Selfish genetic elements break the rules of Mendelian inheritance to bias their transmission to following generations, often with negative fitness consequences. A striking example involves selfish X chromosomes that operate in males and interfere with the production of sperm that carry a Y chromosome. Only X chromosome-bearing sperm are produced, and this can result in extraordinary female-biased sex-ratio distortions. Most studies have focused on how selfish X chromosomes operate in and affect males, and there has been relatively little work on their consequences in females. In this thesis, I characterize fitness effects and transmission in females, in a recently discovered selfish X chromosome system in Drosophila testacea, a common woodland fly. I show that females with two copies of the selfish X chromosome have reduced fitness compared to females carrying zero, or one copy. Specifically, these females have a lower hatch rate and lifetime fecundity. Additionally, I show that heterozygous females are more likely to transmit the selfish X chromosome than the wildtype copy to their offspring. I observe this transmission bias in eggs, larvae, and adults, which suggests that the selfish X chromosome is preferentially segregating into the egg, rather than the polar bodies, during oogenesis. We believe this is the first documented case of a selfish X chromosome acting through both sexes. The negative fitness effects and the biased transmission in males and females will have important consequences on the evolutionary dynamics of the selfish X chromosome. In addition, the phenomenon of biased transmission in both sexes has the potential to yield interesting insights in the mechanism of meiotic drive. / Graduate / 2022-05-12

We have used Chinese hamster ovary (CHO)-human hybrid cells containing chromosomes 16, 18, X, and 21 to test the ability of human kinetochores to successfully bind to spindle microtubules and to be distributed to the daughter cells. We have established the intrinsic rate of non-disjunction among these human chromosomes noted above and compared these rates with those in cells presented with mitotic challenges such as taxol, nocodazole, and mitosis with unreplicated genomes (MUG). Cells were grown on culture slides, fixed and processed for immunofluorescence and fluorescence in situ hybridization (FISH). Daughter pairs were identified by staining with anti-á-tubulin to identify midbodies. Human centromere DNA probes were used for FISH in order to test for the successful passage of human kinetochores to daughter cells during anaphase. Our data indicate that different human kinetochores vary in their ability to properly engage the spindle and to be successfully distributed. In addition, mitotic challenges have been shown to affect the rate of non-disjunction. The mechanism of this effect is not yet known.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

In eukaryotes, the defined loci on each chromosome, the centromeres, accomplish the critical task of correct cell division. In some organisms, centromeres are composed of a euchromatic central core region embedded in a stretch of heterochromatin and the inheritance and maintenance of centromeres are controlled by dynamic epigenetic phenomena. Although the size of centromeres differs between organisms, its organization, and the placement of euchromatic and heterochromatic regions is conserved from the fission yeast, Schizosaccharomyces pombe, to humans, Homo sapiens. However, relatively little is known about centromeres in the filamentous fungi from the Ascomycota, representing the largest group of fungi and fungal pathogens. Further, studies from humans, flies, yeast and plants have shown that the inheritance of centromeres is not strictly guided by centromeric DNA content, which is highly AT-rich, repetitive and constantly evolving. Therefore, it is difficult to align ans assemble the sequenced contigs of centromeric regions of higher eukaryotes, including most filamentous fungi. A genetic technique, tetrad (or octad) analysis has helped to map the centromeres of the filamentous fungus Neurospora crassa early on. The research presented in this dissertation used N. crassa as a model to focus on characterizing different features of centromeres with an emphasis on the centromere-specific histone H3 (CenH3) protein. Data included here represent the first study on centromere-specific proteins in Neurospora, and demonstrate that the central core of the centromeres are heterochromatic, showing enrichment of silent histone marks, which is in contrast to the centromere arrangement in fission yeast. The CenH3 protein, whose deposition on the genome licenses formation or maintenance of centromeres, shows highly divergent N-terminal regions and a conserved histone fold domain (HFD) in all eukaryotes. This bipartite nature of CenH3 is also observed in the Ascomycota, which provides an opportunity for functional complementation assays by replacing Neurospora CenH3 (NcCenH3) with CenH3 genes from other species within the Ascomycota. The results from this experimental approach provide good measures for (1) determining the specific regions of CenH3 required for the assembly of centromeres during meiotic and mitotic cell divisions and (2) analyzing the resistance to changes in the organization of centromeres in N. crassa. The genetic analysis showed that the divergent N-terminal region is essential for the proper assembly of centromeres, and that the conserved carboxy-terminus of CenH3 is important for the process of meiosis but not mitotic cell division. ChIP-seq analyses suggest that the observed loss of Podospora anserina CenH3 (PaCenH3- GFP) from certain N. crassa centromeres does not result in obvious phenotypic defects, e.g. diminished growth or evidence for aneuploidy. Further, the low enrichment of PaCenH3-GFP at certain centromeres is possibly predetermined during meiosis, which results in irreversible and progressive decreases in enrichment. It remains to be determined if this process is random as far as selection of centromeres is concerned. Together the results presented here suggest that during meiosis more stringent structural requirements for centromere assembly apply and that these are dependent on CenH3, and that depletion of CenH3 from centromeres does not critically affect mitosis in the asynchronously dividing nuclei of Neurospora hyphae. / Graduation date: 2013

<p>The centromere is essential for chromosome segregation and genome stability. It is the site of kinetochore assembly and chromosome attachment to the spindle microtubules, and it is important for chromosome movement during mitosis and meiosis. Normal human chromosomes have one centromere, but genome rearrangements that occur with instability, aging, and disease often result in chromosomes with two centromeres, called dicentrics. Nearly seventy-five years ago, Barbara McClintock demonstrated that dicentric chromosomes in plants are associated with instability through mitotic "breakage-fusion-bridge" cycles. However, human dicentrics are unusually stable due to the poorly understood phenomenon of centromere inactivation. Centromere inactivation has been primarily studied in patient-derived dicentrics, limiting the derivation of a molecular pathway. Key centromere and kinetochore proteins are not present at inactive centromeres, but beyond these observations, the process of centromere inactivation is unclear. Epigenetic and sequence-dependent factors are known to contribute to centromere specification, but requirements for centromere assembly, maintenance, and suppression remain obscure. The aims of this research were to (1) determine the mechanism(s) by which de novo dicentric chromosomes are stabilized, (2) ascertain the factors influencing the involvement of specific chromosomes in de novo fusions, and (3) establish the epigenomic, temporal, and mechanistic basis of centromere inactivation. To uncover the mechanistic foundations of these processes, we developed in vitro cell culture systems to study the formation and stabilization of de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. This finding is notable since the most prevalent rearrangement in humans involves the acrocentrics and is called Robertsonian translocation (ROB). In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the centromeric DNA array is reduced compared to the same array before dicentric formation. Many functional dicentrics persist for months after formation. Our results indicate that dicentric human chromosomes undergo alternative fates after formation across a broad temporal window. During transient telomere disruption, we observed a dramatic change in nucleolar appearance. Nucleolar proteins did not coalesce into condensed structures, but appeared dispersed throughout the nucleus. This surprising alteration in nucleolar organization and nuclear architecture suggests remodeling of the nucleolus and subsequent effects on nucleolar-associated chromosomes, such as the acrocentrics, could contribute to the high incidence of ROB formation. Further studies and development of additional cell culture systems will allow us to evaluate current models of centromere assembly and disassembly and the importance of chromatin organization to centromere function and genome architecture.</p> / Dissertation

Pour assurer une ségrégation correcte des chromosomes, la cohésion entre les deux chromatides sœurs doit être protégée au centromère contre la vague de phosphorylation du "prophase pathway", depuis la prophase jusqu'à la transition métaphase-anaphase. Cette protection est sous contrôle de la shugoshine (Sgo1), une protéine recrutée au centromère par la thréonine 120 de l'histone H2A phosphorylée par la kinase Bub1. Mon équipe d'accueil a montré que la déplétion de la désacétylase HDAC3 conduit à l'acétylation et la perte de la di-méthylation de la lysine 4 de l'histone 3 au centromère. Cette acétylation forcée de H3K4 est corrélée avec un défaut de la protection de la cohésion et une perte de la localisation des acteurs majeurs de cette protection. L'objectif général de ma thèse est de déterminer le rôle de la protéine kinase CDK11p58 dans la protection de la cohésion. Nous avons pu confirmer que CDK11p58 est nécessaire à la protection de la cohésion centromérique. Des analyses de déplétion de CDK11 montrent une séparation précoce des chromatides sœurs. Cette séparation est corrélée à une perte de la localisation de Bub1, de la phosphorylation de H2A-T120 et de Sgo1 au centromère, mais la diméthylation de H3K4 reste intacte. Grâce à des expériences de FISH en utilisant des sondes qui ciblent la région centromérique du chromosome 11, nous avons démontré que CDK11 protège la cohésion des chromatides sœurs à partir de la mitose mais pas en interphase. En utilisant des lignées exprimant la forme sauvage ou mutée sur le domaine kinase de CDK11p58, nous avons démontré que l'activité kinase de cette protéine est nécessaire pour ce processus de protection. Les résultats de ma thèse documentent le rôle de l'activité kinase de CDK11p58 dans la protection de la cohésion des chromatides sœurs. Ces résultats montrent l'existence d'un substrat de CDK11p58 impliqué dans le recrutement au centromère des facteurs de cohésion qui assurent la protection des cohésines centromériques contre le "prophase pathway". / Sister chromatid cohesion during the early stages of mitosis is essential to ensure faithful chromosome segregation. Sister chromatid cohesion is established in S phase and is maintained at centromeres until the metaphase to anaphase transition. Protection of cohesion at centromeres is under the control of the Bub1 kinase which phosphorylates histone H2A on threonine 120. Phosphorylated H2AT120 recruits the cohesion protection factor shugoshin (Sgo1) at centromeres. We had previously reported that depletion of the HDAC3 deacetylase induces acetylation of histone H3 lysine 4 at the centromere and loss of dimethylation at the same position. Forced acetylation of H3K4 at centromeres correlates with impaired Sgo1 recruitment and loss of sister chromatid separation. Cdk11p58, a member of the p34cdc2 related protein kinase family, is a G2/M specific protein, involved in different cell cycle events such as centrosome maturation, spindle formation or centriole duplication. It has also been reported as being involved in sister chromatid cohesion. Here we report that, upon cdk11p58 depletion, sister chromatids do not prematurely separate until the early stages of mitosis. We confirm that Cdk11p58 depletion induces a loss of Bub1 and Sgo1 from the centromeres and we show that H3K4 dimethylation is not affected by Cdk11p58 depletion. We report that depletion of endogenous Cdk11p58 in a cell line expressing a kinase-dead version of Cdk11p58 do not rescue the premature sister chromatid separation phenotype. Thus, phosphorylation of an unknown susbtrate by Cdk11p58 is necessary to maintain Bub1 at centromeres and our efforts are now directed towards its identification.

Le syndrome de Bloom (BS) est une maladie génétique rare caractérisée par une forte augmentation du taux d’échanges entre chromatides soeurs, des anomalies de ségrégation des chromosomes et une prédisposition au développement de tous types de cancers. Ce syndrome est la conséquence de mutations dans les deux copies du gène BLM, codant pour une 3’-5’ ADN hélicase de type RecQ. La ou les fonctions de la protéine BLM sont encore mal définies mais les données de la littérature convergent vers un rôle de BLM dans des mécanismes de surveillance et/ou maintien de l’intégrité du génome. La protéine BLM serait impliquée dans le redémarrage de fourches de réplication bloquées pendant la phase S et serait nécessaire à la résolution de ponts anaphasiques en mitose, notamment de ponts particuliers appelées « UltraFine anaphase Bridges » (UFBs). Ces UFBs, qui relient les chromatides soeurs entre elles, ne sont pas détectables par les colorants classiques et leur présence ne peut-être révélée que par la détection des protéines PICH (Plk1-Interacting Checkpoint Helicase) ou BLM. A l’état basal, ces UFBs sont essentiellement d’origine centromérique (cUFBs).Tout l’enjeu de mon projet était de déterminer si BLM était également impliquée dans la prévention de la formation de ces cUFBs et donc si BLM jouait un rôle avant l’anaphase. Nous avons montré que BLM est recrutée aux centromères de la phase G2 jusqu’en mitose. BLM, en coopération avec la protéine PICH, est nécessaire (1) à l’organisation structurale de l’ADN centromérique, (2) à la disjonction complète des centromères, indépendamment de la voie des cohésines, suggérant une implication de ces protéines dans le processus de décaténation des centromères et (3) au recrutement de la topoisomérase IIa (Topo IIa) active aux centromères.Nos résultats révèlent ainsi une nouvelle localisation et une nouvelle fonction de la protéine BLM aux centromères et montrent pour la première fois l’implication des protéines BLM et PICH dans la décaténation centromérique avant l’anaphase. Nous proposons que BLM et PICH, par leurs activités respectives hélicase et de remodelage de la chromatine, modifient la structure des centromères pendant la pré-métaphase, rendant ainsi certaines caténations accessibles à la Topo IIa avant l’anaphase. La défaillance de ce mécanisme entraînerait la persistance de caténations centromériques non résolues avant l’anaphase. Ainsi, dans les cellules BS, la fréquence élevée de cUFBs aurait deux origines différentes : une partie correspondrait à des cUFBs formés du fait d’une décaténation défaillante des centromères avant l’anaphase, et l’autre partie correspondrait à des cUFBs « physiologiques » non résolus en anaphase. Afin de distinguer l’origine des cUFBs, nous avons appelé ceux issus de caténations non résolues avant l’anaphase les UFBs centromériques surnuméraires (SC-UFBs pour Supernumerary Centromeric UFBs). / Bloom syndrome (BS) is a rare genetic disease characterized by a sharp increase in the rate of sister chromatid exchanges, chromosome segregation abnormailities and a predisposition to the development of all types of cancers. This syndrome is caused by mutations in both copies of the BLM gene, which encodes BLM, a RecQ 3'-5 DNA helicase. The specific function(s) of BLM remain unclear, but the data from the literature converge towards a role for BLM in mechanisms monitoring and / or maintaining genome integrity. The BLM protein may be involved in restarting stalled replication forks during S phase and necessary to resolve anaphase bridges in mitosis, including particular bridges called "Ultrafine Anaphase Bridges" (UFBs). These UFBs, which link sister chromatids together, are not detectable by conventional stains and their presence can only be revealed by the detection of the proteins PICH (PLK1-interacting checkpoint helicase) or BLM. In untreated cells, UFBs originate mostly from centromeres (cUFBs).The challenge of my project was to determine whether BLM was also involved in preventing the formation of cUFBs and so, if it played a role before anaphase.We showed that BLM is recruited at centromeres from G2 phase to mitosis. BLM, in cooperation with PICH, is required for (1) structural organization of centromeric DNA, (2) completion of centromere disjunction, independently of the cohesin pathway, suggesting an involvement of these proteins in centromere decatenation process, and (3) recruitment of active topoisomerase IIα (Topo IIα) to centromeres. Thus, we report a new localization and a new function of BLM at centromeres, revealing for the first time a new role for BLM and PICH in a previously unknown centromeric decatenation mechanism, crucial for complete centromere disjunction.We propose that the combined action of BLM and PICH promotes, through their helicase and chromatin remodelling activities, respectively, the organization of centromeric chromatin, thereby rendering some centromeric catenates accessible to Topo IIa before the onset of anaphase. The failure of this mechanism may lead to the persistence of some centromeric catenations not resolved before anaphase. Thus, the increase in the frequency of centromeric UFBs in BLMdeficient cells has two different origins: cUFBs arising from catenations not resolved before anaphase and physiological cUFBs not processed at anaphase onset. Two distinguish the two cUFB origins, we defined the former as supernumerary centromeric UFBs (SC-UFBs).

DNA methylation is a key epigenetic mark which undergoes global reprogramming during early mammalian embryonic development, resulting in almost complete erasure of the mark after fertilisation of the zygote. Genome-wide patterns of DNA methylation are subsequently re-established in the implanting blastocyst by de novo DNA methyltransferases Dnmt3a and Dnmt3b along with their catalytically inactive co-factor Dnmt3l, while these DNA methylation patterns are maintained through cell divisions by maintenance methyltransferase Dnmt1. The exact mechanisms by which these DNA methyltransferase enzymes are targeted to specific genomic regions remain unclear, but may involve interaction with modified histones and/or the participation of co-factors. Lsh (lymphoid specific helicase), a putative chromatin remodelling helicase, has been implicated in facilitating de novo methylation, as Lsh knockout embryos and derived somatic cell lines display substantial but specific DNA methylation losses at repetitive elements and single copy genes. This study aims to define the requirement for Lsh in establishing de novo DNA methylation and gene expression patterns during the early stages of mouse embryonic development. The '2i' culture system using two small molecule kinase inhibitors was harnessed to convert lsh-/- mouse embryonic stem cells (mESCs) to a hypomethylated 'ground state' of pluripotency. Culture conditions were then altered to transition these ground state mESCs to cells representing later, more methylated stages of development ('serum' mESCs, epiblast stem cells and embryoid bodies). Implementation of this model system suggests that Lsh does not contribute to DNA methylation establishment in a pluripotent context, but rather is important for facilitating de novo DNA methylation during differentiation to culture models representing later developmental stages. These investigations also reveal that Lsh differentially regulates DNA methylation at major and minor satellite repeats depending on cellular context, and that this regulation may involve a role for Lsh in maintenance of DNA methylation.

Understanding the role of microtubule-associated proteins is the key to understand the complex mechanisms regulating microtubule dynamics. This study employs the model system Dictyostelium discoideum to elucidate the role of the microtubule-associated protein TACC (Transforming acidic coiled-coil) in promoting microtubule growth and stability. Dictyostelium TACC was localized at the centrosome throughout the entire cell cycle. The protein was also detected at microtubule plus ends, however, unexpectedly only during interphase but not during mitosis. The same cell cycle-dependent localization pattern was observed for CP224, the Dictyostelium XMAP215 homologue. These ubiquitous MAPs have been found to interact with TACC proteins directly and are known to act as microtubule polymerases and nucleators. This work shows for the first time in vivo that both a TACC and XMAP215 family protein can differentially localize to microtubule plus ends during interphase and mitosis. RNAi knockdown mutants revealed that TACC promotes microtubule growth during interphase and is essential for proper formation of astral microtubules in mitosis. In many organisms, impaired microtubule stability upon TACC depletion was explained by the failure to efficiently recruit the TACC-binding XMAP215 protein to centrosomes or spindle poles. By contrast, fluorescence recovery after photobleaching (FRAP) analyses conducted in this study demonstrate that in Dictyostelium recruitment of CP224 to centrosomes or spindle poles is not perturbed in the absence of TACC. Instead, CP224 could no longer be detected at the tips of microtubules in TACC mutant cells. This finding demonstrates for the first time in vivo that a TACC protein is essential for the association of an XMAP215 protein with microtubule plus ends. The GFP-TACC strains generated in this work also turned out to be a valuable tool to study the unusual microtubule dynamics in Dictyostelium. Here, microtubules exhibit a high degree of lateral bending movements but, in contrast most other organisms, they do not obviously undergo any growth or shrinkage events during interphase. Despite of that they are affected by microtubuledepolymerizing drugs such as thiabendazole or nocodazol which are thought to act solely on dynamic microtubules. Employing 5D-fluorescence live cell microscopy and FRAP analyses this study suggests Dictyostelium microtubules to be dynamic only in the periphery, while they are stable at the centrosome. In the recent years, the identification of yet unknown components of the Dictyostelium centrosome has made tremendous progress. A proteomic approach previously conducted by our group disclosed several uncharacterized candidate proteins, which remained to be verified as genuine centrosomal components. The second part of this study focuses on the investigation of three such candidate proteins, Cenp68, CP103 and the putative spindle assembly checkpoint protein Mad1. While a GFP-CP103 fusion protein could clearly be localized to isolated centrosomes that are free of microtubules, Cenp68 and Mad1 were found to associate with the centromeres and kinetochores, respectively. The investigation of Cenp68 included the generation of a polyclonal anti-Cenp68 antibody, the screening for interacting proteins and the generation of knockout mutants which, however, did not display any obvious phenotype. Yet, Cenp68 has turned out as a very useful marker to study centromere dynamics during the entire cell cycle. During mitosis, GFP-Mad1 localization strongly resembled the behavior of other Mad1 proteins, suggesting the existence of a yet uncharacterized spindle assembly checkpoint in Dictyostelium. / Die Kenntnis der Funktion von Mikrotubuli-assoziierenden Proteinen (MAPs) ist von grundlegender Bedeutung für das Verständnis der Mikrotubuli-Dynamik und deren Regulation. Im Rahmen dieser Arbeit wurde die Rolle des Mikrotubuli-assoziierenden Proteins TACC (Transforming acidic coiled-coil), welches in vielen Organismen an der Stabilisierung und dem Wachstum von Mikrotubuli beteiligt ist, im Modellorganismus Dictyostelium discoideum untersucht. Das Dictyostelium TACC Protein konnte während des gesamten Zellzyklus am Centrosom nachgewiesen werden. Darüber hinaus wurde es an den Mikrotubuli-Plus-Enden vorgefunden, überraschenderweise jedoch ausschließlich während der Interphase. Die gleiche Zellzyklusabhängige Lokalisation wurde für CP224 beobachtet, einem Homologen der XMAP215 Proteine in Dictyostelium. Diese ubiquitären MAPs sind konservierte, direkte Interaktionspartner der TACC Proteine und spielen eine zentrale Rolle bei der Nukleation und der Polymerisation von Mikrotubuli. Durch diese Arbeit konnte erstmals in vivo gezeigt werden, dass TACC und XMAP215 Proteine während der Interphase und Mitose unterschiedlich stark mit Mikrotubuli-Plus-Enden assoziiert sein können. Durch Untersuchungen an Knockdown-Mutanten wurde ersichtlich, dass Dictyostelium TACC eine Rolle beim Mikrotubuli-Wachstum während der Interphase spielt und über weite Strecken der Mitose essentiell für die Ausbildung von astralen Mikrotubuli ist. In anderen Organismen konnte als Ursache instabiler Mikrotubuli in TACC Mutanten häufig unzureichendes Rekrutieren des jeweiligen XMAP215 Proteins an das Centrosom ausgemacht werden. Um entsprechende Auswirkungen auf die Lokalisation von CP224 durch den Knockdown von TACC in Dictyostelium zu untersuchen, wurden Fluorescence Recovery after Photobleaching (FRAP) Experimente durchgeführt. Diese ergaben, dass CP224 auch in Abwesenheit von TACC in vollem Umfang an die Centrosomen und Spindelpole rekrutiert wird. Anders als im Wildtyp, konnte in TACC Mutanten allerdings kein CP224 an den Mikrotubuli-Plus-Enden nachgewiesen werden. Somit konnte erstmals in vivo gezeigt werden, dass ein TACC Protein essentiell für die Assoziation eines XMAP215 Proteins mit den Mikrotubuli-Plus-Enden ist. Im Laufe der genannten Experimente stellte sich heraus, dass sich die GFP-TACC Stämme aufgrund ihrer markierten Plus-Enden sehr gut für Untersuchungen zur ungewöhnlichen Mikrotubuli-Dynamik in Dictyostelium eignen. Zwar weisen Mikrotubuli hier über die gesamte Länge ausgeprägte Krümmungs- und Seitwärtsbewegungen auf, es können jedoch im Vergleich zu anderen Organismen während der Interphase kaum Wachstums- oder Verkürzungsvorgänge beobachtet werden. Dennoch können Dictyostelium Mikrotubuli unter Verwendung von Agenzien wie Thiabendazol oder Nocodazol, welche ausschließlich auf dynamische Mikrotubuli wirken, signifikant verkürzt werden. Durch FRAP Experimente und Einsatz von 5D Fluoreszenz-Mikroskopie an lebenden Zellen konnte in dieser Arbeit erstmalig nachgewiesen werden, dass Dictyostelium Mikrotubuli nur in der Zellperipherie, nicht aber im pericentrosomalen Bereich dynamisch sind. Die Identifikation bislang unbekannter Bestandteile des Dictyostelium Centrosoms erfuhr in den vergangenen Jahren große Fortschritte. Ein von unserer Gruppe durchgeführter Proteomics-Ansatz brachte eine Vielzahl potentiell centrosomaler Proteine zu Tage, von welchen bereits viele am Centrosom nachgewiesen werden konnten. Der zweite Teil dieser Arbeit befasst sich mit der Charakterisierung dreier noch unbekannter Proteine aus dem Proteomics-Ansatz, Cenp68, CP103 und dem Dictyostelium Homologen des Spindle Assembly Checkpunkt Proteins Mad1. Hierbei zeigte sich, dass lediglich CP103 Bestandteil isolierter, Mikrotubuli-freier Centrosomen ist, während Cenp68 an die Centromere und Mad1 an die Kinetochoren lokalisieren. Die Charakterisierung von Cenp68 umfasste außerdem die Herstellung eines polyklonalen anti-Cenp68 Antikörpers, das Suchen nach Interaktionspartnern und die Erzeugung eines Cenp68 Knockout-Stammes. Letzterer wies jedoch keinen offensichtlichen Phänotyp auf. Das Verhalten des Dictyostelium Mad1 Proteins während der Mitose stimmte in großen Teilen mit dem anderer Mad1 Proteine überein, was auf die Existenz eines bislang unerforschten Spindle Assembly Chekpunkts in Dictyostelium hinweisen könnte.

Background: Hybridogenesis (hemiclonal inheritance) is a kind of clonal reproduction in which hybrids between parental species are reproduced by crossing with one of the parental species. European water frogs (Pelophylax esculentus complex) represent an appropriate model for studying interspecies hybridization, processes of hemiclonal inheritance and polyploidization. P. esculentus complex consists of two parental species, P. ridibundus (the lake frog) and P. lessonae (the pool frog), and their hybridogenetic hybrid - P. esculentus (the edible frog). Parental and hybrid frogs can reproduce syntopically and form hemiclonal population systems. For studying mechanisms underlying the maintenance of water frog population systems it is required to characterize the karyotypes transmitted in gametes of parental and different hybrid animals of both sexes. Results: In order to obtain an instrument for characterization of oocyte karyotypes in hybrid female frogs, we constructed cytological maps of lampbrush chromosomes from oocytes of both parental species originating in Eastern Ukraine. We further identified certain molecular components of chromosomal marker structures and mapped coilin-rich spheres and granules, chromosome associated nucleoli and special loops accumulating splicing factors. We recorded the dissimilarities between P. ridibundus and P. lessonae lampbrush chromosomes in the length of orthologous chromosomes, number and location of marker structures and interstitial (TTAGGG)(n)-repeat sites as well as activity of nucleolus organizer. Satellite repeat RrS1 was mapped in centromere regions of lampbrush chromosomes of the both species. Additionally, we discovered transcripts of RrS1 repeat in oocytes of P. ridibundus and P. lessonae. Moreover, G-rich transcripts of telomere repeat were revealed in association with terminal regions of P. ridibundus and P. lessonae lampbrush chromosomes. Conclusions: The constructed cytological maps of lampbrush chromosomes of P. ridibundus and P. lessonae provide basis to define the type of genome transmitted within individual oocytes of P. esculentus females with different ploidy and from various population systems.