My research mainly focused on the functional redundancy and expression divergence of gene duplicates to address currently unsolved problems. Herein, we employed a method based on GO terms to measure functional overlap between paralogs. We established that functional similarity between duplicate genes is the key determinant of their backup capacity. Later, we also investigated expression divergence. Recent studies suggest that only a small proportion of expression variation can be explained by transcriptional variation between paralogs. Here, the contribution from diverged TF-regulations was re-examined and differential promoter chromatin status was also found as an important contributor to expression divergence. To better understand the role of gene duplication in great detail, a case study was performed on the yeast chaperone system, which includes many gene duplicates. Taken together, this study sheds light on the roles of redundancy and divergence in long-term retention of gene duplicates.

My research mainly focused on the functional redundancy and expression divergence of gene duplicates to address currently unsolved problems. Herein, we employed a method based on GO terms to measure functional overlap between paralogs. We established that functional similarity between duplicate genes is the key determinant of their backup capacity. Later, we also investigated expression divergence. Recent studies suggest that only a small proportion of expression variation can be explained by transcriptional variation between paralogs. Here, the contribution from diverged TF-regulations was re-examined and differential promoter chromatin status was also found as an important contributor to expression divergence. To better understand the role of gene duplication in great detail, a case study was performed on the yeast chaperone system, which includes many gene duplicates. Taken together, this study sheds light on the roles of redundancy and divergence in long-term retention of gene duplicates.

The shikimate pathway connects primary metabolism with the biosynthesis of the three aromatic amino acids (phenylalanine, tyrosine and tryptophan), which are essential protein building blocks. This pathway also provides precursors for a wide array of plant secondary metabolites with adaptive functions in plant adaptation and defense. The third and fourth steps of the shikimate pathway (the conversion of shikimate from 3-dehydroquinate via 3-dehydroshikimate) are catalyzed by a bi-functional enzyme called 3-dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH). DQD/SDHs have been biochemically characterized in a few plant species including Arabidopsis thaliana, Solanum lycopersicum and Nicotiana tabacum. The embryo-lethal phenotype of Arabidopsis null mutants lacking DQD/SDH highlights a critical role of shikimate in primary metabolism. Quinate shares high structural similarity with shikimate and is an important secondary metabolite present in many plant species. Quinate and its derivatives (e.g. chlorogenic acid) serve important functions in plant defense due to their astringent (i.e. bitterness) and antimicrobial properties. Quinate can be derived from 3-dehydroquinate, and this reaction is catalyzed by quinate dehydrogense (QDH), the reaction mechanism of which resembles that of SDH. With a functional genomics approach, I demonstrated that two of the five poplar putative DQD/SDHs (Poptr1 and Poptr5, poplar DQD/SDH1 and 2) have exclusive specificity for shikimate, while the other three (Poptr2 to Poptr4, poplar QDH1 to 3) are involved in quinate biosynthesis. Phylogenetic reconstruction of the DQD/SDH/QDH superfamily has identified two distinct clades in seed plants that may act preferentially on either shikimate or quinate, whereas lineages that have diverged prior to the angiosperm/gymnosperm split, only have a single copy DQD/SDH. An evolutionary analysis was carried out, and the sequence of the immediate pre-duplication ancestral DQD/SDH (>300MYA) was estimated and reconstructed. Protein structure modelling and in vitro biochemical characterization of the ancestral recombinant protein was performed along with some extant members of this family (pre-duplication representatives: Rhodopirellula baltica (Rhoba), Chlamydomonas reinhardtii (Chlre), Physcomitrella patens (Phypa) and Selaginella moellendorffii (Selmo); post-duplication species: Pinus taeda (Pinta1 & Pinta2) and Populus trichocarpa (Poptr1 & Poptr3). Together, the results indicate that quinate biosynthetic activity was gained prior to duplication and remained low until it became beneficial and favored by selection. The optimization of quinate biosynthetic activity was at the expense of losing some primary shikimate biosynthetic function creating a pleiotropic conflict. This was then resolved by gene duplication and further specialization leading to genes encoding specialized enzymes (either SDH or QDH). Diversification of the DQD/SDH/QDH superfamily likely occurred through sub-functionalization via a mechanism described as “Escape from Adaptive Conflict.” / Graduate / 0307 / guojia@uvic.ca

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (p. 119-126).

The RNA interference (RNAi) mechanism is a conserved system of nucleic acid manipulation, based on the interaction between small RNA guide molecules and Argonaute effector proteins. RNAi pathways are found in the vast majority of eukaryotes, and have diversified into a broad array of functions including gene regulation, antiviral immunity and transposable element (TE) suppression. Many of these functional innovations coincide with duplication of Argonaute genes, suggesting that gene duplication may be a key driving force in the diversification of RNAi. However, few studies have explicitly investigated Argonaute evolution after duplication. In this thesis, I focused on the impact of gene duplication on the evolution of Argonaute genes. Argonaute genes in different species exhibit a broad array of functions; however, most of our knowledge of Argonaute function in the arthropods is based on studies in D. melanogaster. To compare the rate of duplication and its evolutionary effect between different Argonaute subclades, I quantified gene turnover rates and evolutionary rate change in Argonaute genes from 86 Dipteran species (Chapter 2). I find that duplication rate varies widely between subclades and lineages, and that duplication drives an increase in evolutionary rate, suggesting that functional divergence after Argonaute duplication is prevalent throughout the Diptera. In the obscura group of Drosophila I identified a series of recent duplications of Argonaute2 (Ago2), which has antiviral and anti-TE functions in D. melanogaster. To quantify the extent of functional divergence between these paralogues, I measured the expression of paralogues from three species (D. subobscura, D. obscura and D. pseudoobscura), in different tissues and under viral challenge (Chapter 3). I find that the majority of Ago2 paralogues have specialised to a derived testis-specific role, potentially to suppress TE activity or meiotic drive. While CRISPR-Cas9 mediated knockout of these genes ultimately proved unsuccessful (Chapter 5), the selective importance of their derived function is suggested by its multiple independent origins. Functional novelty, as appears to have evolved in the obscura group Ago2 paralogues, is often driven by strong selection. To quantify the evolutionary rate and positive selection on these paralogues, I gathered intraspecies polymorphism data for all paralogues in D. subobscura, D. obscura and D. pseudoobscura, combining this with publicly-available population genomic data for D. pseudoobscura (Chapter 4). I find that the majority of paralogues in all species have extremely low diversity, indicative of recent selection, and identify recent selective sweeps on three paralogues in D. pseudoobscura. This suggests that the majority of Ago2 paralogues in the obscura group are evolving under strong positive selection. In this thesis I have aimed to quantify the effect of gene duplication on Argonaute evolution. I find that Argonaute genes duplicate frequently in some lineages, resulting in the evolution of derived functions that may be driven by positive selection. This suggests that functional diversification is prevalent in eukaryotic RNAi, and is likely to coincide with expansion of the Argonaute gene family.

No description available.

The shikimate pathway links carbohydrate metabolism to the biosynthesis of the aromatic amino acids and an enormous variety of aromatic compounds with essential functions in all kingdoms of life. Aromatic compounds derived from the plant shikimate pathway have substantial biotechnological value and many are essential to the diet of metazoans whose genomes do not encode shikimate pathway enzymes. Despite its importance to the physiology of plants and human health the regulatory mechanisms of the plant shikimate pathway are not well understood. Shikimate kinase (SK) genes encode an intermediate step in the shikimate pathway and were previously implicated in regulation of the plant shikimate pathway. The distribution of SK genes in higher plants was resolved using phylogenetic and biochemical methods. The two SK isoforms of Arabidopsis thaliana, AtSK1 and AtSK2, were functionally characterized. AtSK1 expression is induced by heat stress and the recombinant enzyme was shown to form a homodimer which is important for maintaining the stability and activity of the enzyme at elevated temperatures. The crystal structure of AtSK2, the first reported plant SK structure, identified structural features unique to plant SKs which may perform important regulatory functions. The resolution of bona fide SKs in higher plants led to the discovery of two novel neofunctionalized homologs - Shikimate Kinase-Like 1 (SKL1) and SKL2. These novel genes evolved from SK gene duplicates over 400 million years ago and are found in all major extant angiosperm lineages, suggesting they were important in the development of biological properties required by land plants. The description of albino and variegated skl1 mutants in Arabidopsis thaliana implicate the SKL1 gene product as an important regulator of chloroplast biogenesis. Functional assays were attempted to determine the biochemical function of SKL1 and recombinant constructs of the Arabidopsis thaliana SKL1 protein were crystallized towards structure determination. The results of this thesis further our understanding of the organization and regulation of the plant shikimate pathway. Furthermore, the discovery of SKL1 may yield important insights into chloroplast biogenesis and function. The evolution of the plant SK superfamily highlights the utility of SKs as scaffolds for functional innovation.

The shikimate pathway links carbohydrate metabolism to the biosynthesis of the aromatic amino acids and an enormous variety of aromatic compounds with essential functions in all kingdoms of life. Aromatic compounds derived from the plant shikimate pathway have substantial biotechnological value and many are essential to the diet of metazoans whose genomes do not encode shikimate pathway enzymes. Despite its importance to the physiology of plants and human health the regulatory mechanisms of the plant shikimate pathway are not well understood. Shikimate kinase (SK) genes encode an intermediate step in the shikimate pathway and were previously implicated in regulation of the plant shikimate pathway. The distribution of SK genes in higher plants was resolved using phylogenetic and biochemical methods. The two SK isoforms of Arabidopsis thaliana, AtSK1 and AtSK2, were functionally characterized. AtSK1 expression is induced by heat stress and the recombinant enzyme was shown to form a homodimer which is important for maintaining the stability and activity of the enzyme at elevated temperatures. The crystal structure of AtSK2, the first reported plant SK structure, identified structural features unique to plant SKs which may perform important regulatory functions. The resolution of bona fide SKs in higher plants led to the discovery of two novel neofunctionalized homologs - Shikimate Kinase-Like 1 (SKL1) and SKL2. These novel genes evolved from SK gene duplicates over 400 million years ago and are found in all major extant angiosperm lineages, suggesting they were important in the development of biological properties required by land plants. The description of albino and variegated skl1 mutants in Arabidopsis thaliana implicate the SKL1 gene product as an important regulator of chloroplast biogenesis. Functional assays were attempted to determine the biochemical function of SKL1 and recombinant constructs of the Arabidopsis thaliana SKL1 protein were crystallized towards structure determination. The results of this thesis further our understanding of the organization and regulation of the plant shikimate pathway. Furthermore, the discovery of SKL1 may yield important insights into chloroplast biogenesis and function. The evolution of the plant SK superfamily highlights the utility of SKs as scaffolds for functional innovation.

<p>Gene duplication is an important evolutionary tool for fostering diversification and expanding gene families. However, while this concept is well understood and accepted in a theoretical capacity, the particular changes that lead to the functional diversification of gene duplicates are less well understood and documented. Additionally, little work has been done to understand how functions are gained or lost, which leads to the diversification of orthologous genes. The Sir2 family of NAD+-dependent deacetylases is an excellent gene family to study questions of duplication and diversification as it is ubiquitous throughout all kingdoms of life, and it has expanded through a number of gene duplications so that while most bacteria have a single sirtuin/species, mammals have seven sirtuins/species. Sirtuins also have a wide array of biological functions and targets, but some of these functions are conserved in eukaryotes.</p><p>In this study, Sir2 is used to investigate the principles behind gene duplication and functional diversification in a molecular context. Sir2 function is studied in multiple species of budding yeast, the model organism Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida lusitaniae using a combination of genetic, biochemical, and high-throughput methods. Sir2 and its paralog Hst1 from S. cerevisiae were used with their non-duplicated ortholog Sir2 from K. lactis to examine the type of molecular changes that occur after gene duplication and lead to subfunctionalization. Then Sir2 from the more divergent C. lusitaniae was used to study how functions are gained or lost.</p><p>To study the molecular mechanism of subfunctionalization in the duplicated deacetylases ScSir2 and ScHst1 with the non-duplicated KlSir2 used as a proxy for the ancestral state, we hypothesized that the basis for subfunctionalization in this case was in the interaction domains. ScSir2 and ScHst1 act in distinct complexes that target them to the genomic loci they regulate. KlSir2 interacts with the same complexes as both ScSir2 and ScHst1. Therefore, we first identified the minimal regions of ScSir2 and ScHst1 necessary for each to interact with its respective complex. Then we identified mutations in those interaction domains that eliminated those interactions. Those mutations were then tested in KlSir2 for their impact on its interactions with the same complexes. We found that the interaction domains in ScSir2 and ScHst1 were conserved in KlSir2, demonstrating that Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.</p><p>To understand better how Sir2 has gained or lost functions, we studied the Sir2 function in C. lusitaniae to serve as an intermediate between the fission yeast Schizosaccharomyces pombe Sir2, whose functions have been identified, and K. lactis and S. cerevisiae. Interestingly, ClSir2 was localized to the rDNA, which is also the case in S. pombe, K. lactis, and S. cerevisiae, but not at the telomeres, which is another locus at which Sir2 is found in other yeast. Additionally, ClSir2 was not found to have an impact on gene expression unlike Sir2 and Hst1 in other yeast where they repress transcription.</p> / Dissertation

The substantial difference in ionic concentration and osmotic pressure between marine and freshwater environments creates a barrier to dispersal that relatively few metazoan lineages have been able to cross during the evolution of life on earth. Only about half of animal phyla have representatives in both marine and freshwater environments. Even within the phyla that contain freshwater species there are often large clades that continue to be exclusively marine. Interestingly, though, among some of the clades with freshwater species, this transition has occurred repeated. In order to begin to better understand the mechanisms that have allowed some marine lineages to colonize freshwater environments, I investigated the role of gene duplications in this process. First, using published annelid genomes I compared the gene copy number of the Na+/K+-ATPase alpha subunit gene family, the plasma membrane Ca2+ ATPase (PMCA) gene family, and the sarcoplasmic reticulum Ca2+ (SERCA) gene family between marine and freshwater species. I also used gene tree/species tree reconciliation to infer the time of those duplication events. There was a burst of duplications of the Na+/K+-ATPase alpha subunit gene that coincides with the colonization of freshwater habitats by annelids. The evidence of such a burst of duplications for the PMCA or SERCA gene families is inconclusive. Next, in order to increase the sample size and look for more gene families that were involved in the transition to freshwater habitats I downloaded 11 genomes from spiralian animals. I looked for specific gene families that showed a significant increase in size in freshwater species compared to marine species and identified the Na+/K+-ATPase alpha subunit gene family among others. I also used GO enrichment analysis to determine which GO terms were overrepresented in gene families that expanded along freshwater lineages and found terms related to ion transfer to be most common. Finally, I examined available mollusk genomes to compare size of the gene families of interest from the spiralian analyses between marine and freshwater mollusk species. I again found the Na+/K+-ATPase alpha subunit gene family to show a significant increase in size in the freshwater species. How marine animals were able to colonize freshwater habitats is one of the great questions in metazoan evolution and this work represents an important early step in understand this process.