<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

To shed light on forces that shape the molecular evolution of bird genomes, and in turn avian adaptations, comparative analyses of avian DNA sequences are important. Moreover, contrasting findings in birds to those of other organisms will lend a clearer view on general aspects of molecular evolution. However, few such analyses have been conducted in birds. Progress is presented in this thesis. Theories predict a reduction in the mutation rate of the Z chromosome as the harmful effects of recessive mutations are exposed in female birds. We find no evidence for this. Instead, the substitution rates of sex chromosomes and autosomes are largely compatible with expectations from male-biased mutation. This suggests that a majority of mutations arise during DNA replication. Substitution rates also vary across chicken autosomes. For instance, microchromosomes accumulate ~20% more substitutions than macrochromosomes. We show that a majority of the autosomal variation in substitution rate can be accounted for by GC content, mainly due to the incidence of mutable CpG-dinucleotides. Sequence comparisons also show that the pattern of nucleotide substitution varies in the chicken genome and this reinforces regional differences in base composition. The level of selective constraint in at least some avian lineages is higher than in mammalian lineages as indicated by low dN/dS – ratios. Larger historical population sizes of birds relative to mammals could explain this observation. Within the avian genome, the dN/dS is lower for genes on micro- than macrochromosomes, potentially owing to a higher incidence of house-keeping genes in the former category. Contrasting data on non-synonymous and synonymous substitution for divergence and polymorphism shows that positive selection has contributed more to the evolution of Z-linked than autosomal genes. This is likely explained by the full exposure of beneficial recessive mutations on Z when in female birds.

Evolutionary biology is dependent on accurate phylogenies. In this thesis two branch support methods, Bayesian posterior probablities and bootstrap frequencies, were evaluated with simulated data and empirical data from the chloroplast genome. Bayesian inference was found to be more powerful and less conservative than maximum likelihood bootstrapping, but considerably more sensitive to choice of parameters. Bayesian inference increased in power when data were underparameterized, but the associated increase in type I error was comparatively larger. The chloroplast DNA phylogeny of the tribe Sileneae (Caryophyllaceae) was inferred by analysis of 33,149 aligned nucleotide bases representing 24 taxa. The position of the SW Anatolian taxa Silene cryptoneura and S. sordida strongly disagreed with previous studies on nuclear DNA sequence data, and indicate a possible case of homoploid hybrid origin. Silene atocioides and S. aegyptiaca formed a sister group to Lychnis and remaining Silene, thus suggesting that Silene may be paraphyletic, despite recent revisions based on molecular data. Several nodes in the phylogeny remained poorly supported, despite large amounts of data. Additional sequence sampling is not expected to solve this problem. The main reason for poor resolution is probably a combination of rapid radiation and substitution rate hererogeneity. Apparent incongruent patterns between different regions of the chloroplast genome are evaluated with ancient interspecific chloroplast recombination as explanatory model. Extremely elevated substitution rates in the exons of the plastid clpP gene was documented in Oenothera and three separate lineages of Sileneae. Introns have been lost in some of the lineages, but where present, intron sequences have a markedly slower substitution rate, similar to the rates found in other introns of their genomes. Three branches in the phylogeny show significant whole gene positive selection. In two of the lineages multiple partial copies of the gene were found.

The process of evolution is of both scientific and medical interest. This thesis presents several studies using complete genomic reference sequences, comparative genomic data, and intraspecific diversity data to study the two key processes of evolution: mutation and selection. Large duplications, deletions, inversions, and translocations of DNA contribute to genomic variation both between and within species. Human chromosomes 15 and 17 contain a high percentage of dispersed, recently duplicated sequences. Examination of the relationships between these sequences showed that the majority of all duplications within each chromosome could be linked through core sequences that are prone to duplication. Comparison to orthologous sequences in other mammals allowed a reconstruction of the ancestral state of the human chromosomes, revealing that regions of rearrangement specific to the human lineage are highly enriched in chromosome-specific duplications. Comparison to copy number variation data from other studies also shows that these regions are enriched in current human structural variation. One specific region, the MAPT locus at 17q21.31, known to contain an inversion polymorphism in Europeans, was resequenced completely across both human orientation haplotypes and in chimpanzee and orangutan, revealing complex duplication structures at the inversion breakpoints, with the human region being more complex than chimpanzee or orangutan. Fluorescent in-situ hybridization analysis of human, chimpanzee, and orangutan chromosomes showed inversion polymorphisms of independent origin in all three species, demonstrating that this region has been a hotspot of genomic rearrangement for at least twelve million years. These results reveal a mechanistic relationship between sequence duplication and rearrangement in the great apes. We also generated a draft sequence of the chimpanzee genome and compared it to that of the human. Among other findings, this showed that CpG dinucleotides contribute 25% of all single base mutations, with a rate of mutation ~10-fold that of other bases, and that the male mutation rate in great apes is ~5-6 times the female rate, a higher ratio than had been observed in comparisons of primates and rodents. We detected six regions of probable recent positive selection in humans with a statistical method relying on chimpanzee sequence to control for regional variation in mutation rates. Finally, resequencing of several lines of domestic chicken and comparison to the reference chicken genome identified a number of gene deletions fixed in domestic lines and also several potential selective sweeps. Of particular interest are a missense mutation in TSHR nearly fixed in all domestic chickens and a partial deletion of SH3RF2 fixed in a high growth line. The TSHR mutation may play a role in relaxation of seasonal reproduction. A high-resolution QTL mapping experiment showed that the SH3RF2 deletion is significantly associated with increased growth. This work provides important new insights into the mechanics of evolutionary change at both the single nucleotide and structural level and identifies potential targets of natural and artificial selection in humans and chickens.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

Made available in DSpace on 2016-06-02T20:21:24Z (GMT). No. of bitstreams: 1 3224.pdf: 2177320 bytes, checksum: 89cae5d787c7083f95c6ec6ff9f87e2a (MD5) Previous issue date: 2010-08-31 / Financiadora de Estudos e Projetos / The genus Anastrepha which is endemic to the Neotropical region has big economic importance to cause great losses in fruit production. We studied three species of the cryptic group fraterculus: Anastrepha fraterculus, A. obliqua and A. sororcula. Although no single marker is able to distinguish these species, some morphological and behavioral markers are able to distinguish them. There is evidence that these markers do not match the patterns of genetic variation and divergence of these flies. We investigated the role of genes related or not to reproduction in the speciation of these flies. We used a cDNA library of the reproductive tissues of females of A. fraterculus, to isolate expressed genes from those tissues. From a framework of divergence population genetics, we estimated relevant demographic parameters that reported the genetic architecture of speciation in this group. To study the separation of these species, we used gene trees as a framework to be compared and to infer eventual species trees. We amplified 12 genes isolated from the cDNA library. The analysis of population divergence and levels of polymorphism were made in DNAsp and showed high levels of polymorphism for the vast majority of loci. Neutrality tests reveal that only Fs Fu was significantly negative for the combination of species here considered. These results indicate a possible evidence of population expansion on this group of species. These data were corroborated by the values of ancestral and current theta estimated from the analysis undertaken on the software IMa2 program. These analysis also showed relevant levels of migration in some contrasts, but particularly among species which are more distant in the tree from the other two. However, the same analyses performed amongst species pairs fail to reveal high levels of migration rate among species, though t e theta values were close to those estimated in the analysis with all three species together. We also tested for the presence of selection at different loci, using the program PAML and obtained evidence of positive selection for three genes CG7203, CG8064 and MLC, whereas the gene CG7009 showed evolution by a mild purifying selection. The haplotype networks showed the grouping of haplotypes of the species for five of the 12 genes CG7009, CG8064, Elp, TCTP and Cyclophylin. CG7009 revealed possible species specific markers for A. sororcula. We carried out the deep coalescence analysis in the program MESQUITE, comparing gene trees simulated with real trees for each gene locus, placing the species as an outgroup for each tree model and obtained significant results, which would be compatible to simulated scenarios only for the genes V Cyclophylin and TCTP. The tree obtained by minimizing the deep coalescence estimated showed that A. sororcula is farther from the other two species of the genus Anastrepha. It is necessary, however, a larger investigation of different genes, particularly genes with higher evolutionary rates and an expansion of the samples of these species to consider geographical distribution and assist in studies of speciation of this group. / O gênero Anastrepha, endêmico da região Neotropical, tem grande importância econômica por causar grandes prejuízos na produção de frutos. Neste trabalho, estudamos três espécies do grupo críptico fraterculus: Anastrepha fraterculus, A. obliqua e A. sororcula. Apesar de não haver um único marcador capaz de diferenciar essas espécies, alguns marcadores morfológicos e comportamentais, são capazes de distinguí-las. Há evidências de que esses marcadores não correspondam aos padrões de variação e divergência genética dessas moscas. Investigamos o papel de diversos genes nucleares na especiação dessas moscas. Usamos uma biblioteca de cDNAs do aparelho reprodutivo de fêmeas de A. fraterculus, para a amplificação de genes expressos desse tecido. A partir de um quadro de divergência genética populacional, estimamos parâmetros demográficos relevantes que informaram a arquitetura genética da especiação neste grupo. Para estudar a separação dessas espécies, utilizamos árvores de genes como base e as comparamos com árvores de espécies. Foram amplificados 12 genes expressos na biblioteca de cDNAs. As análises de divergência populacional e nível de polimorfismo foram feitas no DNAsp e mostraram valores bem altos do nível de polimorfismo para a grande maioria dos loci e os testes de neutralidade foram significativos apenas para Fs de Fu. Esses resultados indicam uma possível expansão populacional das espécies estudadas. Estes dados foram corroborados pelos valores de q das análises realizadas pelo programa IMa2, que também mostrou uma taxa de migração alta entre a espécie mais distante na árvore para uma das outras duas. No entanto, as mesmas análises feitas com as espécies par a par não revelaram uma taxa de migração significativa entre as espécies, os valores de t e theta foram próximos dos obtidos para as análises com as espécies juntas. Fizemos também testes para detectar a presença de seleção nos diferentes loci, usando o programa PAML e obtivemos indício de seleção positiva para três genes CG7203, MLC e CG8064; o gene CG7009, no entanto, mostrou evoluir por uma seleção purificadora branda, pois não rejeitamos somente o primeiro dos dois testes. As redes haplotípicas, feitas no TCS, mostraram o agrupamento dos haplótipos das espécies para cinco dos 12 genes CG7009, CG8064, Elp, TCTP e Cyclophylin. Para o CG7009 notamos possíveis marcadores específicos para A. sororcula. Realizamos, pelo programa MESQUITE, análises de coalescência profunda comparando árvores de genes simuladas com árvores de genes reais para cada locus, colocando uma das espécies como grupo externo em cada modelo de árvore e III obtivemos resultados significativos apenas para os genes Cyclophylin e TCTP. A árvore obtida pela minimização das coalescências profundas mostrou que A. sororcula se encontra mais distante das outras duas espécies do gênero Anastrepha. Faz-se necessária, no entanto, uma investigação maior de diferentes genes, particularmente genes com taxas evolutivas mais altas e uma expansão das amostras destas espécies para considerar sua distribuição geográfica e auxiliar nos estudos da especiação deste grupo.

Orientadores: Ana Maria Lima de Azeredo Espin, Pablo Fresia Coronel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T09:56:03Z (GMT). No. of bitstreams: 1 Bergamo_LuanaWalravens_M.pdf: 4822096 bytes, checksum: 6731d49afdda09d9b33086d944c30301 (MD5) Previous issue date: 2014 / Resumo: A pecuária é uma importante atividade econômica do Brasil, mas tem sofrido perdas significativas devido ao impacto de parasitas. Neste cenário destaca-se a mosca-da-bicheira, Cochliomyia hominivorax, que é um importante ectoparasita causador de miíase primária e endêmico das Américas. Sua distribuição geográfica sofreu redução a partir da implementação da técnica do inseto estéril (SIT), sendo considerada erradicada nos Estados Unidos e países continentais da América Central. Na América do Sul, o controle desta espécie é realizado através de inseticidas, cujo uso indiscriminado pode acarretar na seleção de indivíduos resistentes. Em estudos anteriores, as mutações denominadas Gly137Asp e Trp251Leu foram observadas no sítio ativo da enzima carboxilesterase E3 e associadas à resistência a inseticidas dietil e dimetil-organofosforados, respectivamente. A caracterização molecular da região desse gene que compreende desde o final do éxon 2 até o final do éxon 4 para C. hominivorax revelou que o íntron I2 apresenta um tamanho maior que em outras espécies de Muscomorpha. A análise da composição nucleotídica e comparações por métodos estatísticos entre modelos de mutação-seleção de sequências do cDNA da carboxilesterase E3 de C. hominivorax e outros Muscomorpha mostraram sinais de seleção restritiva sobre as substituições sinônimas. Porém, o padrão observado não é exclusivo deste gene, sendo observado em outras regiões do transcriptoma. As pressões seletivas que modelaram a evolução do gene E3 do ponto de vista das mutações não-sinônimas foram investigadas a partir de uma estratégia hierárquica, considerando dados interespecíficos e populacionais. Primeiramente, na investigação de resposta à longo prazo, foram utilizadas as sequências dos éxons das espécies C. hominivorax, Cochliomyia macellaria, Chrysomya megacephala e sequências públicas de outros Muscomorpha. Os testes branch-site relaxado e branch-site estrito, em conjunto com o método Bayes Empirical Bayes (BEB), não indicaram sítios sob seleção positiva no ramo de C. hominivorax. A análise pelo método MM01, que leva em conta as propriedades físico-químicas dos aminoácidos, também não detectou nenhum sinal de seleção. Já na investigação com base em dados populacionais, amostras de C. hominivorax de 21 localidades da América do Sul foram sequenciadas para um fragmento do gene E3, previamente caracterizado. Os resultados da AMOVA e Fst-par-a-par indicam que há estruturação entre as localidades quando consideradas as sequências do gene E3 juntamente com sequências dos genes mitocondriais CR, COI e COII. Porém, os resultados da SAMOVA mostraram uma baixa correlação entre os dados genéticos e geográficos, indicando que esta espécie apresenta uma complexa estrutura populacional. Através do DAPC foi possível distinguir três grupos genéticos entre as localidades. Testes de desequilíbrio de ligação foram significativos entre as duas mutações que são relacionadas à resistência a inseticidas organofosforados, indicando uma associação negativa entre elas. Uma análise baseada na detecção de locos outliers recuperou um dos sítios do primeiro códon associado à resistência com sinal de seleção positiva / Abstract: Livestock production is an important economic activity in Brazil, but has been suffering significant losses due to the impact of parasites. The New World screwworm fly (NWS), Cochliomyia hominivorax, is an important ectoparasite and myiasis causing fly endemic from the Americas, which stands out in this scenario. The geographic distribution of NWS has been reduced after the implementation of the sterile insect technique (SIT), being considered eradicated in North and part of Central America. In South America, NWS is controlled by chemical insecticides, which indiscriminate use can cause the selection of resistant individuals. The Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 have been associated to resistance of diethyl and dimethyl organophosphates, respectively. The molecular characterization of this gene region comprising from the end of exon 2 to the end of exon 4 for C. hominivorax revealed that the intron I2 has a larger size than in other Muscomorpha species. The analysis of nucleotide composition and the comparisons between mutation-selection models by statistical methods of cDNA sequences of carboxylesterase E3 from C. hominivorax and other Muscomorpha showed signs of restrictive selection on synonymous substitutions. However, the observed pattern is not exclusive for this gene, being observed in other regions of the transcriptome. The selective pressures that have shaped E3 gene evolution from the point of view of non-synonymous mutations were investigated from a hierarchical strategy, considering interspecific and populational data. At first, in the investigation of long term response, the sequences of exons of C. hominivorax, Cochliomyia macellaria, Chrysomya megacephala and public sequences from other Muscomorpha were used. The relaxed branch-site and strict branch-site tests, together with the Bayes Empirical Bayes (BEB) method, indicated no sites under positive selection in the C. hominivorax branch. The analysis by MM01 method, which takes into account the physicochemical properties of amino acids, also detected no sign of selection. Secondly, in the investigation based on populational data, samples of C. hominivorax from 21 sites in South America were sequenced for a fragment of E3 gene previously characterized. The results of AMOVA and pairwise Fst indicate that there is structure between sites when considering sequences of E3 gene and CR, COI and COII mitochondrial genes. However, SAMOVA results showed a low correlation between genetic and geographic data, indicating that this specie has a complex population structure. Three genetic groups were distinguished between the sites by DAPC. Linkage disequilibrium tests were significant between the two mutations related to organophosphate insecticides resistance, indicating a negative association between them. An analysis based on outlier loci detection recovered sign of positive selection in one of the sites from the first codon associated with resistance / Mestrado / Genetica Animal e Evolução / Mestra em Genética e Biologia Molecular

Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo;s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa

<p>Synthetic biology reprograms organisms to perform non-native functions for beneficial reasons. An important practice in synthetic biology is the ability to edit DNA to change a base pair, disrupt a gene, or insert a new DNA sequence. DNA edits are commonly made with the help of homologous recombination, which inserts new DNA flanked by sequences homologous to the target region. To increase homologous recombination efficiency, a double stranded break is needed in the middle of the target sequence. Common methods to induce double stranded breaks use nucleases, enzymes that cleave ribonucleotides (DNA and RNA). The most common nucleases are restriction enzymes, which recognize a short, fixed, palindromic DNA sequence (4-8 base pairs). Because of the short and fixed nature of the recognition sites, restriction enzymes do not make good candidates to edit large chromosomal DNA. Alternatively, scientists have turned to programmable endonucleases which recognize user-defined DNA sequences, often times much larger than the recognition sites of restriction enzymes (15-25 base pairs). Programmable endonucleases such as CRISPR-based systems and prokaryotic Argonautes are found throughout the prokaryotic kingdom and may differ significantly in activity and specificity. To compare activity levels among endonuclease enzymes, activity assays are needed. These assays must clearly delineate dynamic activity levels of different endonucleases and work with a wide variety of enzymes. Ideally, the activity assay will also function as a positive selection screen, allowing modifications to the enzymes via directed evolution. Here, we develop an <i>in vivo</i> assay for programmable endonuclease activity that can also serve as a positive selection screen using two plasmids, a lethal plasmid to cause cell death and a rescue plasmid to rescue cell growth. The lethal plasmid houses the homing endonuclease, I-SceI, which causes a deadly double-stranded break at an 18 base pair sequence inserted into an engineered <i>E. coli</i> genome. The rescue plasmid encodes for a chosen endonuclease designed to target and cleave the lethal plasmid, thereby preventing cell death. With this, cell growth is directly linked to programmable endonuclease activity. Three endonucleases were tested, SpCas9, eSpCas9, and xCas9, displaying recovered growth of 49.3%, 26.1%, and 16.4% respectively. These values translate to kinetic enzymatic activity and are congruent with current literature findings as reported values find WT-SpCas9 to have the fastest kinetics cleaving around 95% of substrate within 15 seconds, followed closely by eSpCas9 cleaving 75% of substrate within 15 seconds and finally trailed by xCas9 cleaving 20% of substrate in about 30 seconds. The differences between each endonuclease’s activity is exacerbated in our <i>in vivo</i> system when compared to similar <i>in vitro</i> methods with much lower resolution. Therefore, slight differences in activity between endonucleases within the first few minutes in an <i>in vitro</i> assay may be a few percentages different whereas in our <i>in vivo</i> assay, these differences in activity result in a more amplified signal. With the ability to display the dynamic response of enzymes, this assay can be used to compare activity levels between endonucleases, give insight into their kinetics, and serve as a positive selection screen for use in directed evolution applications. </p>

In my doctoral project, I studied the evolution of retroviruses and long-term interactions with their hosts. Retroviruses infect a broad range of species including possibly all vertebrates. They are unique in their ability to efficiently create endogenous retroviruses (ERVs) - viral copies integrated into the host genomes and consequently inherited by successive generations as usual genomic locus. ERVs represent a significant portion of vertebrate genomes and play an important role in a variety of cellular processes and pathologies; however, their sequences are still largely unexplored. The results of my work contributed to the uncovering of ancient evolutionary history of retroviruses. In this regard, I employed the ERV sequences, as they represent "genetic fossils" of viral infections that occurred throughout entire retroviral evolution. By discovery and analysis of ancient ERV lineages, I shed light on the deep history of retroviruses and revealed how the past infections shaped the evolution of vertebrate antiviral defense. In addition to the investigation of retroviral evolution, I also studied process of ongoing endogenization and fixation of newly emerged ERVs in a mammalian host population. In this part of my work, I focused on a unique model of ERV that have been recently invading mule deer genome.