No description available.

During female meiotic prophase in many organisms, a specialized chromatin structure is formed in the oocyte nucleus. This structure is known as the karyosome, and has been proposed to be important for the formation of the female meiotic bipolar spindle. However, how the karyosome is formed and maintained is not very well understood. To identify proteins involved in the formation and maintenance of the karyosome, I carried out a cytological screen on a collection of 220 mutant fly lines for mutants that were defective in karyosome morphology. The screen identified 46 mutants on the X and 2nd chromosome with abnormal karyosomes. Genetic analysis of these 46 mutants, followed by molecular analysis of one mutant, identified SRPK (SR Protein Kinase) as a protein that is important for the proper formation of the karyosome. NHK-1 (Nucleosomal Histone Kinase 1) was previously identified as a protein that is essential for the formation of the karyosome via its phosphorylation of BAF (Barrier-to-Autointegration Factor). NHK-1 phosphorylation of BAF leads to the release of chromatin from the nuclear membrane, an essential step for the formation of the karyosome, however, the regulation of this process is unclear. In order to identify genes that interact with NHK-1, I carried out a genetic modifier screen using a semi-lethal allele of NHK-1, NHK-1trip. After screening a collection of 44 deficiencies located on the 2nd chromosome, I identified a genetic region (44B8-44D1) containing a gene that interacts with NHK-1 and, when gene dosage is halved, enhanced the semi-lethal phenotype of NHK-1trip.

To enhance the scientific utility and practical application of the honey bee genome and assign the linkage groups to specific chromosomes, I identified chromosomes and characterized the karyotype of the sequenced strain DH4 of the honey bee. The primary analysis of the karyotype and ideogram construction was based on banding and Fluorescence In Situ Hybridization (FISH) for rDNA detection. FISH confirmed two locations for the NOR on telomeric regions of chromosomes 6 and 12 plus an additional less frequent signal on chromosome 1, all three of which were confirmed with silver staining (AgNO3). 4’6-diamidino-2phenylindole (DAPI), and CBanding methods were used to construct the primary ideograms that served as a basis to further identify the chromosomes and locate important structures. The primary map was compared with Giemsa banding, AgNO3-banding, Trypsin banding, and R-banding. The karyotype of the honey bee was established as two metacentric chromosomes (1 and 10), two submetacentric with ribosomal organizer (6 and 12), four submetacentric heterochromatic chromosomes (16, 15, 4 and 13), four euchromatic subtelocentric chromosomes (2, 8, 11 and 14) and four acrocentric chromosomes (3, 5, 7 and 9). In situ nick-translation banding methods were used to verify the heterochromatin distribution. The cytogenetic maps of the honey bee karyotype represented in the ideograms were subsequently used to place 35 mapped BACs (Solignac et. al. 2004) of Solignac’s BAC library. As the BACs hybridized to multiple sites, the mapping was based on strength and frequency of the signals. Location and position of the BACs was compared with those published in the different version of Map Viewer of the NCBI and BeeBase web sites. 10 BACs were confirmed with the last version of Map Viewer V4, 12 BACs were mapped based on high frequency and agreement with the earlier version of Map Viewer. 14 BACs were mapped as confirmed based on moderate frequency of the signal and agreement with the last version of MVV, most of these BACs hits as a secondary signal.

The Chfr checkpoint is a point at which a cell checks whether it is safe to enter mitosis. Chfr is a protein that functions at this particular checkpoint to ensure safe entry into mitosis, but the molecular mechanism by which this protein functions is not entirely clear. The hypothesis in this thesis is that Ubc13, Chfr, and Uev1/Mms2 function together in mitosis. The results were observed using immunocytochemistry, the mitotic shake off procedure, Western blot analysis, and coimmunoprecipitation. High Ubc13, Mms2, and Chfr-Ub levels at the interphase-early prophase transition, indicate that these proteins function together at the Chfr checkpoint. Localization of Chfr to decondensed chromatin in interphase cells and to decondensing chromatin in telophase cells indicates a decondensing function for Chfr. Interaction between Chfr and Ubc13, Chfr and phosphorylated histone H3, as well as Ubc13 and phosphorylated histone H3, further indicates that these proteins may function together at the Chfr checkpoint, because phosphorylated histone H3 is a mitotic protein at that particular point in mitosis. Localization of Chfr, Ubc13, and Mms2 to the centrosomes, indicates that they function together at these sites to target substrates important in centrosome maturation, separation, and spindle formation. Furthermore, there are two molecular states of Chfr: Chfr and Chfr-Ub. Chfr is predominant at late prophase, whereas, Chfr-Ub is predominant at interphase-early prophase. Chfr increases in level upon nocodazole exposure at late prophase to counteract the mitotic stress; and it also looses its ubiquitin signal upon passage into mitosis. High Ubc13 and Mms2 levels coincide with high Chfr-Ub levels at the interphase-early prophase transition, indicating that they function together at the Chfr checkpoint. The ubiquitin signal could be either K-48-linked or K-63-linked in nature. The Chfr, Ubc13, and Mms2 protein complex could function through a self ubiquitination-decondensation-Chfr destruction-recondensation mechanism. Chfr could bind to pH3 and its auto-ubiquitin signal to serve as a bulky modification that hinders chromosome condensation.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13537号 / 論医博第2277号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 近藤 玄, 教授 齋藤 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract Genetic information is stored in the base sequence of DNA. As DNA is often damaged by radiation or reactive chemicals, cells have developed mechanisms to correct the DNA lesions. These mechanisms involve recognition of damage, DNA repair and cell cycle delay until DNA is restored. Failures in the proper processing of DNA lesions may lead to mutations, premature aging, or diseases such as cancer. In this thesis study the human topoisomerase IIβ binding protein 1 (TopBP1) was identified as the homolog of budding yeast Dpb11 and fission yeast Cut5. TopBP1 was found to be necessary for DNA replication and to associate with replicative DNA polymerase ε. TopBP1 localised to the sites of DNA damage and stalled replication forks, which suggests a role in the DNA damage response. TopBP1 interacted with the checkpoint protein Rad9, which is a part of a protein complex whose function includes tethering proteins to sites of DNA damage. This supports a role for TopBP1 in the early steps of checkpoint activation after DNA damage. TopBP1 also interacted with the tumour suppressor protein p53 in a phosphorylation dependent manner. In addition, the data support a role for TopBP1 outside of S-phase. During M-phase, TopBP1 was found to localise to centrosomes along with the tumour suppressor proteins Brca1 and p53. Analysis of the expression of TopBP1 in mouse tissues suggested that TopBP1 may also play a role during meiosis. The localisation pattern of TopBP1 in mouse meiotic spermatocytes resembled that of many proteins functioning during meiotic recombination. For example, co-localisation of ATR kinase and TopBP1 was observed during meiotic prophase I. In accordance with the findings from mouse studies, the analysis of a cut5 mutant during yeast meiosis showed that Cut5 is essential for the meiotic checkpoint. These results strongly suggest that TopBP1 operates in replication and has checkpoint functions during both the mitotic and meiotic cell cycles.

L’objectif de cette thèse a été de comprendre deux caractéristiques majeures des divisions méiotiques chez la femelle: le blocage en prophase de 1ère division méiotique qui permet à l’ovocyte d’accumuler des réserves énergétiques et des déterminants nécessaires au développement embryonnaire ; et l’absence de phase-S entre les deux divisions méiotiques ce qui permet de former des cellules haploïdes aptes à la fécondation. Pour cela, j’ai choisi comme modèle d’étude l’ovocyte de Xénope qui permet de suivre ces processus in vitro en réponse à la progestérone. L’ovocyte subit les deux divisions méiotiques grâce à l’activation du facteur universel de la division cellulaire, le MPF, et se bloque en métaphase de 2ème division méiotique dans l’attente d’être fécondé. Chez tous les vertébrés, le 1er arrêt en prophase dépend de l’activité de la protéine kinase dépendante de l’AMPc, PKA, dont l’inactivation est nécessaire pour la reprise de la méiose. Le substrat de PKA dans l’ovocyte était resté inconnu. Nous avons découvert que la protéine Arpp19, jusqu’alors connue pour son rôle positif dans l’activation du MPF, est phosphorylée par PKA de cette phosphorylation bloque l’activation du MPF nécessaire pour la levée du blocage en prophase. ARPP19 possède donc un double rôle, le 1er exercé comme substrat de PKA et responsable de l’arrêt en prophase, le second dans l’activation du MPF suite à un changement dans sa phosphorylation. Dans un second temps, nous avons étudié la protéine Cdc6, un acteur majeur de la réplication de l’ADN. Absente en prophase, Cdc6 s’accumule entre les deux divisions méiotiques ce qui permet à l’ovocyte d’acquérir la compétence à répliquer l’ADN. Cette compétence ne s’exprime pas ce qui permet de réduire de moitié la ploïdie. Nous avons montré que Cdc6 est un inhibiteur puissant du MPF capable de bloquer les divisions méiotiques et d’induire la réplication de l’ADN. Pour éviter ces effets délétères l’accumulation de Cdc6 est strictement régulée lors des deux divisions méiotiques, ce qui est absolument requis pour assurer l’enchainement des deux divisions cellulaires sans phase-S intercalaire. / The goal of my PhD project was to understand two main features of the female meiotic division: the arrest in prophase of the 1st meiotic division that allows the accumulation of nutrients and determinants necessary for the embryonic cell cycles; and the absence of S-phase between the two meiotic divisions in order to produce haploid gametes. For this purpose, I studied Xenopus oocytes, a powerful model system that allows the biochemical analysis of these two processes in vitro. In ovary, oocytes are arrested in prophase I and resume meiosis in response to progesterone. The oocytes then proceed through the 1st and the 2nd meiotic divisions and halt at metaphase II, awaiting for fertilization. These two consecutive divisions are controlled by two waves of Cdk1 activation, the universal factor responsible for the entry into mitosis. I analysed the mechanisms responsible for arresting the oocyte in prophase I. In all vertebrates, this arrest depends on a high activity of the cAMP-dependent protein kinase, PKA, whose downregulation is required for the release of the prophase block. The substrate of PKA had never been identified up to date. I discovered that the small protein Arpp19, already known for positively regulating entry into M-phase, is phosphorylated by PKA in prophase I and is dephosphorylated upon progesterone addition, an event required for Cdk1 activation. Hence, Arpp19 has a dual function, responsible of the prophase arrest as a PKA substrate, and then converted into an activator of Cdk1 by changes of its phosphorylation pattern. The second part of my thesis has been dedicated to understanding the role and the regulation of the Cdc6 protein during meiotic divisions. This protein is essential for DNA replication in somatic cells. It is accumulated between the two oocyte meiotic divisions and restores the competence to replicate DNA in oocyte. However, this competence is repressed before fertilization, allowing formation of haploid cells. I found that the accumulation of Cdc6 is tightly controlled during meiotic maturation by the Cyclin B accumulation and the Mos/MAPK pathway. I further demonstrated that Cdc6 is a strong inhibitor of Cdk1 in Xenopus oocytes and that the timely accumulation of Cdc6 is required to coordinate the two meiotic divisions with no intercaling S-phase.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 139-152.

La progresión meiótica y el desarrollo embrionario temprano están programados, en parte, por la activación tradcuccional de mRNAs maternos como lo son los que codifican para las proteinas de ciclina B1 o mos. Estos mRNAs no son traducidos al mismo tiempo ni en el mismo lugar. Por lo contrario, su traducción está especificamente regulada por elementos de poliadenilación citoplasmática (CPEs) presentes en sus 3'UTRs. Los elementos CPEs reclutan a la proteina de unión a CPE (CPE-binding protein CPEB (Colegrove-Otero et al., 2005; de Moor et al., 2005; Mendez and Richter, 2001; Richter, 2007)). Esta proteina de unión al RNA no sólo determina cuándo y en qué medida un mRNA será activado traduccionalmente por poliadenilación citoplasmática (Mendez et al., 2000a; Mendez et al., 2000b; Mendez et al., 2002) sino que también participa, junto con el represor de la traducción Maskin, en el transporte y la localización de sus mRNAs diana hacia los sitios de localización subcelular donde su traducción ocurrirá (Huang et al., 2003; Huang and Richter, 2004). Durante el desarrollo embrionario de Xenopus, CPEB se encuentra localizada en el polo animal de los oocitos y más tarde, sobre el huso mitótico y centrosomas en el embrión (Groisman et al., 2000). Se ha demostrado que embriones de Xenopus inyectados con agentes que interrumpen la traducción dependiente de poliadenilación citoplasmática, detienen la división celular y presentan estructuras mitóticas anormales (Groisman et al., 2000). En este trabajo que derivó en mi tesis doctoral, hemos demostrado que la activación traduccional localizada en el huso mitótico de mRNAs regulados por CPEB que codifican para proteinas con una conocida función en aspectos estructurales del ciclo celular como la formación del huso mitótico y la segregación cromosómica, es esencial para completar la primera división meiótica y para la correcta segregación cromosómica en oocitos de Xenopus.