Gene & Genome Duplication in Acanthamoeba Polyphaga Mimivirus

Gene duplication is key to molecular evolution in all three domains of life and may be the first step in the emergence of new gene function. It is a well recognized feature in large DNA viruses, but has not been studied extensively in the largest known virus to date, the recently discovered Acanthamoeba Polyphaga Mimivirus. Here we present a systematic analysis of gene and genome duplication events in the Mimivirus genome. We find that one third of the Mimivirus genes are related to at least one other gene in the Mimivirus genome, either through a large segmental genome duplication event that occurred in the more remote past, either through more recent gene duplication events, which often occur in tandem. This shows that gene and genome duplication played a major role in shaping the Mimivirus genome. Using multiple alignments together with remote homology detection methods based on Hidden Markov Model comparison, we assign putative functions to some of the paralogous gene families. We suggest that a large part of the duplicated Mimivirus gene families are likely to interfere with important host cell processes, such as transcription control, protein degradation, and cell regulatory processes. Our findings support the view that large DNA viruses are complex evolving organisms, possibly deeply rooted within the tree of life, and oppose the paradigm that viral evolution is dominated by lateral gene acquisition, at least in what concerns large DNA viruses

Tandemly Arrayed Genes in Vertebrate Genomes

Tandemly arrayed genes (TAGs) are duplicated genes that are linked as neighbors on a chromosome, many of which have important physiological and biochemical functions. Here we performed a survey of these genes in 11 available vertebrate genomes. TAGs account for an average of about 14% of all genes in these vertebrate genomes, and about 25% of all duplications. The majority of TAGs (72–94%) have parallel transcription orientation (i.e., they are encoded on the same strand) in contrast to the genome, which has about 50% of its genes in parallel transcription orientation. The majority of tandem arrays have only two members. In all species, the proportion of genes that belong to TAGs tends to be higher in large gene families than in small ones; together with our recent finding that tandem duplication played a more important role than retroposition in large families, this fact suggests that among all types of duplication mechanisms, tandem duplication is the predominant mechanism of duplication, especially in large families. Finally, several species have a higher proportion of large tandem arrays that are species-specific than random expectation

Cellular and Synaptic Organization of the Human Olfactory Bulb

The distribution of cell types and synapses is well characterized in the rodent olfactory bulb (OB), and from that plausible models of odor processing have been constructed. Individual olfactory sensory neurons (OSNs) express only 1 of ~1000 odorant receptors (ORs) and send their axons to specific synaptic targets in the OB glomerular neuropil. Each glomerulus is innervated exclusively by OSN axons expressing the same OR. The distribution of these glomeruli is conserved across animals, as is the numerical relationship between number of expressed ORs and number of glomeruli in the OB. Our objective is to extend such results to the level of the human OB to determine how its cellular and synaptic organization, and more specifically how the number and distribution of its glomeruli, compare to what has been elucidated in mice. As there are ~2,000 glomeruli for ~1,000 ORs in mice, we predicted ~700 glomeruli in humans based on the ~350 intact OR genes identified in the human through genomic studies. Using immunohistochemistry, the organization of cells and synapses in human OBs was evaluated and quantified. While the laminar structure of the OB is broadly conserved between species, in the human OB the laminar organization as well as additional structural features suggest a less rigorously organized OB than in rodents, perhaps suggesting that odor processing in the human OB may be less efficient than in mice. Of particular note, the total number of glomeruli in the human OB differs significantly from predicted and demonstrates a high degree of variability amongst specimens, thus far ranging from approximately 3000 - 9000/OB. These results indicate that the principles of OR-homotypic axon convergence developed from mouse studies may not be readily applicable to the human, and that central processing of odor signals in the human may differ from those characterized in the mouse

Axiales Patterning und seine Steuerung in der Embryogenese eines basalen Metazoen, Hydractinia echinata

In dieser Arbeit konnten erstmals Transgene von Hydractinia echinata unter der Kontrolle der nativen regulatorischen Regionen von unterschiedlichen Genen erzeugt und Expressions-studien mithilfe des Reportergen eGFP durchgeführt werden. Es wurden zwei Arten von Konstrukten erzeugt: a) solche unter möglichst ubiquitär aktiven Promotoren und b) solche, die den Promotor eines funktionell zu untersuchenden Gens erhielten. Im Genom von Hydractinia existieren multiple Gene für Aktin, die trotz der Identität der Proteinstruktur (> 99%) unterschiedliche 5`- und 3`regulatorische Regionen aufweisen. Die Aktivität der regulatorischen Regionen der drei zytoplasmatischen Beta-Aktingene HeActI, HeActII und HeActIII konnten durch erfolgreiche Expression von eGFP gezeigt werden. Die Expressionsmuster belegen, dass diese drei ß-Aktine unterschiedlich in Hydractinia exprimiert werden. Ausschließlich HeActII zeigte eine ubiquitäre Expression. Als ein weiteres ubiquitär exprimiertes Gen wurde HeEF1alpha verwendet. Die vorliegende Studie hat gezeigt, dass in Hydractinia Kontrollgene konserviert sind, die in den Hauptsignalwegen der höheren Metazoen Entwicklung kontrollieren. Deren mögliche Funktion in diesem basalen Metazoen wurde mithilfe von Funktionsanalysen durch den Einsatz transgener Techniken nachgegangen. Damit gelang im Rahmen dieser Arbeit ein Einstieg in die Funktionsanalyse von Proteinen in Hydractinia. Dafür wurden die regulatorischen Regionen des ubiquitär exprimierten Gens HeActII zur Entwicklung eines Expressionskonstruktes ausgewählt, um die mögliche Funktion eines Proteins durch ektopische Expression in transgenen Hydractinia zu untersuchen. Manche der standardmäßig im Labor eingesetzten transgenen Techniken zur Funktionsanalsye konnten in Hydractinia nicht verwendet werden. Zwei experimentelle Ansätze mittels Überexpression der analysierten Gene Heß-Cat bzw. HeGsc führten zu Gestaltbildungseffekten während der larvalen Entwicklung und in post-metamorphen Stadien von Hydractinia. Mithilfe transgener Technik gelang es erstmals, das Nervensystem von Hydractinia echinata in vivo darzustellen. Es wurden unterschiedliche Nervenzelltypen durch eGFP-Markierung detektierbar. Ein Teil dieser Zellen war bereits aus ICC-Studien bekannt. Hinzu kamen Nervenzellen, die zuvor noch nie beobachtet wurden. Darunter befanden sich sog. giant bipolar neurons mit einer Länge von mindestens 250μm, die im Stolon und auch in der Planulalarve nachgewiesen wurden. Die Ergebnisse weisen auf eine weit grössere Komplexität des Nervensystems dieses einfach gebauten Metazoen hin, als bisher bekannt. Des Weiteren zeigte sich, dass das Nervensystem nicht nur longitudinal, sondern auch transversal zur anterior-posterioren Körperachse der Larve vernetzt ist. Diese neuen Einblicke in das Nervensystem wurden durch Expressionsstudien unter der Kontrolle der genregulatorischen Regionen der beiden ubiquitär exprimierten housekeeping genes HeActII und HeEF1α, sowie durch die des neuralspezifischen Gens HeELAV möglich. Die Expressionsstudien zeigten, dass sich HeELAV ein hochgeeignetes neuralspezifischers Markergen ist, um in vivo die Entwicklung des Nervensystems von Hydractinia zu untersuchen

Mechanisms of change in protein architecture

Proteins are the basic building blocks and functional units in all living organisms. Moreover, differences between species can frequently be explained with differences in their protein complements. Importantly, proteins are often composed of segments, i.e. domains that have a certain level of evolutionary, structural and/or functional independence. The majority of proteins in nature contain two or more domains, and an individual domain can often occur in combinations with different domain partners. In the first part of my thesis, I traced the history of animal gene families and the proteins these genes encode. By this means, I was able to infer events where changes in protein domain architectures took place. This showed that both insertions and deletions of single copy domains preferentially occur at protein termini, but also that changes are more likely to occur after gene duplication than organism speciation. Finally, domains that were most frequently gained were the ones that are related to an increase in organismal complexity, thus underlining the important role of domain shuffling in animal evolution. In the second part of my thesis, I focused on a set of high confidence domain gain events and investigated the evidence for molecular mechanisms that caused these domain gains. In agreement with observations from the first part - that changes preferentially occur at the termini - I have found that the strongest contribution to gains of novel domains in proteins comes from gene fusion through the joining of exons from adjacent genes into a novel gene unit. Two other mechanisms that have been suggested to play a major role in the evolution of animal proteins, retroposition and middle insertions through intronic recombination, have a smaller role in comparison to gene fusions. Since the majority of these domain gains are again observed after gene duplication, this suggests a powerful mechanism for neofunctionalization after gene duplication. iii Finally, in the last part of my thesis, I address a mechanism that increases the number and variety of proteins in an organism – alternative splicing. In particular, I investigate the functional consequences of tissue-specific alternative splicing events. I found that tissue-specific splicing tends to affect exons that encode protein regions without defined secondary or tertiary structure. Importantly, it is known that these disordered regions frequently play a role in protein interactions. In agreement with this, I observed significant enrichment of tissue-specifically encoded protein segments in disordered binding peptides and posttranslationally modified sites. A possible result of the finely regulated alternative splicing of these segments is a tissue-specific rewiring of protein network. In conclusion, both alternative splicing and domain shuffling can increase proteome diversity. However, a protein with a new function can often directly or indirectly shape the functions of other proteins in its environment

Evolution of Tandemly Repeated Sequences

Despite being found in all presently sequenced genomes, the evolution of tandemly repeated sequences has only just begun to be understood. We can represent the duplication history of tandemly repeated sequences with duplication trees. Most phylogenetic techniques need to be modified to be used on duplication trees. Due to gene loss, it is not always possible to reconstruct the duplication history of a tandemly repeated sequence. This thesis addresses this problem by providing a polynomial-time locally optimal algorithm to reconstruct the duplication history of a tandemly repeated sequence in the presence of gene loss. Supertree methods cannot be directly applied to duplication trees. A polynomial-time algorithm that takes a forest of ordered phylogenies and looks for a super duplication tree is presented. If such a super duplication tree is found then the algorithm constructs the super duplication tree. However, the algorithm does not always find a super duplication tree when one exists. The SPR topological rearrangement in its current form cannot be used on duplication trees. The necessary modifications are made to an agreement forest so that the SPR operation can be used on duplication trees. This operation is called the duplication rooted subtree prune and regraft operation (DrSPR). The size of the DrSPR neighbourhood is calculated for simple duplication trees and the tree shapes that maximize and minimize this are given

Epistasis in wheat breeding

Doctor of PhilosophyGenetics Interdepartmental ProgramAllan K. FritzThe core objective of wheat breeding is to develop superior varieties to the target population of environments. As most agronomic traits are quantitative, the underlying genetic architecture is assumed to be additive, implying that the superiority of parental lines is transmitted to the offspring. However, often rather than rarely, wide crosses between elite genotypes yield poor breeding populations, indicating that additive might not be the major type of gene action. The inbreeding nature of wheat suggests that epistasis could be the underlying genetic architecture. The work presented here describes conventional and innovative methods to test the existence and extent of epistasis in wheat breeding. The first chapter presents a comprehensive literature review that discusses the theoretical framework for the creation of epistasis from gene duplication events and the expansion of this construct to the population levels. Then, the history of wheat is utilized to discuss the evolution of epistasis into a complex genetic system and the strategies utilized by breeding programs to manage this complexity and attain satisfactory levels of genetic gain. The second chapter utilizes an innovative approach to test the existence of sign epistasis in wheat breeding. Sign epistasis occurs when the effect of an allele can be beneficial or detrimental upon variation in the genetic background. Through the coupling of interchromosomal linkage disequilibrium analysis with the calculation of allele frequencies, 19 candidate interactions of sign epistasis were identified. Although the validation analyses attributed to random genetic drift the sign epistasis patterns observed in 11 candidate interactions, eight interactions presented strong evidence for sign epistasis and the main hypothesis could not be refuted. These findings imply that the effect of alleles can vary from positive to negative depending on the genetic background and explain the often poor combining ability of high performing lines. The third chapter explores the genetic architecture of wheat grain yield by modeling additive and additive-by-additive epistatic effects, using data from 3740 experimental lines. Modeling epistasis in whole genome or sub genome models marginally improved prediction accuracy in genomic selection models and resulted in non-orthogonal partition of genetic variance components. The estimation of sub genome additive effects showed that the best lines did not have the greatest additive effects in more than one sub genome, suggesting the possibility of making targeted crosses that combine genotypes with the highest additive effects in each subgenome