It has been theorized that most proteins are under selection pressure to be soluble in crowded cellular spaces. To maintain solubility a proteins’ aggregation propensity should be inversely proportional to their maximum likely concentration. This theory was examined by comparing the proteome of the model organism S. cerevisiae, which has previously undergone a Whole Genome Duplication (WGD) event to the proteome of the closely related yeast K. waltii, which has not undergone WGD. This comparison revealed the following: 1) Predicted aggregation propensities are higher in S. cerevisiae than K. waltii. 2) Aggregation propensity does not predict which genes reverted to a single copy after WGD. 3) In genes which were retained as duplicates in S. cerevisiae after WGD, aggregation propensities rose from the inferred common ancestral protein. 4) Genes retained as duplicates showed less of an increase relative to their homologues in K. waltii than genes which were not retained as duplicates. 5) The relationship between the log predicted aggregation propensity and log mRNA expression level or log protein abundance was not linear as previously predicted. These results suggest that while there is broad selection pressure for reduced aggregation pressure for genes which have been duplicated, the precise relationship between aggregation propensity and gene expression is more complicated than previously predicted. These results also allow speculation that the whole genome duplication in S.cerevisiae may have been made possible by a general relaxation of aggregation-related selection pressure. / text

The yeast Saccharomyces cerevisiae genome is descendant from a whole genome duplication event approximately 150 million years ago. Following this duplication many genes were lost however, a certain class of genes, termed ohnologs, persist in duplicate. In this thesis we investigate network centrality as it relates to ohnolog re- tention with the goal of determining why only certain genes were retained. With this in mind, we compare physical and genetic interaction networks and genetic and pro- tein sequence data in order to reveal how network characteristics and post-duplication retention are related. We show that there are two subclasses of ohnologs, those that interact with their duplication sister and those that do not and that these two classes have distinct characteristics that provide insight into the evolutionary mechanisms that affected their retention following whole genome duplication. Namely, a very low ratio of non-synonymous mutations per non-synonymous site for ohnologs that retain an interaction with their duplicate. The opposite observation is seen for ohnologs that have lost their interaction with their duplicate. We interpret this in the fol- lowing way: ohnologs that have retained their interaction with their duplicate are functionally constrained to buffer for the other ohnolog. For this reason they are retained; ohnologs that have lost their interaction with their duplicate are retained because they are functionally divergent to the point of being individually essential. Additionally we investigate small scale duplications and show that, generally, the mechanism of duplication (smale scale or whole genomes) does not affect the distri- bution of network characteristics. Nor do these network characteristics correlate to the selective pressure observed by retained paralogous genes, including both ohnologs and small scale duplicates. In contrast, we show that the network characteristics of individual genes, particularly the magnitude of their physical and genetic network centralities, do influence their retention following whole genome duplication. / Graduate / mjrimrie@gmail.com

The Hox gene complement of zebrafish, medaka, and fugu differs from that of other gnathostome vertebrates. These fishes have seven to eight Hox clusters compared to the four Hox clusters described in sarcopterygians and shark. The clusters in different teleost lineages are orthologous, implying that a “fish-specific” Hox cluster duplication has occurred in the stem lineage leading to the most recent common ancestor of zebrafish and fugu. The timing of this event, however, is unknown. To address this question, we sequenced four Hox genes from taxa representing basal actinopterygian and teleost lineages and compared them to known sequences from shark, coelacanth, zebrafish, and other teleosts. The resulting gene genealogies suggest that the fish-specific Hox cluster duplication occurred coincident with the origin of crown group teleosts. In addition, we obtained evidence for an independent Hox cluster duplication in the sturgeon lineage (Acipenseriformes). Finally, results from HoxA11 suggest that duplicated Hox genes have experienced diversifying selection immediately after the duplication event. Taken together, these results support the notion that the duplicated Hox genes of teleosts were causally relevant to adaptive evolution during the initial teleost radiation.

In this study, full coding sequences of Atlantic salmon (Salmo salar L.) muscle genes were cloned, including myogenic regulatory factors (MRFs) (myod1c, myog, mrf4, myf5), inhibitors of Myostatin (fst, decorin), markers of myogenic progenitor cell (MPC) proliferation (sox8) and fusion (calpastatin), a marker of slow muscle fibre differentiation (smlc1) and a novel eukaryotic gene involved in regulating growth (cee). Several of these genes were then characterised using a range of experimental and computational analyses with the aim to better understand their role in myogenesis and their evolution in teleosts. A series of experiments supported previous findings that teleosts have extra copies of many genes relative to tetrapods as a result of a whole genome duplication (WGD) event that occurred some 320-350 Mya. For example, it was shown that genes for myod and fst have duplicated in a common teleost ancestor, but were then specifically lost or retained in different lineages. Furthermore, several characterised Atlantic salmon genes were conserved as paralogues, likely from a later WGD event specific to the salmonid lineage. Phylogenetic reconstruction and comparative genomic approaches were used to characterise the evolution of teleost paralogues within a framework of vertebrate evolution. As a consequence of one experiment, a revised nomenclature for myod genes was proposed that is relevant to all diploid and polyploid vertebrates. The expression patterns of multiple myogenic genes were also established in Atlantic salmon embryos using specific complementary RNA probes and in situ hybridization. For example, co-ordinated embryonic expression patterns were revealed for six salmon MRFs (myod1a, myod1b, myod1c, myog, mrf4, myf5), as well as markers of distinct MPC populations (pax7, smlc1), providing insight into the regulatory networks governing myogenesis in a tetraploid teleost. Furthermore, it was shown that Atlantic salmon fst1 was expressed concurrently to pax7 in a recently characterised MPC population originating from the anterior domain of the epithelial somite, which is functionally analogous to the amniote dermomyotome. In another experiment, the individual expression domains of three Atlantic salmon myod1 paralogues were shown to together recapitulate the expression of the single myod1 gene in zebrafish, consistent with the partitioning of ancestral cis-acting regulatory elements among salmonid myod1 duplicates. Additionally, the in situ expression of cee a novel and highly conserved eukaryotic gene was revealed for the first time in a vertebrate and was consistent with an important role in development including myogenesis. Additionally, Atlantic salmon were reared at 2, 5, 8 or 10 ºC solely to a defined embryonic stage, which was just subsequent to the complete pigmentation of the eye. After this time, animals were provided an equal growth opportunity. Remarkably, changing temperature during this short developmental window programmed the growth trajectory throughout larval and adult stages. While 10 and 8 ºC fish were larger than those reared at 2 and 5 ºC at the point of smoltification, strong compensatory growth was subsequently observed. Consequently, after 18 months of on growing, size differences among 5, 8 and 10 ºC fish were not significant, although each group was heavier than 2 ºC fish. Furthermore, significant embryonic-temperature induced differences were observed in the final muscle fibre phenotype, including the number, size distribution and myonuclear density of muscle fibres. A clear optimum for the final muscle fibre number was observed in 5 ºC fish, which was up to 17% greater than other treatments. In a sub-sample of embryos, temperature induced heterochonies were recorded in the expression of some MRFs (myf5, mrf4) but not others (myod1a, myog). These results allowed the proposition of a potential mechanism explaining how temperature can program the muscle phenotype of adult teleosts through modification of the somitic external cell layer, a source of MPCs throughout teleost ontogeny.

Les duplications de gènes et de génome sont considérées comme des moteurs de l’évolution des génomes eucaryotes. Trois duplications de génome complet (ou polyploïdisations) sont survenues au cours de l’évolution des vertébrés, dont deux à la base des vertébrés, et une troisième chez l’ancêtre commun des poissons téléostéens. La diversité morphologique, anatomique et écologique des espèces qui partagent un ancêtre commun polyploïde chez les chordés suggère un rôle des duplications de génome dans la diversification des espèces. En particulier, les duplications de génome semblent avoir facilité l’émergence du plan d’organisation des vertébrés, et être à l’origine de la radiation évolutive survenue chez les poissons téléostéens. Cependant, la portée évolutive des duplications de génome, et notamment les deux hypothèses majeures formulées ci-Avant, restent des questions ouvertes et en grande partie non résolues. Le groupe des téléostéens, qui compte plus de la moitié des espèces vertébrés existantes et partage un ancêtre commun polyploïde, constitue un modèle pertinent pour évaluer la contribution des duplications de génome dans l’expansion des familles multigéniques chez les vertébrés, pour comprendre les mécanismes évolutifs qui façonnent l’évolution des familles de gènes, et finalement tester les hypothèses moléculaires qui peuvent relier duplication de génome et biodiversité. Ainsi, nous avons étudié l’impact de la duplication de génome survenue à la base des téléostéens sur l’évolution de la famille multigénique sox, essentielle pour le développement et l’homéostasie des vertébrés. Notre analyse du contenu et de l’organisation des gènes sox dans 15 génomes de vertébrés, dont 10 téléostéens, révèle une importante expansion de l’ensemble de la famille des gènes sox dans ce vaste groupe de vertébrés, et démontre que cette expansion est essentiellement due à la duplication de génome survenue à la base des téléostéens. Les gènes sox dupliqués par duplication de génome semblent avoir été perdus par non-Fonctionnalisation dans certaines lignées, et préservés en deux copies par sous-Fonctionnalisation et/ou néo-Fonctionnalisation dans certaines autres lignées. Notre étude indique en effet une divergence lignée-Spécifique des patrons d’expression entre les gènes sox dupliqués chez différentes espèces de téléostéens. Ainsi, l’expansion du répertoire des gènes sox à la base des téléostéens semble avoir été suivi d’une évolution lignée-Spécifique du contenu et des fonctions de la famille des gènes sox chez les poissons téléostéens. Cette étude supporte l’hypothèse d’un rôle des duplications de génome dans l’enrichissement et la diversification subséquente des répertoires de gènes du développement tels que les gènes sox, et son rôle potentiel dans la diversification des espèces vertébrés. / Gene and genome duplications are major engines of eukaryotic genome evolution. Three rounds of whole genome duplication (WGD) have occurred during vertebrate evolution, two rounds at the base of the vertebrate lineage, and a third round in the common ancestor of the teleostean fish (the so-Called teleost-Specific WGD). In chordates, species that share a polyploid ancestor are characterized by a huge morphological, anatomical and ecological diversity suggesting a role of WGDs in species diversification. For instance, it is considered that these drastic genomic events provided the raw material for the emergence of the vertebrate body plan, and facilitated speciation processes during the teleost radiation. However, how WGD is related to phenotypic diversification or to major evolutionary transitions are fundamental questions that remain largely unsolved. Teleostean fish constitute more than half of all extant vertebrates and share a polyploid ancestor. Thus, they provide a relevant model to study the importance of WGDs in gene families expansion, to understand evolutionary mechanisms that drive the evolution of these families and, finally, to test molecular hypotheses that might relate WGD and biodiversity. In this project, we studied the impact of the teleost-Specific WGD on the evolution of the sox gene family which are involved in development and homeostasis in vertebrates. Our analysis of the content and the genomic organization of the sox genes in 15 vertebrate genomes, including 10 teleosts, reveals an important expansion of this family in the teleost lineage, and demonstrates that this expansion is mainly due to the teleost-Specific WGD. The duplicated sox genes seem to have been lost by non-Functionalization in certain lineages, and preserved in two copies in others by neo-Functionalization and/or sub-Functionalization. Indeed, this study indicates lineage-Specific divergence in expression patterns between duplicated sox genes in different teleostean species. Hence, the sox family expansion that occurred in the last common ancestor of teleostean fish seems to have been followed by a lineage-Specific evolution of the content and functions of the sox family in this group. Our study supports the hypothesis for a role of WGDs in the enrichment and diversification of developmental genes repertories and its potential role in species diversification in vertebrates.

Les phénomènes évolutifs qui perturbent l’organisation des gènes dans les génomes eucaryotes sont de deux types : les changements dans l’ordre des gènes, ou réarrangements, et les modifications du contenu en gènes du génome, par duplications, délétions ou gains de gènes. Ces processus sont mal connus, tant au niveau de leurs mécanismes d’apparition que de leur impact fonctionnel et sélectif. Ce travail de thèse s’articule autour de deux projets. Le premier s’intéresse à la distribution des points de cassure de réarrangements évolutifs entre un génome ancestral et ses descendants modernes. Cette distribution a été modélisée en fonction des caractéristiques locales du génome pour mettre en évidence quels facteurs influencent la probabilité de cassure. Nos résultats montrent que la distribution des cassures peut s’expliquer simplement comme une fonction de la longueur des espaces intergéniques, fonction qui est cependant non-linéaire contrairement aux attentes sous un régime aléatoire classique. La répartition des points de cassure dans les génomes semble principalement liée à des propriétés de structure, et n’est que peu soumise à des contraintes de sélection. Elle pourrait être liée à la structure chromatinienne du génome. Le second projet s’inscrit dans le cadre du séquençage du génome du poisson zèbre, et fournit un aperçu global de l’organisation de ce génome. Les génomes de poissons téléostéens sont anciennement dupliqués : l’analyse est axée sur les conséquences de cette duplication. Les résultats montrent que le génome du poisson zèbre présente une organisation assez typique d’un génome téléostéen. Les gènes retenus en deux copies après la duplication du génome appartiennent à des catégories fonctionnelles particulières, et sont biaisés vers des gènes déjà conservés après les duplications 1R et 2R ayant eu lieu au début de l’histoire des vertébrés. / Evolutionary processes disrupting the gene organisation in eukaryotic genomes belong to two categories: changes in the order of the genes, known as rearrangements, and changes in the content of the genome by gene duplications, deletions and gains. The mechanisms through which these events arise, and their functional and selective impact on genomes, are poorly understood. This thesis covers two different projects. Firstly, we investigated the distribution of rearrangement breakpoints between an ancestral genome and its modern descendants. This distribution was modelled according to local genomic characteristics to highlight factors influencing the breakage process. Our results show that the distribution of breakpoints can be simply explained as a function of intergenic spacers length, although in a non-linear fashion differing from classical random expectations. The repartition of breakpoints in genomes seems to be linked to structural properties, and is only marginally affected by selective constraints. It might in fact reflect local chromatin structure in the genome. The second project is part of the joint sequencing effort for the zebrafish genome, and provides an overview of the organisation of this genome. Teleost fish genomes are anciently duplicated: the analysis focuses on the consequences of this duplication. Results show that the zebrafish genome displays a typical teleost fish genome organisation. Genes retained in two copies after the whole genome duplication belong to specific functional categories, and are biased towards genes already conserved as duplicates after the 1R and 2R duplication events that have taken place early in vertebrate history.

Two whole genome duplications (2R) occurred early in vertebrate evolution. By using combined information from phylogenetic analyses and chromosomal location of genes, this thesis delineates the evolutionary history of two receptor-ligand systems that expanded by these large scale events. A third whole genome duplication (3R) took place in the teleost fish lineage and has also contributed to the complexity of the gene families. New members of neuropeptide Y (NPY) peptide and receptor families were generated in 2R and 3R. Evolutionary comparisons show that the ancestral teleost fish had four peptides; subsequently, differential losses of the peptide genes occurred. In zebrafish the peptides and receptors display differences in tissue distribution and have  evolved binding preferences. In the frog Silurana tropicalis three peptides and six receptors werev identified, also displaying some differences in tissue distribution and receptor-ligand preferences. The findings in these experimental animals highlight both evolutionary conservation and lineage-specific features of the NPY system. The opioid system consists of four receptors and several peptides originating from four precursors. These results show that the receptor family was formed in 2R and 3R and that 2R together with one local duplication gave rise to the peptide family. The ancestral receptor and peptide genes were located on the same chromosome, suggesting coevolution. The Hox gene clusters, important in early development, provided the first strong evidence for 2R. Several neighboring gene families were analyzed and found to have expanded in 2R and 3R. In depth analyses of insulin-like growth factor binding protein (IGFBP) and voltage-gated sodium channel (SCN) gene families illustrates the importance of local duplications in combination with whole genome duplications in the formation of gene families. These findings provide additional strong evidence for two genome duplications in early vertebrate evolution and show that these events generated many new genes that could evolve new or more specialized functions.

Polyploidy, or whole genome duplication (WGD), is a fundamental evolutionary mechanism for diverse organisms including many plants and some animals. Duplicate genes from WGD are a major source of expression and functional diversity. However, the biological and evolutionary mechanisms for gene expression changes within and between species following WGD are poorly understood. Using genome-wide gene expression microarrays and high-throughput sequencing technology, I studied the genetic and evolutionary mechanisms for gene expression changes in synthetic and natural allopolyploids that are derived from hybridization between closely related species. To investigate evolutionary fate of duplicate genes, I tested how duplicate genes respond to developmental and environmental changes within species and how ancient duplicate genes contribute to gene expression diversity in resynthesized allopolyploids. We found that expression divergence between gene duplicates was significantly higher in response to environmental stress than to developmental process. Furthermore, duplicate genes related to external stresses showed higher expression divergence between two closely related species and in resynthesized and natural allotetraploids than single-copy genes. A slow rate of expression divergence of duplicate genes during development may offer dosage-dependent selective advantage, whereas a high rate of expression divergence between gene duplicates in response to external changes may enhance adaptation. To investigate molecular mechanisms of expression diversity among allopolyploids, I analyzed high-throughput sequencing data of small RNAs in allopolyploids and their progenitors. Small interfering RNAs (siRNAs) induce epigenetic modification and gene silencing of repeats, while microRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) induce expression modulation of protein coding genes. Our data showed that siRNA populations in progenitors were highly maintained in allopolyploids, and alteration of miRNA abundance in allopolyploids was significantly correlated with expression changes of miRNA target genes. These results suggest that stable inheritance of parental siRNAs in allopolyploids helps maintain genome stability in response to genome duplication, whereas expression diversity of miRNAs leads to interspecies variation in gene expression, growth, and development. Results from these research objectives show that genome-wide analysis of high throughput gene expression and small RNAs provides new insights into molecular and evolutionary mechanisms for gene expression diversity and phenotypic variation between closely related species and in the new allopolyploids. / text

Duplicated genes are present in the teleost fish lineage owing to a whole-genome duplication (WGD) event that occured ~ 230-400 million years ago. In the duplication-degeneration-complementation (DDC) model, partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) have been proposed as principal processes for the retention of duplicated genes in the genome. The DDC model was tested by analyzing the differential tissue-specific distribution of transcripts for the duplicated fatty acid-binding protein 10 (fabp10) genes in embryos, larvae and adult zebrafish (Danio rerio). The distribution of zebrafish fabp10a and fabp10b transcripts show a strikingly different tissue-specific pattern leading us to suggest that the zebrafish fabp10 duplicates had been retained in the genome owing to neofunctionalization. In another experiment to test the DDC model, transcriptional regulation of duplicated fabp genes was analyzed in zebrafish fed clofibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. Clofibrate increased the steady-state level of both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b mRNA and heteronuclear RNA (hnRNA), but in different tissues of zebrafish. The steady-state level of fabp10a and fabp11a mRNA and hnRNA was elevated in liver of zebrafish, but not for fabp10b and fabp11b. We also investigated the effect of dietary fatty acids (FAs) and clofibrate on the transcriptional regulation of single copy fabp genes, fabp2, fabp3 and fabp6 in zebrafish. The steady-state level of fabp2 transcripts increased in intestine, while fabp3 mRNA increased in liver of zebrafish fed diets differing in FA content. In zebrafish fed clofibrate, fabp3 mRNA in intestine, and fabp6 mRNA in intestine and heart, was elevated. Whether the regulation of fabp gene transcription by clofibrate is controlled either directly or indirectly, the regulatory elements in the zebrafish fabp genes have diverged markedly since the WGD event, thereby supporting the DDC model.

The expansion of the voltage-gated calcium channel alpha 1 subunit families (CACNA1) of L and N types was investigated by combining phylogenetic analyses (neighbour-joining and maximum likelihood) with chromosomal data. Neighbouring gene families were analysed to see if the chromosomal regions duplicated through whole genome doublings in vertebrates. Results show that both types of CACNA1 expanded in two ancient whole genome duplications as parts of larger genomic regions. Many gene families in these regions obtained copies in an additional teleost-specific genome duplication. This diversification of CACNA1 genes probably contributed to evolutionary innovations in nervous system function.