Dissertations / Theses on the topic 'Molecular evolution'

The current dissertation looks at the molecular evolution of protein-coding genes in the flowering plant Arabidopsis thaliana and within two RNA viruses, humanimmunodeficiency virus (HIV) and Astroviridae. We analyzed members of the receptor-like kinase (RLK) gene family in Arabidopsis thaliana for positive selection. Likelihood analysis found evidence for positive selection in 12 of the 52 RLK family sequences groups. These 12 groups represent 97 of the 403 sequences analyzed. The majority of genes in groups subject to positive selection have not been functionally characterized, but sites under selection are predominantly located in the extracellular region. In HIV we use Akaike Information Criteria (AIC) based model averaging for models of nucleotide evolution to examine estimates of genetic distance and the ratio of transition/transversion (ts/tv). AIC weighted estimates of distance and ts/tv were shown to be robust relative to model assumptions. AIC weighted estimates of the ts/tv ratio in simulated HIV sequences generally had less variance than similar estimates made by selecting the single best scoring AIC model. Astroviruses are a leading cause of viral gastroenteritis in infants worldwide and little is known about the mechanisms of astrovirus-induced diarrhea or the virally encoded components responsible for disease. We report the genomic sequence of nine novel TAstV-2 isolates. Nucleotide and amino acid identities for the isolates were generally > 90% conserved. Phylogenies constructed using genomic RNA and the individual open reading frames (ORF) provide evidence for recombination and indicate differences in substitution rates between non-structural and structural genes. Analysis of the viral capsid genes using codon models of evolution indicate site-specific positive selection in both turkey and human astroviruses.

Speciation is fundamental to genetical evolution in being the process that separates reproductively isolated populations of genes into two or more independently replicating communities of genes. The theory of Punctuated Equilibrium and the Red Queen hypothesis are two enduring and controversial macro-evolutionary ideas proposed by palaeontologists from examination of the fossil record through time. One has implications for the nature of genetical evolution at speciation, the other for its timing.

Unraveling the molecular etiology of a novel phenotype is still a major challenge. Mammalian pregnancy, a novel phenotype, preserves its stepwise evolution in extant species, which gives us additional tools to use to begin to unravel its evolution. Within this thesis, the evolution of three components of pregnancy are explored- the regulation of TAP2 expression, the regulation of HLA-F expression, and the possible role of transposable elements in rewiring the regulatory networks underlying major gene expression shifts at the maternal-fetal interface.

Evolution is an ubiquitous feature of living systems. The genetic composition of a population changes in response to the primary evolutionary forces: mutation, selection and genetic drift. Organisms undergoing rapid adaptation acquire multiple mutations that are physically linked in the genome, so their fates are mutually dependent and selection only acts on these loci in their entirety. This aspect has been largely overlooked in the study of asexual or somatic evolution and plays a major role in the evolution of bacterial and viral infections and cancer. In this thesis, we put forward a theoretical description for a minimal model of evolutionary dynamics to identify driver mutations, which carry a large positive fitness effect, among passenger mutations that hitchhike on successful genomes. We examine the effect this mode of selection has on genomic patterns of variation to infer the location of driver mutations and estimate their selection coefficient from time series of mutation frequencies. We then present a probabilistic model to reconstruct genotypically distinct lineages in mixed cell populations from DNA sequencing. This method uses Hidden Markov Models for the deconvolution of genetically diverse populations and can be applied to clonal admixtures of genomes in any asexual population, from evolving pathogens to the somatic evolution of cancer. To understand the effects of selection on rapidly adapting populations, we constructed sequence ensembles in a recombinant library of budding yeast (S. cerevisiae). Using DNA sequencing, we characterised the directed evolution of these populations under selective inhibition of rate-limiting steps of the cell cycle. We observed recurrent patterns of adaptive mutations and characterised common mutational processes, but the spectrum of mutations at the molecular level remained stochastic. Finally, we investigated the effect of genetic variation on the fate of new mutations, which gives rise to complex evolutionary dynamics. We demonstrate that the fitness variance of the population can set a selective threshold on new mutations, setting a limit to the efficiency of selection. In summary, we combined statistical analyses of genomic sequences, mathematical models of evolutionary dynamics and experiments in molecular evolution to advance our understanding of rapid adaptation. Our results open new avenues in our understanding of population dynamics that can be translated to a range of biological systems.

Thesis (Ph. D.)--University of Aarhus, 2004.
Title from PDF title page (viewed on Jan 3, 2007). Includes articles and manuscripts co-authored with others. Includes bibliographical references. Also available in PostScript format.

Analyzing regions in the genome with low levels of recombination helps understand the prevalence of sexual reproduction. Here, I show that variability in regions of reduced recombination in Drosophila can be explained by interference among strongly deleterious mutations; selection becomes progressively less effective in influencing the behaviour of neighbouring sites as the number of closely linked sites on a chromosome increases. I also show that the accumulation of loss-of-function mutations on the neo-Y chromosome of Drosophila miranda is compatible with a model of selection against such mutations alone, without the need to invoke the action of selective sweeps. I describe the discovery of two new sex-linked genes in the plant Silene latifolia, SlCyt and SlX9/SlY9. SlCyt has been recently translocated from an autosome to the X and shows signs of a selective sweep. Its possible role in having caused recombination arrest between the evolving X and Y chromosome is discussed. SlX9 still has an intact Y-linked copy that is presumably functional. Nucleotide diversity at SlY9 is very low, whereas SlX9 has an unusually high diversity and shows signs of introgression from S. dioica into S. latifolia, but the effect of this seems very localized.

Molecular formation and destruction processes are explored in rapidly evolving, non-equilibrium astrophysical environments. First, a semi-classical calculation is made for the rate coefficients of excited atom radiative association to form molecular hydrogen and of the process C⁺ + O → CO⁺ + hν. The latter process may be important to the formation of CO in the core of Supernova 1987A. It is shown that the excited atom process may have been important to the formation of H$\sb2$ during the early part of the epoch of recombination in the early Universe. The equations of ionization balance and molecular formation and destruction have been integrated through the epoch of recombination. Other processes are examined in detail. These include heating and cooling of the primordial plasma, damping of fluctuations prior to decoupling, and the possibility of a radiation-driven instability at the onset of recombination. A calculation is presented of the time-dependent chemical evolution in the rapidly expanding outer envelope of SN 1987A. Various cooling rates and hydrogen abundances in the envelope have been examined. It is found that large molecular abundances, in particular CO, form rapidly, while hydrogen remains mostly in its atomic forms. Near-infrared observations of the proto-planetary nebula CRL 618 are presented and discussed. Images acquired in the H and K bandpasses are consistent with a bipolar axis highly inclined to the plane of the sky. From the spectrum, a visual extinction of Aᵥ = 5.3 magnitudes to the molecular hydrogen emitting lobes is found. It is shown by comparison with spectral models that the near-infrared H₂ spectrum exhibits emission from thermally excited molecules at Tₑₓ ∼ 2000 K. A component of fluorescent emission may also be present. The suggestion is explored that large molecules, in particular polycyclic aromatic hydrocarbons (PAHs), are formed in stellar winds. It is asserted that the primary source of interstellar PAHs is mass-losing asymptotic giant branch carbon stars. It is apparent that the known numbers of the most extreme mass-losing carbon stars are able to produce PAHs in sufficient quantities to maintain an interstellar medium well mixed in such molecules at the inferred abundance.

Evolution of Piperales – matK gene and trnK intron sequence data reveal lineage specific resolution contrast. Piperales are one of the largest basal angiosperm orders with a nearly worldwide distribution. The order includes three species rich genera, Piper (ca. 1,000 species), Peperomia (ca. 1,500-1,700 species), and Aristolochia s. l. (ca. 500 species). Sequences of the matK gene and the non-coding trnK group II intron are analysed for a dense set of 105 taxa representing all families (except Hydnoraceae) and all generic segregates (except Euglypha within Aristolochiaceae) of Piperales. A large number of highly informative indels are found in the Piperales trnK/matK dataset. Within a narrow region approximately 500 nt downstream in the matK coding region (CDS), a length variable simple sequence repeat (SSR) expansion segment occurs, in which insertions and deletions have led to short frame-shifts. These are corrected shortly afterwards, resulting in a maximum of 6 amino acids being affected. Furthermore, additional non-functional matK copies were found in Zippelia begoniifolia, which can easily be discriminated from the functional open reading frame (ORF). The trnK/matK sequence data fully resolve relationships within Peperomia, whereas they are not effective within Piper. The resolution contrast is correlated with the rate heterogenity between those lineages. Parsimony, Bayesian and likelihood analyses result in virtually the same topology, and converge on the monophyly of Piperaceae and Saururaceae. Lactoris gains high support as sister to Aristolochiaceae subf. Aristolochioideae, but the different tree inference methods yield conflicting results with respect to the relationships of subfam. Asaroideae. In Piperaceae, a clade formed by the monotypic genus Zippelia and the small genus Manekia (=Sarcorhachis) is sister to the two large genera Piper and Peperomia. Systematics of pipevines – Combining morphological and fast-evolving molecular characters to investigate the relationships within subfamily Aristolochioideae (Aristolochiaceae) A combined phylogenetic analysis of the Aristolochioideae was conducted based on 72 morphological characters and molecular datasets (matK gene, trnK intron, trnL intron, trnL-trnF spacer). The analysis sampled 33 species as the ingroup, including two species of Thottea and 30 species of Aristolochia and the monotypic genus Euglypha, which represent all the infrageneric taxa formally described; Saruma henryi and Asarum caudatum were used as the outgroup. The results corroborate a sister-group relationship between Thottea and Aristolochia, and the paraphyly of Aristolochia with respect to Euglypha that consequently should be included into Aristolochia. Two of the three subgenera within Aristolochia (Isotrema and Pararistolochia) are shown to be monophyletic, whereas the signal obtained from the different datasets about the relationships within subg. Aristolochia is low and conflicting, resulting in collapsed or unsupported branches. The relationship between the New World and the Old World species of subgenus Aristolochia is conflictive because morphological data support these two groups as monophyletic, whereas molecular data show the monophyletic Old World species of Aristolochia nested within the New World species. A sister group relationship is proposed between A. lindneri and pentandrous species, which suggests that a group of five species from central and southern South America (including A. lindneri) could be monophyletic and sister to Aristolochia subsection Pentandrae, a monophyletic taxon consisting of about 35 species from southern USA, Mesoamerica, and the West Indies. Colonisation, phylogeography and evolution of endemism in Mediterranean Aristolochia (Aristolochiaceae). This study provides evidence for a multiple colonisation of the western Old World from Asian ancestors within Aristolochia section Diplolobus (subsection Aristolochia and Podanthemum). Within subsection Podanthemum it is assumed, that the colonisation of the African continent happened at least two times independently. In contrast, for subsection Aristolochia, a rapid morphological radiation in the Near East (or close to this area) with subsequent star like colonisation of the different current distribution areas, which is not paralleled on the molecular level, appears to be more likely. Phylogenetic tree reconstruction is unsupported for these clades, but most clades are highly supported as monophyletic. Interestingly the Mediterranean and temperate Eurasian species, which are morphologically distinct (A. pistolochia, A. clematitis) are not clustering within the main clades, but are independent lineages. Analogue, A. rigida a species from Somalia is well-supported sister to the subsection Aristolochia. Within subsection Podanthemum the colonisation event from an Asian ancestor is clearly traceable, whereas in subsection Aristolochia the path is not traceable, since the ancestors are extinct or not present in the connecting areas. Within the Mediterranean, Near East and Caucasian species of subsection Aristolochia two morphologically and biogeographically well supported groups can be identified: the Near East/Caucasian species and the West Mediterranean species. The previous groupings for the latter, based on morphological characters, could be substantiated only partly by our results. This study provides the first phylogeny of all West Mediterranean species. In addition an independent complex is established including some micro endemic species. The phylogenetic results are discussed with respect to biogeography, and morphology, to give a first insight into the radiation and colonisation of the genus Aristolochia in the Mediterranean region. Universal primers for a large cryptically simple cpDNA microsatellite region in Aristolochia. We provide a new and valuable marker to study species relationships and population genetics in order to trace evolutionary, ecological, and conservational aspects in the genus Aristolochia. Universal primers for amplification and subsequent sequencing of a chloroplast microsatellite locus inside the trnK intron are presented. Utility of the primers has been tested in 32 species representing all clades of Aristolochia, including population studies within the A. pallida complex, A. clusii and A. rotunda. The microsatellite region is characterized as a (AnTm)k repeat of 22–438 bp containing a combination of different repeats arranged as ‘cryptically simple’. Trapped! Pollination of Aristolochia pallida Willd. in the Mediterranean A first study of the pollination biology of a Mediterranean Aristolochia species in its natural habitat is presented. 183 flowers of Aristolochia pallida were investigated, which in total contained 73 arthropods, dominated by two groups of Diptera, Sciaridae (37%) and Phoridae (19%). However, only Phoridae are regarded as potential pollinators, since pollen has been found exclusively on the body surfaces of these insects. All Phoridae belong to the genus Megaselia and are recognised as four undescribed species. The measurements of flower and insect dimensions suggest that size is an important constrain for successful pollination: 1) the insects must have a definitive size for being able to enter the flower and 2) must be able to get in touch with the pollen. Only very few insect groups found in Aristolochia pallida fulfil these size requirements. However, size alone is not a sufficient constrain as too many fly species of the same size might be trapped but not function as pollinators. Instead, specific attraction is required as otherwise pollen is lost. Since all trapped Phoridae are males, a chemical attraction (pheromones) is proposed as an additional constrain. Since A. pallida flowers are protogynous, the record of Megaselia loaded with pollen found in a flower during its female stage proves that this insect must have been visited at least one different flower during its male stage before. Further on, this observation provides strong evidence that the flowers are cross-pollinated. All these factors indicate a highly specialised pollination of Aristolochia pallida by Megaselia species.

Evolution of Piperales – matK gene and trnK intron sequence data reveal lineage specific resolution contrast. Piperales are one of the largest basal angiosperm orders with a nearly worldwide distribution. The order includes three species rich genera, Piper (ca. 1,000 species), Peperomia (ca. 1,500-1,700 species), and Aristolochia s. l. (ca. 500 species). Sequences of the matK gene and the non-coding trnK group II intron are analysed for a dense set of 105 taxa representing all families (except Hydnoraceae) and all generic segregates (except Euglypha within Aristolochiaceae) of Piperales. A large number of highly informative indels are found in the Piperales trnK/matK dataset. Within a narrow region approximately 500 nt downstream in the matK coding region (CDS), a length variable simple sequence repeat (SSR) expansion segment occurs, in which insertions and deletions have led to short frame-shifts. These are corrected shortly afterwards, resulting in a maximum of 6 amino acids being affected. Furthermore, additional non-functional matK copies were found in Zippelia begoniifolia, which can easily be discriminated from the functional open reading frame (ORF). The trnK/matK sequence data fully resolve relationships within Peperomia, whereas they are not effective within Piper. The resolution contrast is correlated with the rate heterogenity between those lineages. Parsimony, Bayesian and likelihood analyses result in virtually the same topology, and converge on the monophyly of Piperaceae and Saururaceae. Lactoris gains high support as sister to Aristolochiaceae subf. Aristolochioideae, but the different tree inference methods yield conflicting results with respect to the relationships of subfam. Asaroideae. In Piperaceae, a clade formed by the monotypic genus Zippelia and the small genus Manekia (=Sarcorhachis) is sister to the two large genera Piper and Peperomia. Systematics of pipevines – Combining morphological and fast-evolving molecular characters to investigate the relationships within subfamily Aristolochioideae (Aristolochiaceae) A combined phylogenetic analysis of the Aristolochioideae was conducted based on 72 morphological characters and molecular datasets (matK gene, trnK intron, trnL intron, trnL-trnF spacer). The analysis sampled 33 species as the ingroup, including two species of Thottea and 30 species of Aristolochia and the monotypic genus Euglypha, which represent all the infrageneric taxa formally described; Saruma henryi and Asarum caudatum were used as the outgroup. The results corroborate a sister-group relationship between Thottea and Aristolochia, and the paraphyly of Aristolochia with respect to Euglypha that consequently should be included into Aristolochia. Two of the three subgenera within Aristolochia (Isotrema and Pararistolochia) are shown to be monophyletic, whereas the signal obtained from the different datasets about the relationships within subg. Aristolochia is low and conflicting, resulting in collapsed or unsupported branches. The relationship between the New World and the Old World species of subgenus Aristolochia is conflictive because morphological data support these two groups as monophyletic, whereas molecular data show the monophyletic Old World species of Aristolochia nested within the New World species. A sister group relationship is proposed between A. lindneri and pentandrous species, which suggests that a group of five species from central and southern South America (including A. lindneri) could be monophyletic and sister to Aristolochia subsection Pentandrae, a monophyletic taxon consisting of about 35 species from southern USA, Mesoamerica, and the West Indies. Colonisation, phylogeography and evolution of endemism in Mediterranean Aristolochia (Aristolochiaceae). This study provides evidence for a multiple colonisation of the western Old World from Asian ancestors within Aristolochia section Diplolobus (subsection Aristolochia and Podanthemum). Within subsection Podanthemum it is assumed, that the colonisation of the African continent happened at least two times independently. In contrast, for subsection Aristolochia, a rapid morphological radiation in the Near East (or close to this area) with subsequent star like colonisation of the different current distribution areas, which is not paralleled on the molecular level, appears to be more likely. Phylogenetic tree reconstruction is unsupported for these clades, but most clades are highly supported as monophyletic. Interestingly the Mediterranean and temperate Eurasian species, which are morphologically distinct (A. pistolochia, A. clematitis) are not clustering within the main clades, but are independent lineages. Analogue, A. rigida a species from Somalia is well-supported sister to the subsection Aristolochia. Within subsection Podanthemum the colonisation event from an Asian ancestor is clearly traceable, whereas in subsection Aristolochia the path is not traceable, since the ancestors are extinct or not present in the connecting areas. Within the Mediterranean, Near East and Caucasian species of subsection Aristolochia two morphologically and biogeographically well supported groups can be identified: the Near East/Caucasian species and the West Mediterranean species. The previous groupings for the latter, based on morphological characters, could be substantiated only partly by our results. This study provides the first phylogeny of all West Mediterranean species. In addition an independent complex is established including some micro endemic species. The phylogenetic results are discussed with respect to biogeography, and morphology, to give a first insight into the radiation and colonisation of the genus Aristolochia in the Mediterranean region. Universal primers for a large cryptically simple cpDNA microsatellite region in Aristolochia. We provide a new and valuable marker to study species relationships and population genetics in order to trace evolutionary, ecological, and conservational aspects in the genus Aristolochia. Universal primers for amplification and subsequent sequencing of a chloroplast microsatellite locus inside the trnK intron are presented. Utility of the primers has been tested in 32 species representing all clades of Aristolochia, including population studies within the A. pallida complex, A. clusii and A. rotunda. The microsatellite region is characterized as a (AnTm)k repeat of 22–438 bp containing a combination of different repeats arranged as ‘cryptically simple’. Trapped! Pollination of Aristolochia pallida Willd. in the Mediterranean A first study of the pollination biology of a Mediterranean Aristolochia species in its natural habitat is presented. 183 flowers of Aristolochia pallida were investigated, which in total contained 73 arthropods, dominated by two groups of Diptera, Sciaridae (37%) and Phoridae (19%). However, only Phoridae are regarded as potential pollinators, since pollen has been found exclusively on the body surfaces of these insects. All Phoridae belong to the genus Megaselia and are recognised as four undescribed species. The measurements of flower and insect dimensions suggest that size is an important constrain for successful pollination: 1) the insects must have a definitive size for being able to enter the flower and 2) must be able to get in touch with the pollen. Only very few insect groups found in Aristolochia pallida fulfil these size requirements. However, size alone is not a sufficient constrain as too many fly species of the same size might be trapped but not function as pollinators. Instead, specific attraction is required as otherwise pollen is lost. Since all trapped Phoridae are males, a chemical attraction (pheromones) is proposed as an additional constrain. Since A. pallida flowers are protogynous, the record of Megaselia loaded with pollen found in a flower during its female stage proves that this insect must have been visited at least one different flower during its male stage before. Further on, this observation provides strong evidence that the flowers are cross-pollinated. All these factors indicate a highly specialised pollination of Aristolochia pallida by Megaselia species.

The concepts involved with the experimental techniques of Electric Field Induced Second Harmonic generation (EFISH), dipole moment measurement, and solvatochromism, are introduced with particular application to the properties of organic molecules. A number of organic chromophores are introduced, but emphasis is applied to the study of tetracyanoquinodimethane (TCNQ) derivatives which are expected to possess large dipole moments (µ) and large hyperpolarisabilities (β). Furthermore, the behaviour of and with respect to the local environment of the molecule is discussed where a novel evolution is predicted from theoretical calculations. The measurement of µ, is discussed with particular reference to the geometry of the local field factor and the size of the molecule. Consequently, the choice of local field is found to be critical when dipole moments are large, as is the case with the TCNQ derivatives. The EFISH experimental technique is introduced where the calibration of the experiment is discussed. The measurement of the molecular figure of merit, µβ at 1.064 µm and 1.907 µm in chloroform and dichloromethane is presented for the TCNQ derivatives, again paying attention to the geometry of the local field factor. µβ is found to be moderate for most of the compounds, but β is found to be unexpectedly small. This is partly due to the fact that µ is large. A novel evolution of the transition frequency with solvent polarity is found for three of the chromophores under study, where the solvatochromic shift reverses. Solvatochromism experiments are conducted with binary solvent mixtures to ascertain the position of the cyanine limit (β=0) with respect to reaction field. It is found that the materials reside close to the cyanine limit in chloroform and dichloromethane. This is attributed as a reason for the low β measurements. Comparisons of µ and β are also made with Sum-Over-State calculations. A better correlation is found for ellipsoidal local field factors.

Se ha predicho teóreticamente que la epistasis, es decir, las interacciones genéticas entre diferentes mutaciones, cumple un rol sustancial en procesos evolutivos, tales como la emergencia de la reproducción sexual, la recombinación, la especiación y la evolución adaptativa. Sin embargo, existe poca evidencia experimental o estadística de la ubicuidad de las interacciones epistáticas en la naturaleza. Aquí, estudiamos la evolución de las proteínas a largo plazo, y demostramos que el modelo constante de selección independiente, no es capaz de describir las tasas y patrones de divergencia encontrados en las proteínas: las proteínas divergen mas allá de los límites teóricos y la tasa de divergencia es mucho mas lenta que la esperada. A su vez, demostramos que la evolución de las proteínas se explica mejor bajo la suposición de un intercambio rápido entre los valores de eficacia biológica asociados con aminoácidos individuales. Mas aún, extendemos nuestro estudio computacional y construimos un modelo teórico que captura el efecto de la selección inconstante sobre la evolución molecular.
Epistasis, or genetic interactions between different mutations, is theoretically predicted to play a substantial role in such evolutionary processes as emergence of sexual reproduction and recombination, speciation, adaptive evolution. However, there is little experimental or statistical evidence of the ubiquity of epistatic interactions in nature. Here, we study long-term protein evolution and show that the constant independent selection model cannot describe rates and patterns of protein divergence: protein sequences diverge beyond theoretical limits and the rate of divergence is much slower than predicted. We show that protein evolution is best explained under the assumption of rapid turnover of fitness values associated with individual amino acids. We further extend this computational study and build a theoretical model to capture the effect of non-constant selection on molecular evolution.

Social insects are ecologically dominant because of their specialized, cooperative castes. Reproductive queens lay eggs, while workers take part in brood rearing, nest defense, and foraging. These cooperative castes are a prime example of phenotypic plasticity, whereby a single genetic code gives rise to variation in form and function based on environmental differences. Thus, social insects are well suited for studying mechanisms which give rise to and maintain phenotypic plasticity. At the molecular level, phenotypic plasticity coincides with the differential expression of genes. This dissertation examines the molecular evolution of genes with differential expression between discrete phenotypic or environmental contexts, represented chiefly by female queen and worker castes in social insects. The studies included herein examine evolution at three important levels of biological information: (i) gene expression, (ii) modifications to DNA in the form of methylation, and (iii) protein-coding sequence. From these analyses, a common theme has emerged: genes with differential expression among castes frequently exhibit signatures of relaxed selective constraint relative to ubiquitously expressed genes. Thus, genes associated with phenotypic plasticity paradoxically exhibit modest importance to overall fitness but exceptional evolutionary potential, as illustrated by the success of the social insects.

In this thesis, I studied molecular evolution of the vertebrate genome, focusing on sex chromosomes, protein coding genes, and genome size variation. The evolutionary history of avian sex chromosomes was analyzed by comparing substitution rate among 12 gametologous gene pairs on the Z and W chromosomes. Divergence estimates were distributed into three discrete clusters, evolutionary strata, implying stepwise cessations of recombination. Stratum 3 and stratum 2 are located the intervals 1-11Mb and 16-53Mb on the chicken Z chromosome, respectively. Stratum 1 was located in the middle of stratum 2, suggesting a chromosomal inversion. Using a molecular clock, the estimated times for cessation of recombination between Z and W are 132–150 (stratum 1), 71–99 (stratum 2), and 47–57 (stratum 3) million years ago. Higher divergence rate in the Z chromosome than in autosomes (faster-Z) can be explained by positive selections on recessive alleles in hemizygous females, or by stronger genetic drift due to the smaller effective population size of the Z chromosomes. I found there was no difference in the intensity of the faster-Z effect among male-biased, female-biased, and unbiased genes, as might have been expected under a selection model. This result therefore supports the hypothesis that faster-Z is predominantly due to genetic drift. Next, I analyzed molecular evolution of protein-coding genes in birds. In the comparison of zebra finch, chicken and non-avian outgroups, I found that neutral substitution rate was highest in zebra finch, intermediate in chicken, and lowest in ancestral birds. This difference seems attributable to differences in generation time, ancestral birds being most long-lived. Several functional categories were overrepresented among positively selected genes in avian lineages, such as transporter activity and calcium ion binding. I also found that many genes involved with cognitive processes including vocal learning were positively selected in zebra finch. I also found evidence for Hill-Robertson interference acting against the removal of slightly deleterious mutations at linked loci. Finally, I studied the impact of recombination on genome size variation. I found that highly recombining regions have a more condensed genome structure, including shorter lengths of intron, intergenic spacer, transposable elements and higher gene density. In chicken and zebra finch I found that recombination rate was positively correlated with deletion bias, estimated by sequence comparisons between individual transposable elements (LINEs) and the corresponding master sequences. These observations indicate that the more compact genome structure in highly recombining region is due to a higher rate of sequence loss. Higher deletion bias in autosomes than in sex chromosomes supports this idea. I also found that sequence loss due to the deletion bias can explain nearly 20% of genome size reduction after the split of birds from other reptiles. In human, the recombination rate was positively correlated with the deletion bias estimated from polymorphic indels. These results support the hypothesis that the recombination drives genome contraction via the mutation process.

Spermatozoa in different species are morphologically and physiologically distinct, as well as under different post-copulatory selection regimes. Here we used published data on sperm cell proteomics and microarray expression profiles of the testis and sequential spermatogenic stages to elucidate trends in the evolution of male-related genes. A comparative proteomic analysis of the Mus musculus and Drosophila melanogaster sperm proteomes demonstrated the conservation of the functional composition of sperm proteomes. Despite this similarity, spermatozoa are known to be a rapidly evolving, highly species specific, cell type. One possible hypothesis that may explain the dichotomy of rapid evolution and conservative constraint is gene duplication. Consistently, a survey of retrotransposition, a RNA-based form of gene duplication, in mammals and D. melanogaster revealed that ~20% of known retrogenes encode novel sperm proteins. Further analysis demonstrated that retrotransposition has an important role in the generation of novel sperm genes that function in metabolism. Of particular interest was the observation that sperm retrogenes in mammals and Drosophila were enriched with functions in disparate metabolic pathways, which mirrored the different pathways underlying processes subject to post-copulatory sexual selection due to their role in sperm competition. In addition to this important role in the evolution of sperm there is a general, documented, trend that retrogene are expressed in the testis. However, little is known about the selection on retrogenes as they initially acquire their ability to be expressed. One hypothesis is that the region into which a retrogene is inserted has an especial influence on the evolution of its expression. To determine whether there is an observable effect of retrogene location on retrogene expression a new model for the identification of genomic regions enriched for genes expressed in the testis was developed. Utilising this method significant co-localisation of testisexpressed genes in the D. melanogaster genome was observed, together with a significant association between retrogene residence in these genomic regions and the acquisition of retrogene testis expression.

The dominantly held view in evolutionary theory focuses on gradual or punctuated change, primarily via natural selection, as the mechanism by which novel traits arise and evolution occurs. Noticeably absent from this portrayal of evolution is mention of the conservation of general characteristics, such as homologous morphological features or conserved nucleotide sequences, commonly observed across even distantly related groups at both the molecular and organismal levels. This raises at least the following questions: a) How does the evolution of conserved traits fit into an evolutionary theory that emphasizes change? b) What components of an evolving system provide the capacity for adaptation in spite of this apparent conservation of general traits? And c) How do these components affect the evolution of lineages? Here I suggest that heritable traits such as DNA methylation and histone modifications provide one place to look when addressing these questions. Current quantitative and population genetic models reflect the dominant view of evolution described above, and act as the foundation for both formal and informal descriptions and predictions of evolutionary change. Using results from recent work in molecular epigenetics, I consider the evolutionary implications for these traits, and show how current models of evolution fail to accurately capture this influence. In doing so, I also address some of the philosophical implications for how we conceptualize evolution, and what potential changes might be necessary for a more complete theory.
Master of Arts

Le carpelle est l’organe reproducteur femelle des plantes à fleurs. Nous présentons une analyse transcriptomique comparative, focalisant particulièrement sur le développement des tissus femelles reproducteurs, entre la plante modèle établie Arabidopsis thaliana et la soeur probable du restant des plantes à fleurs vivantes Amborella trichopoda. Des modules de co-expression de gènes ont été d’abord définis et ensuite comparés statistiquement entre espèces sur la base de relations d’orthologie entre tous les gènes dans les deux génomes sous comparaison. Cette étude a révélé des modules génétiques entiers, plutôt que seulement des gènes individuels, qui auraient conservé leurs patrons d’expression depuis le dernier ancêtre commun des plantes à fleurs. D’autres modules d’origine plus récente ont été également mis en évidence, modules qui ont peut-être contribué à une complexification dans la morphologie des carpelles qu’a eue lieu plus tardivement pendant l’évolution des plantes à fleurs. Le deuxième thème majeur de cette thèse concerne l’un des régulateurs de ce deuxième groupe, le facteur de transcription SPATULA (SPT) de la famille bHLH. Nous présentons des données suggérant que SPT aurait acquis son rôle actuel dans les tissus à l’apex du carpelle chez un ancêtre commun des Brassicacées. Nos données suggèrent également que l’acquisition de ce nouveau rôle dépendait de changements au niveau des séquences cis-régulatrices de SPT. Nous décrivons des expériences en cours qui ont pour objectif d’identifier les séquences d’ADN précises responsables pour ce changement évolutive
The carpel is the female reproductive organ of flowering plants. We present a comparative transcriptomic analysis, focusing in particular on the development of female reproductive tissues, between the established model plant Arabidopsis thaliana and the probable sister of all remaining living flowering plants Amborella trichopoda. Gene co-expression modules were first defined and then compared statistically between species on the basis of orthology relationships between all genes in the two genomes under comparison. This study revealed entire genetic modules, rather than just individual genes, that appear to have retained their expression patterns since the last common ancestor of living flowering plants. Other modules of more recent origin have also been brought to light, which may have contributed to a complexification in the morphology of carpels that occurred later during the evolution of flowering plants. The second major theme of this thesis concerns one of the regulators of this second group, the transcription factor SPATULA (SPT) of the bHLH family. We present data suggesting that SPT acquired its present role in the tissues at the apex of the carpel in a common ancestor of Brassicaceae. Our data also suggest that the acquisition of this new role depended on changes in the cis-regulatory sequences of SPT. We describe ongoing experiments that aim to identify the precise DNA sequences responsible for this evolutionary change

Doutoramento em Biologia
No actual cenário de perda acelerada de biodiversidade, o nosso conhecimento dos ecossistemas marinhos, apesar da sua extensão e complexidade, continua muito inferior ao dos ecossistemas terrestres. A classe Malacostraca (Arthropoda, Crustacea), um grupo dos mais representativos nos ecossistemas marinhos, apresenta um elevado nível de diversidade morfológica e ecológica, mas difícil sua identificação ao nível de espécie requer frequentemente a ajuda de especialistas em taxonomia. A utilização recente do “barcoding” (código de barras do ADN), revelou ser um método rápido e eficaz para a identificação de espécies em diversos grupos de metazoários, incluindo os Malacostraca. No âmbito desta tese foi construída uma base de dados de código de barras de ADN envolvendo 132 espécies de Malacostraca vários locais de amostragem no Atlântico Nordeste e Mediterrâneo. As sequências de ADN mitocondrial provenientes de 601 espécimes formaram, em 95% dos casos, grupos congruentes com as identificações baseadas em características morfológicas. No entanto, foi detectado polimorfismo em seis casos e a divergência intra-específica foi elevada em exemplares pertencentes a duas espécies morfológicas, sugerindo, neste caso, a ocorrência de especiação críptica. Este estudo confirma a utilidade do código de barras de ADN para a identificação de Malacostraca marinhos. Apesar do sucesso obtido, este método apresenta alguns problemas, como por exemplo a possível amplificação de pseudogenes. A ocorrência de pseudogenes e as possíveisabordagens para a detecção e resolução deste tipo de problemas são discutidas com base em casos de estudo: análises dos códigos de barras ADN na espécie Goneplax rhomboides (Crustacea, Decapoda). A análise dos códigos de barras ADN revelou ainda grupos prioritários de decápodes para estudos taxonómicos e sistemáticos, nomeadamente os decápodes dos géneros Plesionika e Pagurus. Neste âmbito são discutidas as relações filogenéticas entre espécies seleccionadas dos géneros Plesionika e Pagurus. Este trabalho aponta para várias questões no âmbito da biodiversidade e evolução molecular da classe Malacostraca que carecem de um maior esclarecimento, podendo ser considerado como a base para estudo futuros. Análises filogenéticas adicionais integrando dados morfológicos e moleculares de um maior número de espécies e de famílias deverão certamente conduzir a uma melhor avaliação da biodiversidade e da evolução dentro da classe.
The biodiversity of many habitats is under threat and although seas cover the majority of our planet’s surface, far less is known about the biodiversity of marine environments than that of terrestrial systems. The complexity of its species and ecosystems is immense. Marine malacostraca are known as a group with a high level of morphological and ecological diversity but are difficult to identify by traditional approaches and usually require the help of highly trained taxonomists. A faster identification method, DNA barcoding, was found to be an effective tool for species identification in many metazoan groups including some malacostraca. Moreover, the generation of a larger comparative database allows additional insights into the tempo and mode of molecular evolution. Indeed, examination of diversity at the COI region yields an informative framework to identify and explore priority issues, demanding in turn a fully integrative approach utilising additional molecular, distributional and ecological information. Here we expand the DNA barcode database with a case study involving more than 132 malacostracan species from the Northeast Atlantic Ocean and Mediterranean Sea. DNA sequences from around 601 specimens grouped into clusters corresponding to known morphological species in 95% of cases. However shared polymorphism between sister-species was detected in six species. Intraspecific divergence was high in specimens belonging to two morphological species, suggesting the occurrence of cryptic speciation, allowing a rapid assessment of taxon diversity in groups that have until now received limited morphological and systematic examination. We highlight taxonomic groups or species with unusual nucleotide composition or evolutionary rates. Such data are relevant to strategies for conservation of existing decapod biodiversity, as well as elucidating the mechanisms and constraints shaping the patterns observed.This study reconfirms the usefulness of DNA barcoding for the identification of marine malacostraca, despite complexities that sometimes arise due to pseudogenes (numts). Here, we study the effect of numts on DNA barcoding based on barcoding analyses in decapoda species: Goneplax rhomboides. DNA barcodes reveal priority groups for taxonomic and systematic focus of decapods. Here we discussed two cases of phylogenetic relationships among selected species of Plesionika and Pagurus, respectively. Issues relating to the molecular biodiversity and evolution of the Malacostraca arising from this study allow identification of future priorities. Further phylogenetic analyses including morphological and molecular data of selected families is required, especially encompassing broad geographic and ecological coverage, will lead to an improved evaluation of the biodiversity and evolution among selected Malacostraca species

Advances in DNA sequencing are creating new opportunities for studying the process of evolution. These measurements can be particularly useful for rapidly evolving microbial organisms, whose small size and fast generation times make them ideal for controlled laboratory experiments and for tracking replicate populations in vivo. However, the interpretation of this new source of data is complicated by the unique ways in which these large microbial populations evolve. The basic problem is that natural selection is forced to do too many things at once. Unlike the classical picture, where new mutations arise one-by-one, rapidly evolving populations often harbor many selected variants at the same time. When recombination is limited, selection cannot act on these mutations individually, but only on combinations of mutations that happen to arise on the same genetic background. These effects, known as clonal interference, create correlations along the genome that are difficult to disentangle. Existing population genetic models often neglect these effects, which leaves us at loss when interpreting data from these populations. In Chapters 2-5, we analyze the effects of clonal interference in a simple ``null model'' of microbial evolution. We focus on the simplest model that is consistent with two empirical observations: (1) many fitness-influencing mutations are created every generation and (2) mutations have a broad range of fitness effects. After analyzing the basic dynamics of this model, we obtain predictions for the substitution rates of individual mutations and the patterns of linked neutral diversity, and we show how these quantities depend on the population size, mutation and recombination rates, and the fitness effects of new mutations. In Chapters 6 and 7, we apply this null model to data from laboratory experiments in S. cerevisiae and E. coli. We develop a statistical framework to infer the underlying parameters (the fitness effects of new mutations), which allows us to quantify deviations from the model over longer evolutionary timescales. Finally, in Chapters 8 and 9, we investigate the behavior of the model when some of the parameters are allowed to evolve or change in time.
Physics

In this study, we investigate the causes and consequences of directional neutral evolution. We believe this is an evolutionary force that has not received the attention its importance to DNA and protein evolution warrants. Traditional sequence analyses rely upon models that assume homogeneity of nucleotide and amino acid compositions across different lineages, but our examinations of modern sequences show that this assumption is often violated. Our findings imply that periods of directional changes to the nucleotide or amino acid compositions of biological sequences have occurred in the past. Moreover, we demonstrate that these directional changes are very common and can be quite extreme. We show that modifications to the genetic code---a phenomenon that is common among mitochondrial genomes---can cause directional changes to the amino acid composition of proteins. Codon reassignments shift the relative number of codons assigned to each amino acid, and when coupled with neutral evolution over a period of time these changes will lead to a corresponding shift in the relative amino acid usages within the protein. More common causes of directional changes to the amino acid composition of proteins are mutation biases in the DNA. In particular, we show that biases to the relative proportions of A+T and G+C are capable of bringing about amino acid composition changes to the proteins the DNA encodes, with AT-rich DNA favouring the encoding of the FYMINK amino acids, and GC-rich DNA favouring the encoding of GARP amino acids. We show that this effect is both pervasive across all kingdoms of life, and that it also affects every protein within the genome---whether highly or loosely conserved. Finally, we show that directional neutral evolution in the DNA can operate in the presence of purifying selection in transmembrane domains. Since selection does not preclude neutral evolution if there are multiple favourable mutations possible, DNA mutation bias is able to influence the direction of evolution of the transmembrane domains without interfering with their fitness. We extend this same notion to include the possibility of mutation biases biasing the outcome of positive selection, as well.

This study aims to understand the key role played by molecular hydrogen in the evolution of galaxies, with a view to constraining its radial distribution in the Galaxy and the CO→H(_2) conversion factor α(_20).The star formation rate is shown to be correlated with the surface density of H(_2). A correlation between the molecular hydrogen fraction and the metallicity of a region allows the time evolution of H(_2) to be described. This leads to a modified 'Schmidt Law' of the SFR which explains quite naturally the production of galactic metallicity gradients and the constancy of the SFR in the absence of infall. A consistent closed model of the chemical evolution of the Galaxy is proposed to solve the G-dwarf problem, the stellar age-metallicity relation and the metallicity gradient, leading to the prediction of some initial amount of pre-disc processing of gas into visible and dark matter. It is found that a constant yield of metals is more appropriate than a yield proportional to metallicity. Possible time variations of the returned fraction, the dark matter fraction and the SFR are also studied. For consistency, we suggest that dark matter in the solar neighbourhood could be totally baryonic provided the Miller-Scalo IMF is modified at the lower end, that is, the dark matter resides in low mass stars or brown dwarfs. The production of metallicity gradients in spiral galaxies is shown to be a direct consequence of the radial variation of the total surface density of matter and the age of the disc. The role of molecular gas in the evolution of the Oort Cloud of comets is examined. It is shown that comet showers with a mean interval of ̴̱ 30My cannot be produced using perturbations of the Oort Cloud by known stars or molecular clouds. If there is indeed an apparent 30My periodicity in the terrestrial mass extinction and geological records, we argue that astronomically induced processes are unlikely to be the primary cause. Evidence is presented that the lifetime of the molecular gas phase is ≤ 2.lO(_8)y, and arguments, particularly from CO observations of the Virgo galaxy cluster, favouring longer lifetimes are shown to be not well founded. We suggest that the ICM in Virgo reduces the value of α(_20) as compared to isolated galaxies. From the above considerations, the radial distribution of in the Galaxy is derived and shown to agree in the inner Galaxy with that derived from ɤ-ray analysis. In the solar neighbourhood we find α(_20) = 2.5±0.5, and present evidence that α(_20) varies as a function of Galactocentric radius and from galaxy to galaxy.

The emergence of life depended on the ability of the first biopolymer populations to thrive and approach larger population sizes and longer sequences. The evolution of these populations likely occurred under circumstances under which Muller's Ratchet in synergism with random drift could have caused large genetic deterioration of the biopolymers. This deterioration can drive the populations to extinction unless there is a mechanism to counteract it. The effect of the mutation rate and the effective population size on the time to extinction was tested on clonal populations of B16-19 ligase ribozymes, evolved with the continuous evolution in vitro system. Populations of 100, 300, 600 and/or 3000 molecules were evolved with and without the addition of Mn(II). The times to extinction for populations evolved without Mn(II) were found to be directly related to the effective size of the population. The small populations approached extinction at an average of 24.3 cycles; while the large populations did so at an average of 44.5 cycles. Genotypic characterization of the populations showed the presence of deleterious mutations in the small populations, which are the likely cause of their genetic deterioration and extinction via mutational meltdown. These deleterious mutations were not observed in the large populations; in contrast an advantageous mutant was present. Populations of 100 and 3000 molecules were evolved with Mn(II). None of the populations showed signs of genetic deterioration nor did they become extinct. Genotypic characterization of the 100-molecule population indicated the presence of a cloud of mutants closely genetically-related, forming a "quasispecies" structure.. The close connectedness of the mutants facilitates the recovery of one from another in the event of being removed from the population by random genetic drift. Thus, quasispecies shift the target of selection from individual to group. The total fitness of the molecules was measured by identifying the fitness component of the system that effect the ligase replication cycles: ligation, reverse transcription and transcription. It was found that the strength of the three components of fitness varied and that each one has a differential effect in the total absolute fitness of the ligases.

Nucleotide composition bias and codon usage bias are commonly observed in a wide range of organisms, but the causes of both are still under debate. One view is that codon usage bias and base composition bias are both the result of a directional mutation pressure in favor of AT or GC; others argue that codon usage bias is a result of natural selection acting to mold the codon choice to match the frequency of the corresponding tRNAs. A number of recent studies have indicated that nucleotide bias is especially marked in gene families that are undergoing concerted evolution. In this thesis, I use the Drosophila trypsin gene family as a model system to study the causes and consequences of concerted evolution in Drosophila, and to investigate the evolutionary forces that may be responsible for the observed nucleotide bias. From each of the two related Drosophila species, D. melanogaster and D. erecta, a 12kb genomic region was sequenced and eight trypsin genes were identified. Some members of this gene family have been evolving independently while the others have been evolving in a concerted fashion. In both species, the nonsynonymous codon positions have a moderate GC content, while the synonymous sites are very GC-rich. For the genes that have been undergoing concerted evolution, due to rapid gene conversion, their synonymous G + C content is much higher than that of the independently evolving genes. In addition, these genes are characterized by an elevated frequency of pyrimidines (C or T) at synonymous sites on the coding strand. A combination of selective constraints, directional DNA mutation pressure, and DNA repair bias could have resulted in the base composition pattern observed in these trypsin genes.

Studying evolution at the level of DNA sequences allows the detection of past and recent natural selection. Natural selection has generally been seen as a force acting on proteincoding nucleotide sequences only. However, a number of studies have recently shown that introns and intergenic sequences can also be subject to natural selection. The main aim of this thesis was to detect natural selection in non-coding sequences using Drosophila species, a widely used population genetics model. I have used different methods to determine if evidence for natural selection could be found in two lesserknown species, Drosophila americana and Drosophila miranda. In Chapter 2, I obtained sequences for a large number of genes in D. miranda from BAC sequences, and compared these with sequences from its close relative, D. pseudoobscura. As in previous studies in D. melanogaster, I found a negative relationship between intron length and intron divergence, suggesting that longer introns are under selective constraint. I also found a negative correlation between the rate of nonsynonymous substitutions and codon usage bias, suggesting that fast-evolving genes have a lower codon usage bias, consistent with strong positive selection interfering with weak selection for codon usage. Secondly, in Chapter 3, I gathered polymorphism data for a smaller number of genes in D. americana in order to distinguish between positive and negative selection using methods that require polymorphism and divergence. I found that introns are subject to similar evolutionary forces as synonymous sites. I failed to detect a significant relationship between intron length and divergence or polymorphism. Surprisingly, the direction of this relationship seems to be the opposite of that in previous findings, with longer introns being more diverged than smaller introns. First introns show lower polymorphism and divergence than non-first introns, suggesting that they may be more constrained, although the difference is not significant. Using the same D. americana dataset, I then focussed, in Chapter 4, on insertions and deletions to test the hypothesis that insertions are favoured to compensate for the deletion bias in Drosophila. I used a maximum-likelihood method that takes into account demographic history, in this case a recent population expansion and then calculates the selection coefficients. Although it was not significant, the values suggest positive selection acting on insertions, as expected. In Chapter 5, using the same maximum-likelihood method, I looked at GC to AT polymorphisms in the D. americana intron dataset. It is expected to observe as many GC to AT changes as AT to GC changes and similar mean frequencies if no selection is acting. I find evidence for a preference for GC in introns in my dataset. I also investigated codon usage bias using preferred and unpreferred codons changes and results suggest that there is selection for codon usage bias. Using LDhat on the D. americana dataset, I find that recombination estimates are not significantly different between introns and coding sequences, which is of significance in relation to interpretations of differences in the apparent strength of selection on non-coding and synonymous sites. Finally, in Chapter 6, I looked at a factor that can affect natural selection: gene expression. I used gene expression data from seven Drosophila species to test the hypothesis that genes on the 4th chromosome or Muller element F, which has low crossing-over, have higher gene expression than genes on other chromosomes as previously found. I find that microarray data yields opposite results to the EST data, suggesting that gene expression is actually lower on Muller element F.

This dissertation examines the problem of quantifying amino acid conservation in proteins molecular evolution. Ideally, this conservation is quantified by inferring the rate of evolution at each amino acid site of a multiple-alignment. However, current rate-inference methods have three problematic assumptions. The methods assume that (a) the rates of all sites are independent, (b) the rates are drawn from a known prior distribution, and (c) the mean rate across sites is approximately one. The problems are two-fold. First, the assumptions of site-rate independence and known mean rate are contradictory. To see the contradiction, consider a two-site alignment with known rate of ~0.5 at site one. The rate at site two is unknown under the independent-sites assumption, but is ~1.5 by the assumption of known mean rate. Second, if the rates are drawn from a known prior distribution, the assumption of known distribution implies the question ?which distribution??. Previous work has focused only on selecting better families of rate distributions, often at the expense of additionally parameterizing the evolutionary model. Herein, I develop a method of inferring rates requiring only the assumption of known mean rate, and not requiring additional parameterization. Thus a model of evolution based on our method is a more general framework for inferring rates than previous work. Since a known mean rate is required to distinguish evolutionary rate from time, our method is arguably the most general possible that allows rate and time to be fully and independently identified. The method is assessed by investigating conservation in the Myc, Max, and p53 transcription-factor families.

The principal aim of this investigation is to sequence IL-1β within a number of non-mammalian vertebrates, in order to establish a better idea of when this molecule arose within vertebrate evolution. IL-1β is a good candidate to study as it has been shown to play many important roles within the immune and neuroendocrine system of mammals. Using an homology cloning approach IL-1β has been sequenced from a cartilaginous fish (Scyliorhinus caniculus), a number of bony fish (Pleuronectes platessa, Dicentrarchus labrax and Carassius auratus), an amphibian (Xenopus laevis) and a primitive mammal (Ornithorhynchus anatinus). All nucleotide sequences are found to contain differing numbers of mRNA instability motifs, characteristic of genes which code for inflammatory mediators. All amino acid sequences contain the IL-1 family signature and show highest conservation in the regions which are predicted to form the 12β-sheets, important for the folding of the active molecules. The O. anatinus IL-1β contains an ICE cut site which is in keeping with all other mammalian sequences, whereas none of the other sequences contain this cut site as found in all non-mammalian vertebrates to date. Like all other IL-1β molecules they contain no signal peptides, indicating that these molecules are not secreted through the classical golgi/endoplasmic reticulum route. Within C. auratus two different IL-1β genes were sequenced, IL-1β and IL-1β2. The second gene contained a different IL-1 family signature to the first and also had regions of coding sequence missing, a region at the 5- end, which is presumed to be due to the loss of an exon and two other regions, one which is 3 bp and the other 12 bp long. The second gene also showed differences in the 3' UTR sequence and position of its stop codon when compared to the first gene.

Doutoramento em Biologia
Durante 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.