Les eucaryotes sont des organismes chimériques issus de l’endosymbiose entre une archéobactérie et une α-protéobactérie. Au cours de ce processus, ces organismes ont évolué de sorte à obtenir un grand nombre de caractéristiques observées chez les eucaryotes modernes, notamment une mitochondrie, un noyau, un système endomembranaire, un système d’épissage ou encore des chromosomes linéaires terminés par un télomère. Bien que les caractéristiques du dernier ancêtre commun des eucaryotes aient majoritairement été identifié, la suite des évènements évolutifs ayant mené à l’apparition de cet organisme demeure peu compris. Afin de mieux reconstruire cette suite d’évènements, l’analyse des génomes d’organismes basals aux eucaryotes sera nécessaire pour identifier des traces de cette évolution. Ainsi, nous proposons que l’analyse d’une collection de génomes d’eucaryotes « primitifs », les jakobides et malawimonades, des eucaryotes unicellulaires flagellés se nourrissant de bactéries, pourrait permettre une meilleure compréhension de ce processus. De plus, il a été supposé que le génome d’un de ces organismes, Andalucia godoyi, pourrait posséder des chromosomes circulaires, une caractéristique atypique chez les eucaryotes, une caractéristique qui pourra être confirmée par la production d’assemblage génomique de haute contigüité. Afin d’obtenir des assemblages génomiques de haute qualité, les jakobides A. godoyi, Jakoba bahamiensis, Seculamonas ecuadoriensis, Stygiella incarcerata et le malawimonades Malawimonas californiana ont été séquencés par nanopore. Le séquençage nanopore a présenté des résultats mitigés et les organismes J. bahamiensis et M. californiana ont présentés un faible rendement de séquençage, possiblement dû à la contamination par des polysaccharides. Pour les autres organismes, nous avons développé un pipeline d’assemblage utilisant les assembleurs Flye et Shasta qui nous a permis de produire des assemblages génomiques. L’analyse du génome de A. godoyi a permis d’identifier la présence de quatre chromosomes circulaires, possiblement localisés dans le noyau, contenant plusieurs gènes liés au métabolisme, au transport et à la signalisation et qui constituent possiblement un type de chromosome circulaire différent de ceux observés précédemment chez les eucaryotes. Dans l’ensemble, ces travaux ont permis la mise en place d’une collection de génome d’eucaryotes « primitifs » qui pourront être utilisés pour des analyse de génomique comparative afin de mieux comprendre l’évolution des eucaryotes. / Eucaryotes are chimeric organisms that are the product of an endosymbiotic event between an archaebacteria and an α-proteobacteria. During the eukaryogenesis, these organisms have gained many characteristics that defines modern eucaryotes such as a mitochondrion, a nucleus, an endomembrane system, the splicing machinery, and linear chromosome with telomeres. While most characteristics of the last common eukaryote ancestor have mostly been identified, most of the evolutionary process that led to this organism is still unknown. To reconstruct this string of event, we must analyse the genome of “primitive” basal eukaryotes with a slow evolutionary rate and a lifestyle like that of the last common eukaryotes ancestor, and thus are most likely to contain remains of ancestral mechanisms that have been lost in most known eukaryotes. We propose that this analysis of the genome of the jakobids and malawimonads, two groups are free-living flagellate that feeds on bacteria, could provide such clues on the evolution of eukaryotes. Using nanopore sequencing, a collection of high-quality genomes has been built to help in this analysis. Furthermore, it has been supposed that the genome of the jakobid Andalucia godoyi could be composed to both linear and circular chromosomes, a genomic structure that have not been identified in other eukaryotes, which was investigated using the high quality nanopore assembly. To generate a collection of high-quality genome assemblies, we have sequenced the genomes of the jakobids A. godoyi, Jakoba bahamiensis, Seculamonas ecuadoriensis and Stygiella incarcerata as well as the malawimonad Malawimonas californiana by nanopore. While the yields were too low for J. bahamiensis and M. californiana, probably due to a contamination by polysaccharides, we were able to assemble chromosome level genome for A. godoyi and S. incarcerata and high-quality draft genome for S. ecuadoriensis et R. americana. Using this assembly, we were able to identify four circular chromosomes in the genome of A. godoyi. The circular chromosomes are likely to be located in the nucleus and encodes genes with functions related to the metabolism, ions and macromolecules transport as well as signaling. Furthermore, these molecules differ from known circular chromosome in eukaryotes as they are unlikely to be selfish DNA elements, such as known eucaryotes plasmids, or circular by-product of replication identified in other eukaryotes. Overall, this work sets the bases for larger scale comparative genomics of the jakobids and malawimonads, by generating a small collection of genomes that will be used in future studies to better understand the origin of the eukaryotes.

Not much is known about the mechanisms affecting the genetic composition of populations of different species. To investi­gate one of these potential mechanisms, mating behaviour, the fruit fly Drosophila melanogaster, was chosen as an experimen­tal animal. To quantify mating behaviour in easily measurable parameters, it was subdivided into several distinct components; mating activity, mating time, mating competition ability and male mating capacity. As behavioural components to a great extent are influenced by environmental conditions all experiments were performed under controlled temperature and humidity. All components of mating behaviour were estimated by introducing females and males into mating chambers. Mating behaviour seems to be one of the major factors affect­ing the genetic composition of Drosophila melanogaster popula­tions. The experiments performed showed that differences in mating properties led to a substantial sexual selection among the genotypes. This selection was of a stabilizing type with regard to characters associated to bristle number and Y chromo­somal chromatin. This selection situation seems to warrant the retention of intermediate phenotypes in a population and will therefore contribute to the genetic variation present. Differences in mating properties were also shown to be able to maintain a balanced polymorphism for allozyme variants in populations. This maintenance was obtained through different forms of balancing selection as heterozygous superiority in sexual activity and balancing selection between female and male genotypes. Heterozygous superiority or overdominance in fitness always leads to balanced polymorphism through segre­gation of individuals with lowered fitness. The balancing selection between the female and male genotypes is best looked upon as a form of marginal overdominance, conferring the aver­aged highest fitness to the heterozygous genotype, thereby maintaining the polymorphism of the population. / <p>Härtill 5 uppsatser</p> / digitalisering@umu

When a cell divides into two, it is imperative that each resultant daughter receives a full complement of chromosomes; DNA is ultimately responsible for all cellular processes. Cohesion between sister chromatids from the moment of their generation in S phase is central to ensuring the fidelity of chromosome segregation. Smc1 and Smc3 proteins interact with each other via their hinges and with a bridging kleisin subunit via their heads to form the cohesin ring. It is cohesin, through entrapment of sister chromatid within its ring, that confers sister chromatid cohesion. The process of cohesin’s loading onto DNA is poorly understood. While it is thought to depend on ATP hydrolysis, opening of the ring at one of its three interfaces, and the as yet undefined action of the kollerin complex, comprising Scc2 and Scc4 proteins, the sequence of events as they occur are yet to be defined. A recent screen for suppressors of a thermosensitive scc4 allele in budding yeast revealed a mutation within Smc1’s hinge that could bypass the kollerin subunit. Here, the Smc1 suppressor mutation is investigated. Through targeted mutagenesis, the Smc1D588Y mutant identified in the screen and two additional point mutants, Smc1D588F and Smc1D588W, are herein proven able to bypass Scc4 function completely. Thus we provide the strongest evidence to date to suggest that cohesin’s hinge is a critical factor in its loading. Biochemical evidence shows that isolated Smc1 hinge mutants are defective in their binding to Smc3 hinges. This, together with the genetic link made between the hinge and loading complex, suggests that hinge opening might be a requisite for loading. Through mutagenesis of Scc2 and Scc4 we show that the N-terminus of each protein is responsible for their dimerisation. Furthermore, the N- terminus of Scc2 confers no function other than in its binding to Scc4. Finally, we show that Scc4 is required for the enrichment of both Scc2 and cohesin at centromeres, but not at arm loci. Our results are therefore indicative of there being two different pathways of cohesin loading.

It is increasingly recognised that individual risk of cancer may be related to genetically determined differences in the ability of cells to identify and repair DNA damage. Cell cycle based assays of chromosomal radiosensitivity provide the greatest power for discriminating differences in response to DNA damage and it has been suggested that individuals who are genetically susceptible to cancer show increased chromosomal radiosensitivity. The relationship between chromosomal radiosensitivity and early onset cancer was investigated in a population of Danish survivors of childhood and adolescent cancer and a control group comprising of their partners using the G₂ assay of chromosomal radiosensitivity. Heritability was also examined in the offspring. No significant differences in radiosensitivity profiles were found between partner controls and either the cancer survivors or offspring. However, when compared to the Westlakes Research Institute control population, significant differences were observed with the cancer survivors (P = 0.002) and offspring (P < 0.001), supporting an association of chromosomal radiosensitivity with cancer predisposition. Heritability studies suggested the majority of phenotypic variance of chromosomal radiosensitivity was attributable to a putative major gene locus with dominant effect. Since G2 chromosomal radiosensitivity indirectly measures the ability of cells to repair DNA damage induced by ionising radiation exposure, variants in DNA repair genes may explain inter-individual variation observed. Sixteen polymorphisms in nine genes from four DNA repair pathways were investigated. Genotype frequencies at the Asp148Glu polymorphism were associated with childhood cancer in survivors. Analysis of variance and FBAT analysis suggested significant associations at both the Thr241Met and Ser326Cys polymorphism sites with G₂ radiosensitivity, but neither remained significant after multiple-test adjustment. This study invites further exploration of the predictive capacity of G₂ chromosomal radiosensitivity in cancer predisposition. Clearly, further work is needed to correlate radiosensitivity with genetic polymorphisms, which may underlie cancer susceptibility and variation in radiosensitivity.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Large-scale changes are common in genomes, and are often associated with pathological disorders. In the work presented in this dissertation, I provide insights into how inverted repeat sequences in budding yeast fuse during replication. Fusion leads to the formation of dicentric chromosomes, a translocation, and other chromosomal rearrangements.Using extensive genetics and some molecular analyses, I demonstrate that dicentric chromosomes are key intermediates in genome instability of a specific chromosome in budding yeast. I provide three pieces of evidence that is consistent with this conclusion. First, I detect a recombination fusion junction that is diagnostic of a dicentric chromosome (using a PCR technique). Second, I show a strong correlation between the amount of the dicentric fragment and the frequency of instability of the entire chromosome. Third, I demonstrate that a mutant known to stabilize dicentric chromosomes suppress instability. Based on these observations, I conclude that dicentric chromosomes are intermediates in causing genome instability in this system.Next, we demonstrate that fusion of inverted repeats is general. Both endogenous and synthetic nearby inverted repeats can fuse. Using genetics, I also show that many DNA repair and checkpoint pathways suppress fusion of nearby inverted repeats and genome instability. Based on our analysis, we propose a novel mechanism for fusion of inverted repeats that we term `faulty template switching.'Lastly, I discuss two genes that are necessary for fusion of nearby inverted repeats. I identified a mutant of the Exonuclease 1 (Exo1) and a mutant of anaphase inhibitor securin (Pds1) that suppress nearby inverted repeat fusion and genome instability. Studies of Exo1 and Pds1 provide us with insights into the molecular mechanisms of fusion.Our finding that nearby inverted repeats can fuse to form dicentric chromosomes that lead to genome instability may have great implications. The generality of this fusion reaction raises the possibility that dicentric chromosomes formed by inverted repeats can lead to genome instability in mammalian cells, and thereby contribute to a cancer phenotype.

Backcross populations (BC) from Medicago sativa L. x M. dzhawakhetica Bordz. hybrids were analyzed for chromosome number, fertility and morphological characteristics. Previously obtained F1 hybrids were recovered when diploid (2n = 2x = 16) M. sativa was crossed with tetraploid (2n = 4x = 32) M. dzhawakhetica. Resulting F1 hybrids were triploid (2n = 3x = 24), completely male sterile and had low levels of female fertility. Subsequent populations were obtained by successive backcrossing to unrelated (4x) M. sativa clones. The BC1 plants were pentaploid (2n = 5x = 40) and both male and female fertile. BC2 populations had chromosome numbers ranging from 2n = 32 to 48, and most plants (94% were male and female fertile. BC3 populations were tetraploid (2n = 32) or near tetraploid (2n = 33) and were morphologically similar to M. sativa. Preliminary analysis of mitochondrial nucleic acids by agarose gel electrophoresis, indicated biparental inheritance of this organelle in the F1 hybrids; however, further analysis provided inconclusive results.