés peuvent être codés par plusieurs codons. Ce code dégénéré est donc utilisé par l'ensemble du vivant mais pas de la même manière, certains codons synonymes étant utilisés préférentiellement chez des espèces et pas d'autres. Pour comprendre l'émergence des biais d'usage du code (BUC) génétique entre espèces, je me place dans un contexte évolutif.Dans ce manuscrit, je présente mes travaux de recherche en quatre parties. La première partie introductive décrit la mise en évidence et les propriétés du code génétique, son biais d'usage et les diverses caractéristiques de précédents modèles de codons. La deuxième partie présente le modèle d'évolution de codons SENCA pour Sites Evolution at the Nucleotides, Codons and Amino-acids layers que j'ai développé durant ma thèse. SENCA prend en compte la structure du code génétique. Je valide sa paramétrisation par des simulations numériques et une étude sur des espèces bactériennes ou archées. La partie suivante décrit deux extensions de SENCA qui modélisent plusieurs hypothèses d'origines évolutives du BUC et une application de SENCA sur les conséquences génomiques d'adaptations environnementales. La dernière partie étudie les origines de variations de BUC le long du génome humain par une approche de génomique comparativeIn this manuscript, I introduce my doctoral research in four parts. The first introductive part highlights the properties of the genetic code and its usage bias but also the caracteristics of previous published codons models. The second part presents an evolutionary codons models named SENCA for Sites Evolution at the Nucleotides, Codons and Amino-acids layers that I developped. SENCA takes into account the genetic code structure. I perform simulations and study prokaryotes species to confirm its parametrization. The following part provides two extensions of SENCA to test the hypotheses concerning the evolutive origins of CUB and an application of SENCA to study the genomic consequences of an environmental adaptation. The last part studies the origins of CUB variation within the human genome using a comparative genomic strategy
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Oliveira, Lariza Laura de. "Algoritmos evolutivos aplicados na investigação da adaptabilidade do código genético." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-24022016-144852/.
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O código genético é altamente conservado e está presente na maior parte dos organismos vivos. Uma questão que tem intrigado os cientistas é se o código genético é fruto do acaso ou de um processo evolutivo. Se qualquer associação entre aminoácidos e códons é possível, então existem cerca de 1, 51 × 1084 códigos possíveis. A hipótese de que o código genético evoluiu é suportada por sua robustez frente a mutações. Duas metodologias tem sido utilizadas para estudar esta hipótese: a abordagem estatística, que estima o número de códigos aleatórios melhores que o código genético padrão, e a abordagem por engenharia, que compara o código padrão com os melhores códigos hipotéticos obtidos por meio de um algoritmo de otimização. A utilização de ambas abordagens têm sido feita considerando-se apenas uma função objetivo, baseada na robustez frente a mutações quando uma determinada propriedade dos aminoácidos é considerada. Neste trabalho, propõe-se considerar mais de um objetivo simultaneamente para a avaliação dos códigos genéticos. Para isso, três abordagens multiobjetivo utilizando Algoritmos Genéticos são empregadas. São elas: abordagem lexicográfica, ponderada e de Pareto. Os resultados indicam que a utilização de mais de um objetivo é promissor, sendo os códigos hipotéticos gerados mais similares ao código genético padrão, quando comparados com os resultados obtidos por outros autores.The genetic code is highly preserved and it is present in most living organisms. If we consider all codes mapping the 64 codes into 20 amino acids and one stop codon, there are more than 1.51 × 1084 possible genetic codes. The main question related to the organization of the genetic code is why exactly the standard code was selected among this huge number of possible genetic codes.The hypothesis that the genetic code has evolved is supported by its robustness against mutations. Many researchers argue that the organization of the standard code is a product of natural selection and that the codes robustness against mutations would support this hypothesis. Two methodologies have been used to investigate this hypothesis: the first one is the statistical approach which estimates the number of random codes which are better than the standard genetic code. The second is the engineering approach, which compare the standard code with the best hypothetical codes obtained by an optimization algorithm. Both approaches have been used considering only one objective function, which is usually based on the robustness against changes using the polar requirement. In this research, we propose to consider more than one objective simultaneously for the evaluation of genetic codes. For this purpose, three approaches using multi-objective genetic algorithms were employed, are they: lexicographic, weighted, and Pareto-based. The results indicate that considering more than one objective function is promising: the hypothetical codes generated are more similar to the standard genetic code, when compared with the results obtained by the monoobjective approach.
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Phung, Viet-Anh. "Input Calibration, Code Validation and Surrogate Model Development for Analysis of Two-phase Circulation Instability and Core Relocation Phenomena." Doctoral thesis, KTH, Kärnkraftssäkerhet, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-202957.
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Code validation and uncertainty quantification are important tasks in nuclear reactor safety analysis. Code users have to deal with large number of uncertain parameters, complex multi-physics, multi-dimensional and multi-scale phenomena. In order to make results of analysis more robust, it is important to develop and employ procedures for guiding user choices in quantification of the uncertainties. The work aims to further develop approaches and procedures for system analysis code validation and application to practical problems of safety analysis. The work is divided into two parts. The first part presents validation of two reactor system thermal-hydraulic (STH) codes RELAP5 and TRACE for prediction of two-phase circulation flow instability. The goals of the first part are to: (a) develop and apply efficient methods for input calibration and STH code validation against unsteady flow experiments with two-phase circulation flow instability, and (b) examine the codes capability to predict instantaneous thermal hydraulic parameters and flow regimes during the transients. Two approaches have been developed: a non-automated procedure based on separate treatment of uncertain input parameters (UIPs) and an automated method using genetic algorithm. Multiple measured parameters and system response quantities (SRQs) are employed in both calibration of uncertain parameters in the code input deck and validation of RELAP5 and TRACE codes. The effect of improvement in RELAP5 flow regime identification on code prediction of thermal-hydraulic parameters has been studied. Result of the code validations demonstrates that RELAP5 and TRACE can reproduce qualitative behaviour of two-phase flow instability. However, both codes misidentified instantaneous flow regimes, and it was not possible to predict simultaneously experimental values of oscillation period and maximum inlet flow rate. The outcome suggests importance of simultaneous consideration of multiple SRQs and different test regimes for quantitative code validation. The second part of this work addresses core degradation and relocation to the lower head of a boiling water reactor (BWR). Properties of the debris in the lower head provide initial conditions for vessel failure, melt release and ex-vessel accident progression. The goals of the second part are to: (a) obtain a representative database of MELCOR solutions for characteristics of debris in the reactor lower plenum for different accident scenarios, and (b) develop a computationally efficient surrogate model (SM) that can be used in extensive uncertainty analysis for prediction of the debris bed characteristics. MELCOR code coupled with genetic algorithm, random and grid sampling methods was used to generate a database of the full model solutions and to investigate in-vessel corium debris relocation in a Nordic BWR. Artificial neural networks (ANNs) with classification (grouping) of scenarios have been used for development of the SM in order to address the issue of chaotic response of the full model especially in the transition region. The core relocation analysis shows that there are two main groups of scenarios: with relatively small (<20 tons) and large (>100 tons) amounts of total relocated debris in the reactor lower plenum. The domains are separated by transition regions, in which small variation of the input can result in large changes in the final mass of debris. SMs using multiple ANNs with/without weighting between different groups effectively filter out the noise and provide a better prediction of the output cumulative distribution function, but increase the mean squared error compared to a single ANN.Validering av datorkoder och kvantifiering av osäkerhetsfaktorer är viktiga delar vid säkerhetsanalys av kärnkraftsreaktorer. Datorkodanvändaren måste hantera ett stort antal osäkra parametrar vid beskrivningen av fysikaliska fenomen i flera dimensioner från mikro- till makroskala. För att göra analysresultaten mer robusta, är det viktigt att utveckla och tillämpa rutiner för att vägleda användaren vid kvantifiering av osäkerheter.Detta arbete syftar till att vidareutveckla metoder och förfaranden för validering av systemkoder och deras tillämpning på praktiska problem i säkerhetsanalysen. Arbetet delas in i två delar.Första delen presenterar validering av de termohydrauliska systemkoderna (STH) RELAP5 och TRACE vid analys av tvåfasinstabilitet i cirkulationsflödet.Målen för den första delen är att: (a) utveckla och tillämpa effektiva metoder för kalibrering av indatafiler och validering av STH mot flödesexperiment med tvåfas cirkulationsflödeinstabilitet och (b) granska datorkodernas förmåga att förutsäga momentana termohydrauliska parametrar och flödesregimer under transienta förlopp.Två metoder har utvecklats: en icke-automatisk procedur baserad på separat hantering av osäkra indataparametrar (UIPs) och en automatiserad metod som använder genetisk algoritm. Ett flertal uppmätta parametrar och systemresponser (SRQs) används i både kalibrering av osäkra parametrar i indatafilen och validering av RELAP5 och TRACE. Resultatet av modifikationer i hur RELAP5 identifierar olika flödesregimer, och särskilt hur detta påverkar datorkodens prediktioner av termohydrauliska parametrar, har studerats.Resultatet av valideringen visar att RELAP5 och TRACE kan återge det kvalitativa beteende av två-fas flödets instabilitet. Däremot kan ingen av koderna korrekt identifiera den momentana flödesregimen, det var därför ej möjligt att förutsäga experimentella värden på svängningsperiod och maximal inloppsflödeshastighet samtidigt. Resultatet belyser betydelsen av samtidig behandling av flera SRQs liksom olika experimentella flödesregimer för kvantitativ kodvalidering.Den andra delen av detta arbete behandlar härdnedbrytning och omfördelning till reaktortankens nedre plenumdel i en kokarvatten reaktor (BWR). Egenskaper hos härdrester i nedre plenum ger inledande förutsättningar för reaktortanksgenomsmältning, hur smältan rinner ut ur reaktortanken och händelseförloppet i reaktorinneslutningen.Målen i den andra delen är att: (a) erhålla en representativ databas över koden MELCOR:s analysresultat för egenskaperna hos härdrester i nedre plenum under olika händelseförlopp, och (b) utveckla en beräkningseffektiv surrogatsmodell som kan användas i omfattande osäkerhetsanalyser för att förutsäga partikelbäddsegenskaper.MELCOR, kopplad till en genetisk algoritm med slumpmässigt urval användes för att generera en databas av analysresultat med tillämpning på smältans omfördelning i reaktortanken i en Nordisk BWR.Analysen av hur härden omfördelas visar att det finns två huvudgrupper av scenarier: med relativt liten (<20 ton) och stor (> 100 ton) total mängd omfördelade härdrester i nedre plenum. Dessa domäner är åtskilda av övergångsregioner, där små variationer i indata kan resultera i stora ändringar i den slutliga partikelmassan. Flergrupps artificiella neurala nätverk med klassificering av händelseförloppet har använts för utvecklingen av en surrogatmodell för att hantera problemet med kaotiska resultat av den fullständiga modellen, särskilt i övergångsregionen.
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Albuquerque, Julio Cesar Holanda de. "Proposta de constelações de sinais para o codigo genetico." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/259781.
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Orientador: Reginaldo Palazzo JuniorDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação
Made available in DSpace on 2018-08-12T13:34:06Z (GMT). No. of bitstreams: 1 Albuquerque_JulioCesarHolandade_M.pdf: 1364323 bytes, checksum: 01181adde228aa4d914d7edabdde4aca (MD5) Previous issue date: 2008
Resumo: A proposta deste trabalho é apresentar uma abordagem aos processos genéticos e moleculares, utilizando a teoria de comunicações e codificação na modelagem do dogma central da biologia molecular. A partir desta modelagem associamos o código genético a um modulador de um sistema de comunicação. Mais especificamente, tal procedimento consiste em construir uma constelação de sinais a partir dos subgrupos de S3 e S4 baseado no código genético. Considerando este método algébrico de construção de sinais, propomos duas possíveis constelações de sinais para o código genético. A representação do código genético em constelações de sinais correlacionadas deu origem à idéia de "constelação de sinais concatenadas", idéia inovadora na teoria de comunicação e codificação. As constelações de sinais concatenadas possui a propriedade de correção de erros, consistindo de novos conceitos úteis para utilização na teoria da comunicação e codificação. Por outro lado, estas representações do código genético não são únicas pois, até o presente momento, desconhecemos uma álgebra que descreva o código genético juntamente com as suas partições geradas pelos aminoácidos.
Abstract: The purpose of this work is to present an approach to the genetic and molecular processes by use of the communication and coding theory in modelling the central dogma of the molecular biology. From this modelling we associate the genetic code to a modulator in the communication system. More specifically, such a procedure consists is in the construction of a signal constellation by use of the S3 and S4 permutation subgroups based on the code genetic. By considering this algebraic method of signal design, we propose two possible signal constellations to the genetic code. The representation of the genetic code as correlated signal constellations provides the idea idea of "concatenated signal constellation", an innovative idea in communication and coding theory. The concatenated signal constellations have the property of error-correction, a new concept being introduced. On the other hand, these representations of the genetic code are not unique for currently, we do not know an algebraic structure capable of describing the genetic code together with the partitioning generated by the amino acids.
Mestrado
Telecomunicações e Telemática
Mestre em Engenharia Elétrica
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Gerding, Hanne Rieke [Verfasser]. "Site-specific incorporation of 3-nitrotyrosine in proteins generated via genetic code expansion in E.coli / Hanne Rieke Gerding." Konstanz : Bibliothek der Universität Konstanz, 2018. http://d-nb.info/1179076869/34.
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Davis, Lloyd Daniel. "The use of the orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair for further enhancement of genetic code expansion technologies." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607872.
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