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.

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. <br /><br /> 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.

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. <br /><br /> 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.

Proteins are diverse polymers of covalently linked amino acids. They play a role in almost every biological process that occurs within an organism. Twenty different amino acids are genetically encoded by mammalian cells to build proteins. The sequence of these amino acids determines the protein’s final shape, structure, and function. Modern molecular cloning techniques allow for the genetic encoding and expression of mutant proteins that have one or more amino acids replaced with one of the others. The roles of individual amino acids in a protein can therefore be studied. Proteins with novel functions have also been designed or evolved using this technology. However, the genetic code is limited to the twenty natural amino acids. Nonnatural amino acids have unique side groups that not found on any of the twenty natural amino acids. They can be site-specifically incorporated using a mutant orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. Each pair only allows for one type of nonnatural amino acid to be genetically encoded. This technology has resulted in the incorporation of over fifty different types of nonnatural amino acids into proteins in prokaryotic and eukaryotic cells. Unfortunately, most of these pairs are not orthogonal outside of prokaryotic systems and only a few have been developed for mammalian cells. To create more mammalian pairs a nonnatural aaRS has to be evolved and screened in a cumbersome process. In this dissertation an approach is outlined that can be used to change the orthogonality of existing nonnatural suppressor tRNA/aaRS pairs. As a result of the orthogonality change many previously unavailable pairs can be shuttled into mammalian cells. The ability to genetically encode a 21st amino acid is a powerful tool in the study and engineering of proteins. / text

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.

Thesis (Ph.D.)--University of Western Sydney, 2008. / "A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences in fulfilment of the requirements for the degree of Doctor of Philosophy." Includes bibliography.

Made available in DSpace on 2015-10-06T13:02:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-04-09. Added 1 bitstream(s) on 2015-10-06T13:19:24Z : No. of bitstreams: 1 000847289_20151210.pdf: 109983 bytes, checksum: 0805da618c3e8c3448f6b9e2d227a33b (MD5) Bitstreams deleted on 2015-12-11T11:22:37Z: 000847289_20151210.pdf,. Added 1 bitstream(s) on 2015-12-11T11:23:16Z : No. of bitstreams: 1 000847289.pdf: 738165 bytes, checksum: af1a367e9d0c1700d0efba7690754147 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Cromossomos B, também conhecidos como supernumerários, são considerados elementos dispensáveis ao organismo. Porém, alguns estudos têm apresentado evidências de uma possível funcionalidade destes cromossomos extras. São encontrados em muitas espécies de eucariotos, entre elas, Astatotilapia latifasciata, ciclídeo africano que possui de 1 a 2 cromossomos B. Os ciclídeos têm sido muito utilizados como organismos modelo para estudos genéticos e genômicos. Análises genômicas em larga escala prévias envolveram sequenciamento por next generation (Illumina HiSeq sequencing) de genomas sem cromossomo B (B-) e com cromossomo B (B+) de A. latifasciata e uma forma variante do gene hnRNP Q-like (Heterogeneous nuclear ribonucleoprotein Q-like) foi identificada. Esta sequência apresenta um alto número de cópias e características de retrogene no cromossomo B. Diante disso, foi objetivo do presente trabalho compreender a organização genômica, identificar os mecanismos de retro-inserção e fazer análises de expressão desta sequência, possibilitando um melhor entendimento da origem, evolução e possíveis efeitos deste cromossomo na espécie estudada. A partir de análises de bioinformática foram identificadas cópias variantes do gene hnRNP Q-like no genoma B+ e as regiões mais representativas foram selecionadas, de acordo com o grau de mutações, para demais estudos. Análises de fragmentos sequenciados e qPCR confirmaram que o gene possui maior número de cópias e ausência de íntrons no cromossomo B. Evidências de resquícios de domínios da transcriptase reversa de elementos transponíveis sugerem que o gene foi retroinserido no cromossomo B. Dados de qPCR em cDNA mostraram que, apesar de possuir mais cópias no genoma B+, o gene hnRNP Q-like não é diferencialmente expresso nos genomas B+ e B- / B chromosomes, also known as supernumerary, are considered dispensable elements to organisms. However, some studies have presented evidences of a possible functionality of this extras chromosomes. They are observed in many eukaryote species, such as in the African cichlid, Astatotilapia latifasciata, which might have one or two B chromosomes. Cichlids have been used as model organisms to genetics and genomics studies. Previous genomic analyses based on next generation sequencing (Illumina HiSeq sequencing) were realized in the whole genome without B chromosome (B-) and with B chromosomes (B+) of A. latifasciata and a variant form of hnRNP Q-like (Heterogeneous nuclear ribonucleoprotein Q-like) gene was identified. This sequence presented a high copy number and characteristics of a retrogene in B chromosomes. Thus, the aim of this study consists in comprehend genomic organization, identify retroinsertion mechanisms and perform expression analyses of the hnRNP Q-like gene, making it possible a better understanding of origin, evolution and possible effects of this chromosomes in the studied species. Bioinformatics analyses identified variant copies of hnRNP Q-like gene in B+ genome and the higher and lower variable regions of the gene were selected, based in mutation rates, for further analysis. Analyses from sequenced fragments and qPCR confirmed gene has a higher number of copies and do not present introns in B chromosome. Evidences of transposable element relics with reverse transcriptase domains suggest gene was retroinserted in B chromosome. The data of qPCR in cDNA showed that the hnRNP Q-like gene is not differentially expressed in both genomes (with and without B chromosome)

O estudo dos processos quebra espontânea de simetria na natureza têm atraído interesse em diversas áreas da física, como por exemplo em física quântica no estudo das energias de um átomo tal como em física de altas energias, no estudo das partículas elementares. Esses processos até então envolviam sistemas físicos microscópicos, em 1993 surge uma proposta de agregar as idéias de quebra de simetria à um sistema macroscópico, o código genético. A idéia básica é que os códons que formam código se diferenciam em um processo de quebra de simetria, preservando suas propriedades de degenerescência, nos dando uma \"picture\" de como se fez essa diferenciação que resultam nos 20 aminoácidos e do sinal de terminação que se conhece nos dias atuais. Esse modelo nos diz por exemplo, quantos eram os aminoácidos primordiais. Nosso interesse está na verificação dessa quebra de simetria e estudar as relações entre os códons do ponto de vista temporal para tanto, usamos aqui um sistema dinâmico. Esse sistema conserva no princípio de sua evolução a simetria proposta pelo modelo e através de um processo de quebra de simetria estudaremos se esse processo reproduz a cadeia de quebra de simetria proposta no modelo. Como primeiro passo estudamos a representação tridimensional do grupo Sp( 6), que serve como ponto de partida no processo de quebra de simetria no modelo, essa representação é conhecida como grupo de Weyl do Sp(6). É possível construir um sistema dinâmico ou mapa, que na verdade é uma função do R3, com as mesmas propriedades de simetria do grupo de Weyl do Sp(6). A construção desse sistema e seu estudo matemático acarreta no segundo passo deste trabalho. O mapa construído depende de parâmetros que variados de forma correta produzem uma cadeia de quebra de simetria. O estudo dessa quebras consiste no terceiro passo deste trabalho. Por fim determinamos a ação ou seja, como esse sistema muda a rotulação dos códons anteriormente proposta no modelo e mais ainda, que informação biológica poderá ser extraída desse sistema. Como resultado obtivemos em grande parte a ratificação do modelo proposto mostrando que a quebra proposta e a rotulação dos códons de acordo com a ordem evolutiva dada pela quebra de simetria segue também uma coerência dinâmica. / The study of natural symmetry breaking processes have attracted interest in many physics areas including energy atoms studies in quantum physics as well elementary particles in high energy physics. These processes were related with microscpics physic systems, in 1993 appears one propose to use the ideas of symmetry breaking in one macroscopic system the genetic code. The basic idea is that the differentiation of the codons wich are components of the code was done in a process of symmetry breaking preserving the degeneracy properties given to us one picture of how this process occour resulting in 20 aminoacids and termination sign known in the present days. With this model for example, we can predict how many aminoacids were primordial\'s. Our interest is in verify this symmetry breaking and study the codon temporal relations for this we use a dynamical system. The preservation of the starting symmetry proposed by the model is the main caracteristic of our system and through of the symmetry breaking we will study what relations between the symmetry breaking proposed by the model and the dynamical symmetry breaking. As a first step we will study the group Sp(6) in its tridimensional representation which is the starting point in the symmetry breaking process in the mode!. This representation in known as Weyl group of Sp(6). It is possible construct this dynamical system or map, which is one function in the R3 with the same symmetry properties of the Weyl group of Sp(6). The construction of this map and its mathematical study is our second step of this work. The map depends on parameter\'s which are changed in a correct way to produce some symmetry breaking chain. The symmetry breaking studies is our third step. At the end we look at the action of our map in the codons, in other words, how this action change the codons labelling proposed by the model Moreover, what kind of biological information can be extract from this action. As a result the symmetry breaking and the labelling of codons proposed by the model are isomophics, with little restrictions, when compared with the dynamical systems.

Neste trabalho resolvemos o problema da classicação dos possíveis esquemas de quebra de simetria que reproduzem as degenerescências do código genético na categoria dos grupos finitos simples, contribuindo assim para a busca de modelos algébricos para a evolução do código genético, iniciada por Hornos & Hornos. / In this work we solve the problem of classifying the possible symmetry breaking schemes based on simple finite groups that reproduce the degeneracies of the genetic code, thus contributing to the search for algebraic models that describe the evolution of the genetic code, initiated by Hornos & Hornos.

An experimental study on randomly generated protein sequences can provide important insights into the origin and mechanism of secondary structure formation and protein folding. In this study we bring biophysical characterization of five protein sequences selected from the in silico generated library of random chains. The sequences were selected on the basis of bioinformatic analysis in order to find the candidates with the maximum potential to possess secondary structure. This study shows that the random polypeptide sequences form stable secondary structures and in some show the signs of tertiary structure, such as hydrophobic core formation and distinctive oligomerization pattern. While the work presented in this thesis is work in progress on a larger study, the data already demonstrate that unevolved protein sequence space provides a lot of potential for secondary and tertiary structure formation that awaits its characterization. Powered by TCPDF (www.tcpdf.org)