Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 98-112). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

The C₄ photosynthetic pathway acts to concentrate CO₂ around the enzyme Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), ensuring that it catalyses a carboxylation rather than oxygenation reaction, which in turn suppresses photorespiration. In nearly all cases C₄ photosynthesis is underpinned by characteristic Kranz anatomy, with concentric wreaths of bundle sheath (BS) and mesophyll (M) cells surrounding closely spaced veins. The increased yields associated with the C₄ pathway have lead to the suggestion that C₃ crops such as rice should be engineered to undertake C₄ photosynthesis, however, this goal is currently held back by a lack of understanding about how the development of Kranz anatomy is regulated. Recently, a number of candidate Kranz regulators have been identified in an RNA-seq study that compared leaf development in maize foliar (Kranz) and husk (non-Kranz) leaves. However, this study did not consider the impact of a recent whole genome duplication in the maize lineage on the gene expression patterns analysed. Therefore, in this thesis maize homeolog gene-pair divergence during early leaf development was assessed. This revealed that expression divergence of homeolog gene-pairs is a significant evolutionary phenomenon. Functional validation of a subset of Kranz candidates revealed that a Zmscr1-1; Zmscr1h-1 double mutant exhibited defects in Kranz patterning, including increased formation of extra BS cells and veins with no separating M cells. Furthermore, Zmnkd1; Zmnkd2 double mutants exhibited a subtle increase in extra BS cell formation. Taken together, this indicates that both ZmSCR1/ZmSCR1h and ZmNKD1/ZmNKD2 function redundantly during Kranz development. No evidence was obtained that two additional genes, ZmSHR2 and ZmRVN1, play a role in Kranz development, and expression of candidate Kranz regulators in rice did not alter leaf anatomy. Together, this work has confirmed roles for a number of genes in Kranz regulation, and has provided insight into the complex regulation underpinning Kranz development in maize.

For many years, researchers have studied the social lives of bacteria to understand intra- and inter-species interactions. Cell-cell communication, also known as quorum sensing (QS), is used by bacteria to coordinate their behaviour in response to environmental conditions. The QS system in Streptococcus species is well known to regulate competence. Studies show that Streptococcus pneumoniae has two homologous QS systems: 1) the competence (Com) system that regulates competence; and 2) a bacteriocin-like peptide (Blp) system that regulates the production of bacteriocins. Both functions are widespread in the genus. In S. pneumoniae, the Blp QS system shares a common ancestor and has similar features to the Com QS system. However, the evolutionary relationship between these QS systems remains obscure. SUCRE methodology was developed to identify the QS homologous genes in the streptococcal species. SUCRE uses four complementary approaches: homology search, putative gene finding, regulon construction, and evolutionary analysis. The performance of SUCRE was assessed in comparison with other orthology detection methods. SUCRE is precise in identifying the QS homologous genes and has similar performance to OrthoMCL. The QS system structures are found to be conserved across the streptococcal species. A streptococcal species phylogeny was constructed from the ribosomal and tRNA synthetase gene families. Using the QS genes identified from SUCRE and the streptococcal species phylogeny, the study infers the evolution of the QS systems in Streptococcus species. The study shows that the QS systems evolved as a regulon unit. The paralogous relationship between each of the QS systems suggests that duplication has a huge influence on functional divergence of the QS systems in the genus. Although, horizontal gene transfer (HGT) is commonly found in bacteria, little evidence is found to support that the effect of HGT on the functional divergence of the QS systems in this genus. However, the QS regulon genes of the same QS system are found to be non- vertically transferred across species that signifies that the HGT event promotes the sequence variation between these genes.

Background: Duplicated genes can indefinately persist in genomes if either both copies retain the original function due to dosage benefit (gene conservation), or one of the copies assumes a novel function (neofunctionalization), or both copies become required to perform the function previously accomplished by a single copy (subfunctionalization), or through a combination of these mechanisms. Different models of duplication retention imply different predictions about substitution rates in the coding portion of paralogs and about asymmetry of these rates. Results: We analyse sequence evolution asymmetry in paralogs present in 12 Drosophila genomes using the nearest non-duplicated orthologous outgroup as a reference. Those paralogs present in D. melanogaster are analysed in conjunction with the asymmetry of expression rate and ubiquity and of segregating non-synonymous polymorphisms in the same paralogs. Paralogs accumulate substitutions, on average, faster than their nearest singleton orthologs. The distribution of paralogs' substitution rate asymmetry is overdispersed relative to that of orthologous clades, containing disproportionally more unusually symmetric and unusually asymmetric clades. We show that paralogs are more asymmetric in: a) clades orthologous to highly constrained singleton genes; b) genes with high expression level; c) genes with ubiquitous expression and d) non-tandem duplications. We further demonstrate that, in each asymmetrically evolving pair of paralogs, the faster evolving member of the pair tends to have lower average expression rate, lower expression uniformity and higher frequency of non-synonymous SNPs than its slower evolving counterpart. Conclusions: Our findings are consistent with the hypothesis that many duplications in Drosophila are retained despite stabilising selection being more relaxed in one of the paralogs than in the other, suggesting a widespread unfinished pseudogenization. This phenomenon is likely to make detection of neo- and subfunctionalization signatures difficult, as these models of duplication retention also predict asymmetries in substitution rates and expression profiles. Reviewers: This article has been reviewed by Dr. Jia Zeng (nominated by Dr. I. King Jordan), Dr. Fyodor Kondrashov and Dr. Yuri Wolf.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20420号 / 農博第2205号 / 新制||農||1047(附属図書館) / 学位論文||H29||N5041(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 田尾 龍太郎, 教授 奥本 裕, 教授 寺内 良平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The seed storage globulins found among virtually all spermatophytes comprise a multi-gene family of proteins with ancient evolutionary origins. The two main groups of storage globulins include the legumins (11S) and vicilins (7S), both of which play a main role in protein deposition and storage in the seed endosperm. Composed of two cupin domains (bicupin), these proteins have been recently noted not only for their close structural relationships among the two subfamilies (7S and 11S) but also for their similarity to other proteins such as germin-like proteins (GLP's), bacterial oxalate decarboxylases, and other cupin containing proteins. Previous studies have investigated the evolutionary relationships among the legumin and vicilin groups, as well as their presumed evolutionary link to other cupin containing proteins; however these have each come short of any comprehensive resolved evolutionary history of the globulin family. This study focuses first on resolving the relationships among the cupin super-family in relation to the storage globulins, as well as the GLP's, which have been postulated to be the single domain ancestors of the bicupin storage globulins. Nucleotide coding sequences for both N-terminus and C-terminus cupin domains of the storage globulins, including conserved non-cupin domain helical repeats and inter-domain spacers were aligned to a comparably sized set of single cupin coding sequences (CDS). The phylogenetic relationships among the two globulin domains as well as the single cupin genes were elucidated using Bayesian inference of tree likelihoods. Further phylogenetic analysis was performed on the complete CDS's for all storage globulin sequences in the study, using an appropriate out-group of similar overall domain architecture determined by the overall topology of the cupin super-family. This globulin muti-gene tree was used, along with an alignment corresponding to structurally resolved portions of the mature globulin peptides, to perform an analysis of patterns of selection among the various lineages of cupin-containing globulins. The results of these analyses provide evidence for a common origin of all cupin containing genes. The GLP and storage globulin domains do not appear to be immediate ancestors of one another, but are grouped with the fungal spherulins as well, suggesting that the single cupin genes which gave rise to these groups had already diverged prior to the rise of land plants. The storage globulin gene tree provides evidence supporting the notion that true legumins and vicilins were recruited as seed storage proteins independent of one another, after their divergence. This is evidenced by the fact that they comprise two separate groups each with basal non-storage 11S/7S-like proteins. Additional insight into the differentiating selection pressures provides a clearer picture of how similar suites of physicochemical properties came under selection after the recruitment of the 11S and 7S families as seed specific proteins. Regions under strong destabilizing selection correspond to regions known to be of importance in the overall structure of storage globulins. Strong destabilizing selection at the pore of the globulin subunit suggests that this region may have undergone more functional diversification than previously thought to have occurred among the legumins and vicilins.

Gene duplications can facilitate genetic innovation, reduce pleiotropy and catalyze reproductive incompatibilities and speciation. Therefore, the molecular and transcriptional fate of duplicate genes plays an important role in the evolutionary trajectory of entire genomes and transcriptomes. Using the polyploid African clawed frog Xenopus, I have investigated mechanisms that promote the retained expression of duplicate genes (paralogs) after whole genome duplication. The studies herein estimated molecular evolution and characterized expression divergence of thousands of duplicate genes and a singleton ortholog from a diploid outgroup. In this thesis, I have discussed the multiple mechanisms for the retention of duplicate genes in a polyploid genome and examined the potential effects that gene characteristics before duplication have on the odds of duplicate gene persistence. I have also explored the use of microarrays for comparative transcriptomics between duplicate genes, and between diverged genomes. The main objectives of my thesis were to better understand the genetic mechanisms that promote the retained expression of gene duplicates. My research utilized the duplicated genome from the allopolyploid clawed frog Xenopus. Genome duplication in clawed frogs offers a compelling opportunity to study factors that influence the genetic fates of gene duplicates because many paralogs in these frogs are of the same age, permitting one to control for the influence of time when evaluating the impact of duplication. My work has major impacts on several biological fronts including evolutionary genomics and comparative transcriptomics, and also on technical aspects of using microarrays. I have provided among the most comprehensive studies of its kind, in terms of examining molecular and regulatory aspects of thousands of expressed duplicates of the same age, and exploring various alternative hypotheses to explain how these genes are retained. / Thesis / Doctor of Philosophy (PhD)

No description available.

The variation of genome content and structure across the tree of life is astounding and can provide clues to understand the process of evolution. Overall, this helps us understand the history of life and how organisms have fundamentally changed and adapted to their environments. Gene duplication is an important mechanism for molecular evolution because it provides opportunity for functional novelty and molecular innovation. Gene duplication creates new functional gene copies with different selective pressures that allow them to take on new or specialized functions. Throughout this work, I explored the interplay between genetic changes, molecular phenotype, and the selection of duplicate gene copies. I particularly focused on the genetic opportunity, consequences, and selective pressures of the mechanisms for short-term and long-term duplicate copy retention. I modeled the stochastic processes of mutation and selection and their effect on duplicate gene copy retention. Specifically, I modeled the interplay between subfunctionalization and dosage balance and found that selection may cause genes that are sensitive to dosage balance effects to experience delayed subfunctionalization, but ultimately lead to higher levels of subfunctionalization. These findings suggest that subfunctionalization may not occur as a purely neutral process. Next, I used survival analysis methods to model patterns of duplicate gene retention in genomes experiencing consecutive whole genome duplication events. I modeled three hypotheses to explain patterns of duplicate gene retention including the Independence Hypothesis, the Gene Duplicability Hypothesis, and a novel Mutational Opportunity Hypothesis. Under the Gene Duplicability and Mutational Opportunity hypotheses, the expected patterns of duplicate gene retention after consecutive whole genome duplication events are greatly affected by the ages of the whole genome duplication events and the functional properties of the genomic content that influence opportunity and selection. Additionally, I describe how statistical model testing techniques can be applied to investigate which hypothesis is consistent with patterns of retention in real-world phylogenetic datasets. I used these described techniques to explore the hypotheses’ parameter space consistent with a modest dataset of fish and plant lineages. These results suggest that a gene duplicate’s retention after whole genome duplication events may be influenced by its functional properties. Key findings underscore the multifaceted nature of duplicate gene retention, influenced by a myriad of factors including genetic opportunity, selective pressures, and evolutionary context. By dissecting the underlying mechanisms driving duplicate gene retention, this dissertation advances our understanding of the evolutionary dynamics shaping genome evolution and functional diversity across diverse biological systems. / Biology

Background: The Atlantic salmon, Salmo salar L., is a prominent member of the Salmonidae family, and has been the focus of intense research because of its environmental and economic significance as an iconic sporting species and its global importance as an aquaculture species. Furthermore, salmonids constitute ideal organisms for the study of evolution by gene duplication as they are pseudotetraploid descendants of a common ancestor whose genome was duplicated some 25 to 100 million years ago. Whole-genome duplication is considered a major evolutionary force capable of creating vast amounts of new genetic material for evolution to act upon, promoting speciation by acquisition of new traits. Recently, large-scale comparison of paralogous genes in Atlantic salmon suggested that asymmetrical selection was acting on a significant proportion of them. However, to elucidate the physiological consequences of gene and genome duplications, studies integrating molecular evolution and functional biology are crucial. To this end, sequence and molecular analyses were performed on duplicated Elovl5 fatty-acyl elongases of Atlantic salmon, as they are responsible for a rate-limiting reaction in the elongation process of long-chain polyunsaturated fatty acids (LC-PUFA), critical components of all vertebrates. The aim of the research presented here was to investigate the role of gene duplication as an evolutionary process capable of creating genetic novelty, and to identify the potential ecological and physiological implications. Results: Linkage analyses indicated that both fatty-acyl elongases segregated independently and located elovl5 duplicates on different linkage groups. Genetic mapping using microsatellites identified in each elovl5 locus assigned elovl5a and elovl5b to chromosomes ssa28 and ssa13, respectively. In silico sequence analysis and selection tests indicated that both salmon Elovl5 proteins were subject to purifying selection, in agreement with previous results showing indistinguishable substrate specificities. Gene expression and promoter analysis indicated that Elovl5 duplicates differed in response to dietary lipids and tissue expression profile. Lipid biosynthesis and metabolic gene expression profiling performed in Atlantic salmon SHK-1 cells, suggested that the control of lipid homeostasis in fish is similar to that described in higher vertebrates, and revealed the particular importance of Lxr and Srebp transcription factors (TFs) in the regulation of LC-PUFA biosynthetic enzymes. Sequence comparison of upstream promoter regions of elovl5 genes showed intense differences between duplicates. Promoter functional analysis by co-transfection and transcription factor transactivation showed that both elovl5 duplicates were upregulated by Srebp overexpression. However, elovl5b exhibited a higher response and its promoter contained a duplication of a region containing response elements for Srebp and NF-Y cofactors. Furthermore, these studies indicated an Lxr/Rxr dependant response of elovl5a, which was not observed in elovl5b. Analysis of the genomic sequences of elovl5 duplicates by comparison to various sequence databases showed an asymmetrical distribution of transposable elements (TEs) in both introns and promoter regions. Further comparison to introns of the single elovl5 gene in pike indicated much higher TE distribution in salmon genes compared to the pike. Conclusions: Although not conclusive, the most parsimonious origin for the salmon elovl5 duplicates is that they are derived from a WGD event. This conclusion is also supported by the close similarity of two elovl5 paralogs in the recently available rainbow trout genome. Regardless of their origin, Atlantic salmon elovl5 genes have been efficiently retained in the genome under strong functional constraints indicating a physiological requirement for both enzymes to be functionally active. In contrast, upstream promoter regions have strongly diverged from one another, indicating a relaxation of purifying selection following the duplication event. This divergence of cis-regulatory regions has resulted in regulatory diversification of the elovl5 duplicates and regulatory neofunctionalisation of elovl5a, which displayed a novel Lxr/Rxr-dependant response not described in sister or other vertebrate lineages. Promoter analysis indicated that the observed elovl5 differential response to dietary variation could be partly attributed to varying transcriptional regulation driven by lipid-modulated TFs. The distribution of TEs in elvol5 genes of Atlantic salmon shows a clear increase in TE mobilisation after the divergence of esocids and salmonids. This must have occurred after the elongase duplication and thus the salmonid WGD event and contributes to the observed regulatory divergence of elovl5 paralogs.