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1
Canales, C. "Characterisation of extra sporogenous cells (ESP) : an avbidopsis gene required for another development." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365861.
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Pasternak, Michał. "RNAi screen for meiotic genes in mammals reveals BTG4 as a novel regulator of meiosis." Thesis, University of Cambridge, 2016. https://www.repository.cam.ac.uk/handle/1810/283984.
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Widger, Alexander David. "Ablating ATR in mouse meiosis and its consequences for synapsis, recombination and meiotic surveillance mechanisms." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10043772/.
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Meiosis is a fundamental part in the life cycle of sexual species. It denotes a specialised cell division that halves chromosome numbers to generate haploid gametes for reproduction. Cells unable to competently progress through meiotic prophase activate cell surveillance mechanisms causing their elimination. Given the importance of DNA damage kinases like ATR in facilitating mitotic cell surveillance mechanisms, I characterized Atr-deficient spermatocytes to determine the importance of ATR for mammalian meiosis. I found that ATR ensures efficient chromosome synapsis, and that that is partially independent of meiotic recombination. In addition, ATR has three distinct roles in meiotic recombination. Firstly, during nucleolytic processing, it acts to regulate SPO11-oligonucleotide size when ATM is deleted. Secondly, it is required for accurate RAD51 and DMC1 recruitment to DSBs. Thirdly, it regulates the timing of DNA DSB repair on both unsynapsed and synapsed chromosomes. Finally I found that the loss of ATR is unable to rescue meiotic arrest in multiple meiotic mutants, including mice deficient for the other DNA damage PIKKs ATM and DNA-PK. My findings reveal multiple roles for ATR in male mouse meiosis.
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Malik, Shehre-Banoo. "The early evolution of meiotic genes." Diss., University of Iowa, 2007. http://ir.uiowa.edu/etd/275/.
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Ye, Jinpei. "Signalling pathways controlling meiosis in porcine oocytes." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273192.
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Wolf, Peter G. [Verfasser], and Olaf [Akademischer Betreuer] Stemmann. "Meiosis made simple : Mechanisms of meiotic chromosome dynamics elucidated in somatic cells / Peter G. Wolf ; Betreuer: Olaf Stemmann." Bayreuth : Universität Bayreuth, 2017. http://d-nb.info/113220092X/34.
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Fazio, Cynthia Marie. "The influence of meiotic onset on and the role of apoptosis in oocyte death during the meiotic prophase /." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97951.
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Loss of germ cells that entered meiosis at different developmental stages was compared. Mice were injected with BrdU at 13.3, 14.3 or 15.3 days post coitum (dpc) and sacrificed either 3 days after BrdU injection or 4 days post partum (dpp). BrdU-labeled germ cells were detected in ovarian sections through double immunofluorescent staining for BrdU and either GCNA-1 or MVH as a germ cell marker. The results show that the loss of germ cells that entered meiosis at 13.3 or 15.3 dpc was excessive compared to the loss of total germ cells. Such preferential elimination was not found for germ cells that entered meiosis at 14.3 dpc. We conclude that oocyte loss during meiotic prophase is influenced by the timing of meiotic onset.The mechanism of germ cell loss during ovarian development was tested by the presence of markers for apoptosis. Mouse ovaries were isolated at 12.5 dpc, 18.5 dpc and 2 dpp and cultured with doxorubicin (DXR) to induce cell death. Ovarian histological sections were double immunofluorescent stained for GCNA-1 and cleaved caspase-3 or PARP-1. The results suggest that caspase-3 is not activated in germ cells throughout ovarian development whereas PARP-1 is activated in germ cells at 12.5 dpc and 2 dpp but not at 18.5 dpc. Thus, no evidence has yet been provided to support the hypothesis that oocyte death during the meiotic prophase is mediated by apooptosis.
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Ferguson, Kyle Akira. "Meiotic defects in infertile men." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/1228.
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While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm.
We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans.
In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.
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Cooper, Timothy J. "Investigating the spatial regulation of meiotic recombination in S. cerevisiae." Thesis, University of Sussex, 2018. http://sro.sussex.ac.uk/id/eprint/74309/.
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Koehn, Demelza Rae Malone Robert E. "Analysis of meiotic recombination initiation in Saccharomyces cerevisiae." Iowa City : University of Iowa, 2009. http://ir.uiowa.edu/etd/303.
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Ernst, Christina. "Transcriptional and developmental consequences of aneuploidy during male meiosis." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/278212.
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Eukaryotes have developed stringent regulatory mechanisms that control cell division and ensure proper chromosome segregation. Maintaining genome integrity is especially important during meiosis, the specialised cell division programme in the germline that generates haploid gametes. As these cells transmit genetic information to the next generation, the consequences of meiotic errors are not restricted to an organismal level, but can directly impact the fitness of the offspring. Mammals display a high degree of sexual dimorphism in meiosis with regard to the stringency of regulatory mechanisms. This manifests in a relatively high degree of maternally-derived aneuploidies due to weaker checkpoint control in females, whereas more rigorous checkpoints in males frequently perturb fertility. Mouse models of aneuploidy often exhibit complete male sterility and early germ cell arrest, preventing the study of aneuploidy during late and post-meiotic stages in males. In this thesis, we have used the trans-chromosomic mouse model, Tc1, which carries a single copy of human chromosome 21 (HsChr21) and show that, unlike other aneuploid mouse strains, the Tc1 mouse can successfully passage the exogenous human chromosome through male meiosis and generate aneuploid offspring. Our investigations have shown that the presence of the aneuploid human chromosome causes spermatogenic defects due to an arrest at the first meiotic division. Despite this impairment, we found an unexpectedly high number of aneuploid gametes in Tc1 males and the majority of males were able to produce aneuploid offspring, albeit at a lower frequency. Transmission of HsChr21 through the male germline was less efficient compared to female germline transmission, but allowed us to study the impact of male germline-associated chromatin remodelling on the transcriptional deployment of HsChr21 in the offspring. This revealed that, despite fundamentally different developmental dynamics, male- versus female-germline passage result in indistinguishable transcriptional and regulatory phenotypes. An important pathway in the male germline involves the expression of piRNAs, a class of small non-coding RNAs that are commonly found in the germline of animals where they defend cells against transposable elements. Profiling the expression of small RNAs in the Tc1 mouse showed that conserved human piRNA clusters can be successfully transcribed by the mouse piRNA machinery. In addition, we detected Tc1-specific piRNA sequences that were neither present in human nor mouse, mapping to a human-specific repeat element. In line with the previously observed activation of human-specific repeat elements in the Tc1 mouse, this suggests that novel transcripts arising from human repeats can trigger an adaptive piRNA response, thereby demonstrating the plasticity of this pathway to newly invading repeat elements. Transcriptional profiling of spermatogenic cell populations on a single-cell level allowed us to generate an atlas of gene expression over the course of spermatogenesis and dissect meiotic silencing dynamics in the presence of aneuploidy. Transcriptional silencing during meiosis occurs in response to unpaired chromosomes and, in male germ cells, affects the sex chromosomes due to their largely unpaired nature. We found that the presence of HsChr21 has no impact on the silencing of chromosome X, however, the two chromosomes display drastically different silencing patterns with HsChr21 showing a much weaker repression. Taken together, this study revealed a higher than expected tolerance for aneuploidy in the mouse male germline thus allowing the characterisation of meiotic checkpoint mechanisms, the meiotic silencing response to unpaired chromosomes as well as piRNA expression in the presence of an exogenous human chromosome.
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Koehn, Demelza Rae. "Analysis of meiotic recombination initiation in Saccharomyces cerevisiae." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/303.
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Meiosis is the unique process in which diploid cells undergo two consecutive divisions to produce haploid daughter cells. It is indispensable for sexual reproduction in all eukaryotic organisms and maintains proper chromosome number through generations. An integral step in the meiotic program is genetic recombination; recombination is required for a successful reductional division. In the yeast Saccharomyces cerevisiae, recombination is initiated by DNA double strand breaks (DSBs) that are created by ten recombination initiation proteins. Similar phenotypes are observed when any of these genes is mutated. This has made the mechanism by which these proteins function to initiate recombination difficult to unravel. One hypothesis is that these proteins form a functional complex for activity, in which all (or most) of them physically interact. The work described in Chapter 2 contributes to understanding the putative DSB-producing recombination initiation complex, suggesting there is substantial flexibility among initiation protein interactions. The results are also consistent with the view that the proteins assemble on the DNA. Studies in Chapter 3 examined the recombination initiation protein interactions during DSB formation in more detail using a novel experimental approach. While the initial experiments using this approach produced unexpected results, the assay is a promising tool for the future.
In addition to creating DSBs, a subset of the initiation proteins perform a second function during early meiosis; they create a recombination initiation signal (RIS) to delay the onset of the reductional division in wild-type cells. Although the signal and the downstream target are well-defined, less is known about how the RIS is transduced to the downstream target. The work in Chapter 4 contributes to defining this transduction, and therefore enhances our understanding of the relationship between the recombination initiation proteins and the reductional division.
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Fabig, Gunar. "Dynamic and ultrastructural characterization of chromosome segregation in C. elegans male meiosis." Technische Universität Dresden, 2018. https://tud.qucosa.de/id/qucosa%3A32727.
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The production of germ cells is an essential process in all sexually reproducing eukaryotes. During male meiosis, four haploid sperm cells are formed from one primary spermatocyte, thereby undergoing two consecutive cell divisions after only one round of chromosome duplication. This process was studied in the nematode Caenorhabditis elegans, as this model organism offers a number of experimental advantages to simultaneously analyze spindle dynamics and ultrastructure. The worm is easy to cultivate, completely sequenced and numerous mutants are available, the worm is small and thus ideal for light and electron microscopic investigations, and the transparent body allows live-cell imaging within living animals. Importantly, meiotic spindles in C. elegans males are organized by centrosomes and show a lagging X-chromosome, which is always segregated after the autosomes have been partitioned to the newly forming secondary spermatocytes.
The aim of this thesis was to systematically investigate this characteristic feature of chromosome segregation in male meiotic spindles. For that, spindle dynamics in the first and second meiotic division was analyzed with fluorescence light microscopy. Furthermore, the spindle ultrastructure was investigated in spindles of various stages of meiosis I using electron tomography.
Light microscopy revealed a shortening of the distance between centrosomes and chromosomes (anaphase A) and an increase in the pole-to-pole distance (anaphase B). Moreover, spindles in male meiosis I and II showed differences in certain aspects of spindle dynamics. In addition it was demonstrated that spindles in metaphase II in the presence of a single X-chromosome were shorter compared to spindles without the X-chromosome. In addition, it was found that the process of aging had an impact on spindle length in both metaphase I and II.
By manipulating the number of unpaired chromosomes, it could be demonstrated that the lagging behavior of univalent chromosomes is caused by the incapability of pairing in meiotic prophase. After performing a quantitative analysis of the light microscopic data it was further shown that a dynamic microtubule bundle is connecting the X-chromosome to the spindle poles. Using laser microsurgery it could be demonstrated that this bundle exerts a pulling force to the univalent chromosome throughout anaphase.
Unexpectedly, electron tomography showed that anaphase-type movements of the autosomes were not accompanied by a shortening of the kinetochore microtubules. Instead, three findings indicated a shortening of the centrosome-chromosome distance itself: (1) upon anaphase onset, tension is released on the beforehand stretched autosomes; (2) centrosomes shrink in preparation for meiosis II and (3) the attachment angle of end-on microtubules changes.
Interestingly, microtubules connecting the X-chromosome to the spindle poles showed a high curvature around the kinetochore region of the X-chromosome, suggesting an involvement of motor proteins in the process of segregation.
Taken together, this thesis gives the first detailed quantitative analysis of spindle dynamics and architecture during male meiosis in the nematode C. elegans. This wild-type data will serve as a basis for future mutant analyses and should help to further understand the complex dynamic and ultrastructural aspects of spindle organization in the meiotic divisions in C. elegans males.
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Letarte, Jocelyne. "Identification and characterisation of early meiotic genes in wheat." Title page, table of contents and summary only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phl645.pdf.
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Errata inserted. Bibliography: leaves 98-120. This study is concerned with the identification of genes related to the very early stages of meiosis when homologous pairing occurs. A cDNA library is prepared at the premeiotic interphase and prophase stages of meioses. Differential screening is used to identify and select clones showing preferential expression in anthers at early meiosis. Two selected clones are chosen for further analysis and to investigate a possible role in chromosome pairing.
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Reini, K. (Kaarina). "Characterisation of the human DNA damage response and replication protein Topoisomerase IIβ Binding Protein 1 (TopBP1)." Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514282787.
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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.
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Nikalayevich, Elvira. "Meiotic spindle organization and chromosome condensation in Drosophila oocytes." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/17908.
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Errors in chromosome segregation during the first division of female meiosis are very common in humans and result in aneuploidy leading to reproduction problems. Chromosome segregation depends on the formation and function of the meiotic spindle as well as the structure of chromosomes, which need to condense to be able to orient and segregate properly. It is important to understand the mechanisms underlying the female meiotic spindle function and chromosome condensation to gain insight into female fertility problems. The female meiotic spindle assembles without centrosomes, so the mechanisms ensuring microtubule nucleation, spindle assembly and establishment of bipolarity act differently from those of mitosis or male meiosis. I identified a set of genes that are required for microtubule nucleation, spindle maintenance and centromere orientation in Drosophila female meiosis. This was accomplished by mapping previously uncharacterized Drosophila mutants and depleting already known genes by RNAi. I discovered that several proteins have a different role in female meiosis as compared to mitosis, which provides insight into the major differences between these systems. Little is known about the molecular mechanisms of chromosome condensation. The roles of only a few factors, such as condensin complexes, have been studied previously, and the evidence suggests that there are more molecular players required for chromosome condensation. To discover molecular mechanisms critical to this process, I depleted various chromosomal proteins by RNAi and screened for abnormalities of metaphase chromosome morphology in Drosophila oocytes by immunostaining and live imaging. I found that the conserved kinase NHK-1 plays a role in chromosome condensation in female meiosis. BAF is a critical NHK-1 substrate in this process and its phosphorylation is required for detachment of the chromosomes from the nuclear envelope to allow proper condensation. Also, I discovered that the nucleosome remodelling complex NuRD is crucial for chromosome condensation, especially for the chromosome arms. As a result of my PhD project I identified multiple factors required for meiotic spindle function. I also discovered two novel pathways of chromosome condensation that require the NuRD complex and NHK-1 activity.
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Diaz, Patrick Loyola. "Genetic strategies to manipulate meiotic recombination in Arabidopsis thaliana." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/271685.
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During meiosis eukaryotes produce four haploid gametes from a single diploid parental cell. In meiotic S-phase homologous chromosomes, which were inherited from maternal and paternal parents, are replicated. Homologous chromosomes then pair and undergo reciprocal crossover, which generates new mosaics of maternal and paternal sequences. Meiosis also involves two rounds of chromosome segregation, meaning that only one copy of each chromosome is finally packaged into the resulting haploid gametes. In this work I sought to genetically engineer two elements of meiosis, in order to generate tools which may be useful for plant breeding. The first project sought to generate a second division restitution (SDR) population, where the second meiotic division is skipped. This is created by crossing an SDR mutant, omission of second division1, which produces diploid pollen due to a defective meiosis-II, to a haploid inducer line, whose chromosomes are lost from the zygote post-fertilisation. This was intended to give rise to diploid plants possessing chromosomes from just the SDR parent. Importantly, the SDR parent used was heterozygous, meaning that SDR progeny should show mostly homozygous chromosomes, but with regions of residual heterozygosity, determined by crossover locations. This project succeeded in creating a small number of plants with the predicted SDR genotype, although a range of aberrant genotypes were also observed. I present several hypotheses that could account for the observed progeny genotypes. In a second project I attempted to direct meiotic recombination using DNA double strand breaks targeted to specific sites. This project used a spo11-1 mutant, which is unable to produce the endogenous meiotic DNA DSBs that normally mature into crossovers. Instead, TALFokI nucleases (TALENs) were expressed from meiotic promoters in order to generate exogenous DSBs at sites determined by the DNA binding specificity of the TAL repeat domains. The project succeeded in transforming TALENs into spo11-1 mutants and confirming their expression. However, this was not sufficient to recover the spo11-1 mutant infertility or direct crossovers. Potential reasons for this non-complementation are discussed, as well as their implications for control of meiotic recombination in plant genomes.
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Novak, Ivana. "Molecular architecture of meiotic chromosomes /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-959-9/.
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Karfilis, Kate V. "Meiotic Insurance: Designing a system to study crossover control in yeast." DigitalCommons@CalPoly, 2010. https://digitalcommons.calpoly.edu/theses/365.
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Meiosis is a specialized form of cell division in which haploid gametes are produced from diploid progenitors. This reduction in ploidy results from proper meiotic chromosome segregation and is ensured by crossover recombination events. Given their importance, it is no surprise that crossover formation is regulated in most eukaryotes. Crossover assurance is a regulatory mechanism that helps to ensure that each pair of chromosomes gets at least one crossover during meiosis.
We seek to better understand how crossover assurance works. To do so, we have developed a system in which crossover formation between a pair of chromosomes is restricted to a defined region. If crossover assurance functions in this context, then crossovers should frequently form in this defined region.
Our experiments involve three yeast strains: Homolog: diploid Saccharomyces cerevisiae. Homeolog: Diploid S. cerevisiae, but with one copy of III derived from S. paradoxus and one from S. cerevisiae. Homo-meolog: The homeolog strain, but with the HIS4 region of the S. paradoxus III replaced with the corresponding S. cerevisiae sequence.
S. cerevisiae and S. paradoxus are largely syntenic and have 80-90% sequence homology. This level of sequence divergence greatly reduces the incidence of meiotic crossing over. Thus, in the Homeolog strain chromosomes III will frequently fail to form crossovers. In the Homo-meolog strain, a defined region of homology surrounding HIS4 (a hotspot for meiotic recombination) exists in a chromosomal context of homeology. In the Homo-meolog strain, crossover assurance should result in a high incidence of crossover formation in the HIS4 region. By comparing the spectrum of meiotic recombination events in the HIS4 region in the three strains, we will gain insight into the means through which crossover assurance is enforced.
These experiments are in the preliminary stage. Strain construction and data collection are ongoing, but our preliminary results demonstrate an elevated incidence of crossing over in the HIS4 region in the homo-meolog strain relative to both the homolog and homeolog strains. Spore viability patterns in the homo-meolog strain are not statistically distinguishable from that of the homolog strain, but are different from that of the homeolog strain. Taken together, these results suggest that the crossovers are targeted to the HIS4 region in the homo-meolog strain, possible through the action of a crossover assurance mechanism. Further analysis of the patterns of recombination in these strains may provide insight into the means through which this regulation is exerted.
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Lawrence, Emma Jane. "Identifying natural modifiers of meiotic crossover frequency in Arabidopsis thaliana." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/289733.
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During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, producing crossovers. This generates genetic diversity and is required for balanced homolog segregation. Despite the critical functions of crossovers, their frequency and distribution varies extensively within and between species. This crossover variation can be caused by trans-modifiers within populations, which encode diffusible molecules that influence crossover formation elsewhere in the genome. This project utilised natural accessions of Arabidopsis thaliana to identify trans-modifying loci underlying crossover variation within the species. I performed Quantitative Trait Loci (QTL) mapping using a fluorescence-based crossover reporter system to measure recombination frequency in a genomic interval on chromosome 3, termed 420. Mapping in a Col-420 × Bur-0 F2 population revealed four major recombination QTLs (rQTLs) that influence crossover frequency. A novel recessive rQTL on chromosome 1 that reduced crossovers within the interval was fine-mapped to a premature stop codon in TATA Binding Protein (TBP)-associated factor 4b (TAF4b) in Bur-0 (taf4b-1). TAF4b is a subunit of the TFIID complex, a multi-protein general transcription factor complex comprising TBP and numerous TAFs that forms a component of the pre-initiation complex that recruits RNA polymerase II to promoters. Transformation-based complementation experiments and the isolation of several independent taf4b alleles provided genetic proof that TAF4b is essential for wild-type levels of crossover within 420. Analysis of the prevalence of the taf4b-1 mutation in the global Arabidopsis accession collection demonstrated its specificity to three accessions in the British Isles. A combination of cytology, genetic analysis using additional fluorescent reporter lines, and sequencing in F2 recombinant populations demonstrated a genome-wide reduction in crossover frequency in taf4b-1. In addition, RNA sequencing identified numerous transcriptional changes in taf4b-1. Both up- and down-regulated gene sets displayed significant enrichment for genes that are predominantly expressed in meiocytes, and several gene ontology terms pertaining to protein modification and meiotic processes. These results further demonstrate the existence of genetic modifiers of crossover frequency in natural populations of A. thaliana, and the characterisation of a novel trans-modifier of recombination, TAF4b. This signifies a novel function for TAF4b in Arabidopsis, and further enhances our understanding of the molecular factors controlling the frequency and distribution of meiotic crossovers in plants.
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Crichton, James Hugh. "Dissecting the meiotic defects of Tex19.1-/- mouse spermatocytes." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/21042.
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The maintenance of genomic stability through suppression of retrotransposon activity is vital for the avoidance of potentially mutagenic genomic disruption caused by retrotransposition. Germline development is a particularly important phase for retrotransposon silencing as retrotransposition events here have the potential for transmission to the entire embryo, threatening the health of offspring. A collection of germline genome defence genes are required for the suppression of retrotransposons in the developing germline of male mice (e.g. Tex19.1, Dazl, Mili, Miwi2, Gasz, Mov10l1, Mael, Dnmt3l), all of which trigger meiotic prophase arrest when mutated. I have analysed the meiotic defects which arise in Tex19.1-/- male mice to contribute to the understanding of the fundamental mechanisms required for successful completion of meiosis and to investigate the involvement of retrotransposon silencing in this process. The absence of TEX19.1 in male mice causes infertility; with failed chromosome synapsis in ~50% of pachytene nuclei and associated apoptosis, as well as individual univalent chromosomes in 67% of remaining nuclei progressing to metaphase I. Where studied, failed chromosome synapsis is a common feature of germline genome defence mutant spermatocytes. One aim of my studies has been to better understand the mechanism responsible for this failed chromosome synapsis. I have demonstrated that unlike Mael-/- spermatocytes, additional SPO11-independent DNA damage potentially attributable to retrotransposition is not detectable in Tex19.1-/- spermatocytes. Rather, the formation of meiotic DNA double strand breaks (DSBs) is dramatically reduced in early prophase to around 50%, resulting in a reduction in nuclear γH2AX signal, production of SPO11- oligonucleotide complexes and foci formation by early recombination proteins RPA, DMC1 and RAD51. Despite this early reduction, DSB frequency recovers to more normal levels shortly after in zygotene. I have shown that defective pairing of homologous chromosomes by meiotic recombination is likely responsible for the asynapsis previously reported. The initial reduction in DSB frequency could be sufficient to cause failed chromosome synapsis in this mutant, assuming that late-forming DSBs cannot participate effectively in promoting homologous pairing. Alternative hypotheses include altered positioning of DSBs in response to altered chromatin organisation relating to retrotransposon upregulation, misguiding the pairing of homologous chromosomes. Such a model of disruption could also extend to other germline genome defence mutants. I have demonstrated that despite successful pairing of homologous chromosomes in a sub-population of Tex19.1-/- spermatocytes, subsequent progression of these cells through pachytene is delayed. Numerous diverse features of progression are all delayed, including recombination, ubiquitination on autosomes and sex chromosomes, expression of the mid-pachytene marker H1t, and chromosome organisation. The delay identified is related to recombination therefore this feature is likely to stem from the initial defect in DSB formation early in prophase. While some delayed features are probably directly related to recombination, others are not. The coordinated delay observed may suggest the presence of a recombination-sensitive cell-cycle checkpoint operating to regulate progression through pachytene. My research has also aimed to establish the cause of elevated univalent chromosomes not connected by chiasmata in metaphase I Tex19.1-/- spermatocytes. I have demonstrated that that absence of chiasmata is not due to failed crossover formation between synapsed chromosomes. Rather, the frequent observation of individual unsynapsed chromosomes during crossover formation suggests that some spermatocytes with low-level asynapsis are leaking through meiotic checkpoints and are unable to form a crossover before reaching metaphase. Therefore, again this later meiotic defect appears to stem from the initial defect in meiotic DSB formation, the consequences of which vary widely in severity. Remarkably the unsynapsed chromosomes present during crossover formation include both sex chromosomes, and autosomes. Tolerance of an unsynapsed autosome from pachytene into metaphase is an unusual observation in mice and this observation may aid the understanding of spermato cyte quality control mechanisms during this progression. Together these findings have greatly advanced the understanding of the infertility incurred during meiosis in Tex19.1-/- male mice. These findings may also extend to benefit the understanding of other germline genome defence mutants. Diverse observations made during my investigations also reveal a potential system of coordinated progression through pachytene relating to meiotic recombination. The variable severity of the synapsis defects incurred in this mutant appears to have variable effects on spermatocyte survival and could also inform the understanding of meiotic checkpoint sensitivity.
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Blary, Aurélien. "Towards a functional characterization of meiotic recombination in rapeseed : analysis of the meiotic transcriptome and hyper-recombinant mutants." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS576/document.
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La recombinaison méiotique produite par les Crossing Overs (COs) est un facteur limitant pour l’efficacité de la sélection variétale. Une possibilité pour produire des plantes hyper-recombinantes serait d’exploiter la variabilité intraspécifique pour les fréquences de recombinaison. L’identification des polymorphismes causaux, liés à la séquence ou l’expression, représente un travail de longue haleine. Une approche alternative serait de produire des mutants pour des régulateurs négatifs des fréquences de recombinaison. Chez le colza, jeune allotétraploïde (AACC, 2n=38), il est possible de jouer sur ces 2 approches. Dans un premier temps j’ai cherché à vérifier dans quelle mesure pouvait varier le transcriptome méiotique entre 2 variétés ayant servi à cartographier un QTL pour le contrôle de la recombinaison entre chromosome homoéologues (hérités des génomes parentaux). Ce transcriptome méiotique s’est révélé de façon inattendue très variable ; les principales sources de cette variation étant notamment la nature du génome (A ou C) ainsi que l’effet variété. J’ai montré que les HEs (le remplacement d’une région chromosomique par la duplication de la région homoéologue) contribuent de façon importante aux différences d’expression observées à la fois entre variétés ou au sein d’un même génotype. Dans un second temps, j’ai vérifié que FANCM décrit chez Arabidopis thaliana comme un régulateur négatif pour les fréquences de recombinaison avait bien la même fonction chez les Brassica. Chez Brassica rapa j’ai vérifié qu’un mutant fancm complémente comme attendu un mutant déficient pour la voie majoritaire de formation des COs. Chez Brassica napus j’ai observé une faible augmentation à la fois des fréquences de recombinaison entre chromosomes homologues et homoéologues. Ce travail souligne l’importance de la caractérisation des HEs chez les allopolyploïdes. Au-delà de leurs impacts sur le contenu et l’expression génique, les HEs ont très certainement des conséquences phénotypiques. Cette étude présente aussi un exemple de biologie translationnelle pour un trait important en amélioration des plantesMeiotic recombination driven by Crossing-Over (CO) is a limiting factor for the efficiency of plant breeding. One way to produce hyper-recombinant plants is to use the existing interspecific variability for recombination frequencies. Identification of the causal polymorphisms, either link to gene sequence or expression, represents a long-term endeavour. Another possibility is to mutate anti-meiotic CO genes. In rapeseed, a young allotetraploid species (AACC, 2n=38), both of these approaches are possible. First I wanted to check how much varies the meiotic transcriptome between 2 varieties that differ in term of recombination between homoeologous chromosomes (inherited from parental genomes). Unexpectedly, the meiotic transcriptome turned out to be very variable, the main source of this variation being notably the origin of the genome (A or C) and the variety. I also showed that homoeologous exchanges (HEs; the replacement of one chromosomal region with a duplicate of the homeologous region) contributed to this variation and led to large changes in expression both between and within varieties. Then I assessed whether FANCM, an anti-CO protein identified in Arabidopis thaliana had the same function in the Brassica genus. In Brassica rapa, a fancm mutant complements as expected a meiosis mutant defective in the main formation pathway for the formation of meiotic COs. In Brassica napus, I observed a slight increase in both homologous and homoeologous recombination frequencies. This work emphasizes the importance of characterizing HEs in allopolyploids species. Beyond their impact on gene content and expression, HEs most have likely phenotypic consequences. This study also presents an example of translational biology for an important trait in crop breeding
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Severino, Jacqueline 1990. "X chromosome status : a gatekeeper of germ cells meiotic entry." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/671536.
Full textAbstract:
X chromosome reactivation in female mouse germ cells is essential for the transmission of one active
X chromosome to the progeny. However, despite its key role in development, the mechanistic details
and kinetics still remain elusive, as previous studies were restricted by a scarcity of cells in vivo and a
lack of adequate in vitro systems. Here, I present the characterization of X-chromosome dynamics
during germ cell formation, which was possible thanks to the development of a tailor-made in vitro
system which allows the accurate profiling of X-chromosome activity. We recapitulate X-inactivation
starting in epiblast-like cells (EpiLCs), progressing in primordial germ cell-like cells (PGCLCs),
followed by X-reactivation in germ cells upon meiotic entry. We show that PGCLCs undergoing
X-inactivation can enter meiosis more efficiently, while PGCLCs bypassing X-inactivation, with the
consequent lack of X-reactivation, show a reduced meiotic potential. We conclude that tracing the X
chromosome status during germ cell formation facilitates the dissection of the relationship between X
chromosome dynamics and proper germline fate acquisitionEn las hembras de ratón, la reactivación del cromosoma X en las células germinales es esencial para la transmisión de un cromosoma X activo a la descendencia. Sin embargo, a pesar del papel crucial del cromosoma X durante el desarrollo, el mecanismo y la dinámica de su reactivación siguen siendo difíciles de alcanzar, ya que los estudios realizados anteriormente estaban restringidos por la escasez de células in vivo y la falta de sistemas in vitro adecuados. En este estudio he desarrollado un sistema que me permite seguir y caracterizar de manera detallada la actividad del cromosoma X durante la formación de las células germinales femeninas. Empezando por células similares a las células del epiblasto y diferenciandolas hacia células parecidas a células germinales primordiales (PGCLCs), hemos conseguido recapitular la inactivación del cromosoma X. A continuación hemos observado la reactivación del cromosoma X a medida que las células germinales primordiales entran en meiosis. Mostramos que las PGCLCs que se someten a inactivación del cromosoma X pueden entrar en meiosis forma más eficiente, mientras que las PGCLCs que no logran inactivar el cromosoma X, con la consiguiente falta de su posterior reactivación, muestran un menor potencial para entrar en meiosis. Concluimos que el seguimiento del estado del cromosoma X durante la formación de las células germinales nos permitió analizar la relación entre la dinámica del cromosoma X y la correcta especificación y desarrollo de la línea germinal.
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Sarno, Roberta. "Targeting of meiotic recombination in the yeast Saccharomyces cerevisiae." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066326.
Full textAbstract:
La recombinaison méiotique n'est pas distribué de manière aléatoire le long des chromosomes, mais est caractérisée par des domaines froids et chauds qui limitent la diversité génétique transmise par les gamètes. Cependant, le profil de la recombinaison méiotique peut être modifiée, étant donné que la fusion de l’ endonucléase Spo11 au domaine de liaison à l'ADN de Gal4 est suffisante pour favoriser la formation des cassures double brin (CDB) et la recombinaison à proximité des sites de liaison de Gal4, dans la levure et dans les souris. Ici, dans la levure Saccharomyces cerevisiae, nous avons étudié l'effet de la fusion de Spo11 à 8 protéines de liaison à l'ADN lors de la méiose. Comme modules de ciblage, nous avons utilisé des facteurs de transcription de levure et des protéines artificiels de liaison à l'ADN (TALEs et ZFs), qui sont apparus comme des outils efficaces pour faire varier la position et / ou le nombre de sites ciblés. Lors de l'expression de chacun des fusions Spo11, nous avons examiné la progression de la méiose, la formation des CDB dans les sites naturels et ciblées ainsi que le niveau relatif de la recombinaison méiotique. Ce travail dans l’organisme modèle levure ouvre de nouvelles voies pour modifier la recombinaison méiotique chez d'autres organismes, tels que des mammifères et des plantes, pour augmenter la diversité génétique dans les sites d'intérêt et disséquer l'information génétique, en surmontant les limitations dues à la liaison génétiqueMeiotic recombination is not randomly distributed along the chromosomes, but is characterized by hot and cold domains that limit the genetic diversity transmitted by the gametes. However, the recombination profile can be modified, since the tethering of Spo11 endonuclease, upon fusion to the Gal4 DNA-binding domain, is sufficient to enhance DSB formation and recombination near several Gal4 consensus binding sites, in yeast and in mouse. Here, in the yeast Saccharomyces cerevisiae, we studied the effect of Spo11 fusions to 8 different DNA-binding proteins during meiosis. As targeting modules, we used yeast full-length transcription factors and artificial DNA-binding modules (TALEs and ZFs), which emerged to be efficient tools to vary the location and /or the number of targeted sites. Upon expression of each of the Spo11 fusions, we examined meiotic progression, DSB formation at natural and targeted sites as well as the relative level of meiotic recombination. This work in the yeast model opens new avenues to modify meiotic recombination in other organisms, such as mammals and plants, to boost genetic diversity at sites of interest and to dissect the genetic information, overcoming the restrictions due to the genetic linkage
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Joswala, Swetha Ramani. "INVESTIGATION INTO THE MEIOTIC ROLES OF COHESIN AND CENTROMERE PROTEINS IN CAENORHABDITIS ELEGANS." Cleveland State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=csu1611678282003653.
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Fullerton, Donna Lynn. "Investigation of the expression of DMC1, a meiotic gene, in trichomonas vaginalis." Scholarly Commons, 2007. https://scholarlycommons.pacific.edu/uop_etds/664.
Full textAbstract:
T. vaginalis is a protozoan parasite without an observed sexual stage in its life cycle. However, T. vaginalis has genes, such as Dmc1, known to be involved in meiosis in other organisms. In order to look at the expression of these genes in T. vaginalis, RT-PCR was done using purified mRNA. It shows that Dmc1 is expressed in both normal and drug treated cells. However, relative levels are unclear. Localization studies were done in T. vaginalis using immunofluorescence against Dmc1 protein with an HA tag. These studies showed that recombinant Dmc1 remained in the cytoplasm upon treatment with DNA damaging drugs. Additionally, T.vaginalis Dmc1 protein was expressed and purified from E.coli to have polyclonal antibodies made to use in further immunofluorescence studies.
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Hoja, Mary-Rose. "Proteins influencing the integrity of meiotic chromosome dynamics /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-269-8/.
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Susiarjo, Martha. "IDENTIFICATION AND CHARACTERIZATION OF ESTROGEN-MEDIATED EFFECTS ON FEMALE MEIOSIS: STUDIES OF BISPHENOL A AND ESTROGEN RECEPTORS." Connect to text online, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1158685403.
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Brown, Simon D. "Genetic and environmental determinants of meiotic recombination outcome in the fission yeast, Schizosaccharomyces pombe." Thesis, University of Aberdeen, 2017. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=236433.
Full textAbstract:
Meiosis is the process by which sexually-reproducing organisms ensure that precisely half a chromosome set is passed from each parent to the following generation; this circumvents the doubling of the genome that would otherwise occur upon fertilisation. Meiosis occurs via a single round of DNA replication followed by two successive chromosome segregation events. In the first segregation, homologous chromosomes align and become physically linked through the process of meiotic recombination, which is crucial for the accurate segregation of homologous chromosomes. During the second round of segregation, sister chromatids are segregated to produce four haploid daughter cells. Failure to physically tether homologous chromosomes to each other through meiotic recombination can result in the aberrant segregation of homologous chromosomes, which can cause hereditary diseases (aneuploidies) and miscarriages in humans. Meiotic recombination also shuffles alleles of the parental chromosomes, which is crucial for evolution. The study of meiotic recombination, and its regulation, is thus paramount for our understanding of how genetic diversity is generated within populations. The work in this thesis has helped characterise factors, both genetic and environmental, that modulate meiotic recombination in the fission yeast, Schizosaccharomyces pombe. Here, I identify temperature as a major determinant of meiotic recombination outcome; when meiosis is performed at 16°C, significant reductions in meiotic recombination outcome are observed relative to meiosis performed at higher temperatures. Additionally, I present genetic and cytological evidence that the strand resection and strand invasion steps of meiotic recombination are impaired at 16°C relative to higher temperatures, but that double strand break levels appear not to be influenced by temperature. I have also characterised several novel genes predicted to be involved in meiotic recombination, and explored the genetic relationship between several genes already known to be crucial in modulating meiotic recombination. Finally, I have laid the foundations for a future project aiming to map the meiotic recombination landscape across the entire S. pombe genome.
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Griffin, Catherine Helen. "Investigation of natural genetic modifiers of meiotic crossover frequency in Arabidopsis thaliana." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/277143.
Full textAbstract:
Meiotic recombination, known as crossover, is a vital mechanism for generating genetic diversity in sexually reproducing populations. Recombination events are non-uniform across the genome, due to a variety of influences including chromatin structure, DNA-sequence, epigenetic marks and interference from other recombination events. These known factors do not fully explain the distribution of recombination events, and additionally do not account for all the variability in recombination frequency observed both between and within species. Furthermore, of the mechanisms that have been identified, many are not yet fully understood. In Arabidopsis thaliana, considerable variation is observed in recombination frequency and distribution between natural accessions. By investigating recombination events in A.thaliana, this project aimed to identify trans-acting modifiers of recombination frequency that varied between natural accessions. Identification of meiotic recombination modifiers was performed through Quantitative Trait Loci (QTL) mapping in A.thaliana natural-accession cross populations. Populations were generated from crosses between two accessions which differed significantly for recombination frequency as measured across a defined region of the genome flanked by a fluorescent-reporter system. F1 plants were then self-fertilised to produce segregating mosaic F2 populations for mapping. Recombination frequency for specific genomic intervals was determined for each individual in the population through measurement of the segregation of flanking fluorescence-genes expressed in the products of meiosis - seeds or pollen. Individuals were also genotyped using accession-specific markers across the genome, at a marker density of one marker per 2-5Mb, depending on the chromosome. Association of variation in recombination frequency with specific sections of the genome differing between the parental accessions through QTL mapping revealed significant modifiers of meiotic recombination segregating within the populations. This resulted in the identification of three significant large-effect modifiers that differed between Col-0 and Cvi-0 accessions, on chromosomes 1 ,2 and 5, affecting recombination in an interval in the sub-telomere region of chromosome 3. An additional modifier on chromosome 4 affecting the same sub-telomeric interval was identified that differed between the Col-0 and Can-0 accessions. Further fine-mapping of modifiers to improve location resolution was performed by repeated backcrosses into the Col-0 genetic background to remove the influence of other large-effect QTL and possible unknown small-effect modifiers. Improving the resolution provided a number of potential candidates for genes underlying the recombination phenotype for each QTL. Candidate testing was then performed, either through transformation of different accession alleles into the fluorescent-reporter system, or through analysis of T-DNA insertion lines that interrupted candidate genes. Preliminary results from T-DNA insertion mutants crossed to the fluorescent-reporter system suggest a potential role for the AT2G31510 gene in modification of meiotic recombination frequency, though the mode of action remains unknown. These results demonstrate the presence of large-effect modifiers of meiotic recombination frequency that vary between the natural A.thaliana accessions Col-0, Cvi-0 and Can-0. Confirmation of underlying genes or sequence elements and characterisation of their mechanism of action are opportunities for exploration in future experiments.
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Lahouze, Benoit. "Role of DNA methylation in meiotic recombination in Arabidopsis thaliana." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA112128/document.
Full textAbstract:
Pendant la méiose, la division cellulaire qui forme les cellules haploïdes, les chromosomes homologues hérités de chacun des deux parents sont appariés et échangent des segments réciproques appelés crossing-overs (CO). Les CO ne sont pas distribués au hasard dans le génome et leur taux varie le long des chromosomes. Certains des mécanismes responsable ont été décrits chez les mammifères et la levure mais ne sont pas conservés chez les plantes. Les CO sont fortement inhibés dans l'hétérochromatine qui est riche en éléments répétés. Le degré élevé de méthylation d l'ADN qui caractérise les séquences répétées pourrait être un inhibiteur des CO. Cela a été clairement démontré chez le champignon Ascobolus immersus et des études récentes ont montré que la perte de méthylation modifiait la distribution des CO chez Arabidopsis thaliana. Le but de ma thèse a été de décrire plus précisément le rôle de la méthylation de l'ADN dans le contrôle des CO en l'absence de polymorphisme de séquence qui affecte aussi la recombinaison.Pour cela, j'ai mesuré la recombinaison dans différentes plantes dans lesquelles la méthylation de l'ADN a été partiellement ou totalement enlevée grâce à la mutation du gène ddm1. Pour tester l'effet opposé d'un gain de méthylation, j'ai aussi essayé de cibler la methylation de l'ADN à un point chaud de recombinaison connu. Mes résultats montrent que la parte de la méthylation de l'ADN entraîne une augmentation globale de la recombinaison. Paradoxalement, l'heterochromatine qui est normalement très méthylée est moins affectée par la perte de méthylation que le reste du chromosome, probablement car la méthylation de l'ADN a des effets à distance. L'augmentation de CO est accentuée dans les générations successives du mutant ddm1. Cependant, l'effet le plus important est observé dans les hétérozygotes où la moitié du génome seulement est hypométhylée, ce qui suggère un rôle complexe de la méthylation. Finalement, j'ai pu montrer que le polymorphisme affecte la recombinaison surtout dans l'hétérochromatine mais pas dans le sens attendu puisque les plantes homozygotes recombinent moins que les plantes hétérozygotesDuring meiosis, the cellular division that gives rise to haploid cells, homologous chromosomes inherited from each parent are paired and are subjected to reciprocal exchanges of chromosome segments called crossing-overs (COs). COs are not randomly distributed in the genome. Some of the involved mechanisms have recently been described in mammals and yeast bu they are not conserved in plants. Repeat-rich heterochromatin is suppressed for COs. The high level of DNA methylation associated with repeats could be an inhibitor of COs. This was clearly demonstrated in the fungus Ascobolus immersus and recent studies have shown that the loss of DNA methylation also affects COs in Arabidopsis thaliana. The aim of my thesis was to describe more precisely the role of DNA methylation in the control of CO distribution in the absence of any DNA sequence polymorphism which are known to affect recombination. For this purpose, I measured recombination in different plants where DNA methylation has been partially or completely removed thanks to the mutation of the DDM1 gene. To test the opposed effect of a gain of DNA methylation,.I also tried to target DNA methylation at a known recombination hotspot. My results show that the loss of DNA methylation induces a global increase of recombination. Paradoxically, the normally highly methylated heterochromatin is less affected by this loss than the rest of the chromosome, probably because DNA methylation has distal effects. The increased recombination is exacerbated in successive generations of the hypomethylated ddm1 mutants. However, the strongest effect is seen in the heterozygotes where only half of the genome is hypomethylated, suggesting a complex role in the control of CO distribution. Finally, I show that DNA sequence polymorphism affects mainly recombination in the heterochromatin but not in the expected sense, since homozygous plants recombine less than heterozygous
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Runge, Erika. "XGef interacts with and is involved in Ringo's influence on meiotic maturation in Xenopus laevis oocytes." Thesis, Boston College, 2009. http://hdl.handle.net/2345/bc-ir:104432.
Full textAbstract:
Thesis advisor: Laura HakeThe completion of meiosis in Xenopus oocytes requires the coordinated translation of stored mRNAs. CPEB, the cytoplasmic polyadenylation element binding protein, controls the translation of developmentally important early-class maternal mRNAs. Resumption of meiosis through stimulation with progesterone leads to the phosphorylation and activation of CPEB. This results in the lengthening of the poly(A) tails and translation of mRNAs containing the cytoplasmic polyadenylation element (CPE). XGef, a putative guanine nucleotide exchange factor, binds to and is required for CPEB activation. Translation of c-mos, a MAPK kinase kinase, is controlled by CPEB, and activation of the Mos/MAPK pathway is required for meiotic maturation. In addition, the synthesis of Ringo protein, an atypical cdk binding protein and activator, is required for progesterone-induced maturation, though Ringo is able to stimulate resumption of meiosis independent of progesterone. Although much work has been done to understand the key events leading to activation of maturation promoting factor (MPF) and meiotic maturation, the events immediately following progesterone stimulation remain unclear, particularly regarding the role of XGef. The work that follows describes experiments performed to further understand the role of XGef in meiotic maturation through both Ringo and MAPK activity. It was found that XGef and Ringo interact directly and form a complex throughout early meiosis. XGef is involved in Ringo’s influence during meiosis, specifically through MEK-activation of MAPK. Notably, XGef functions in a common pathway and complex with Ringo most likely to influence CPEB phosphorylation and activation
Thesis (BS) — Boston College, 2009
Submitted to: Boston College. College of Arts and Sciences
Discipline: College Honors Program
Discipline: Biology
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Yue, Jicheng. "New insights into the MAPK function in meiotic progression and the regulation of osmostress-induced apoptosis in Xenopus oocytes." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/285548.
Full textAbstract:
En el organismo modelo Xenopus laevis, los oocitos de estadio VI están parados indefinidamente en la profase (G2/M) de la meiosis I hasta que se produce una adecuada estimulación hormonal. Una red de regulación positiva alrededor de la cascada Mos/MEK/ERK asegura la maduración (GVBD) de los oocitos tras la estimulación con progesterona. Sin embargo, el papel de las MAPK de estrés JNK y p38 en la progresión meiótica no es tan claro. Aquí analizamos una proteína de 42 kDa detectada con anticuerpos pJNK (XpJNK-p42) que aparece alrededor del GVBD en oocitos tratados con progesterona. La expresión ectópica de MEKK1 constitutivamente activo acelera la maduración de los oocitos mediante la activación de las vías de señalización de p38 y ERK, pero no de la cascada JNK. Por otra parte, cuatro transcritos diferentes de JNK3 presentes en los oocitos no sintetizan proteína y no se activan durante la maduración inducida por progesterona. El análisis de espectrometría de masas indica que XpJNK-p42 es en realidad ERK2 fosforilado. Curiosamente, el anticuerpo pJNK sólo reconoce pERK2 en oocitos maduros pero no en oocitos expuestos al shock hiperosmótico, lo que sugiere que una modificación post-traduccional de pERK2 tiene lugar durante la progresión meiótica. Es importante destacar que ni la sobreexpresión de ERK2 ni el inhibidor de JNK SP600125 afectan a la fosforilación de c-Jun utilizando extractos de oocitos maduros. En conclusión, las proteínas JNK no están involucradas en la maduración de los oocitos de Xenopus, y la fosforilación de c-Jun detectada en oocitos maduros es independiente de JNK y ERK2.
Hemos descrito previamente que el shock hiperosmótico induce apoptosis en oocitos de Xenopus mediante la activación de cuatro vías independientes: p38, JNK, calpainas y liberación de Smac/DIABLO. También hemos descrito que la activación de p38, JNK1-1 y JNK1-2 es claramente pro-apoptótica. Sin embargo, varias horas después del shock hiperosmótico la isoforma JNK1-2 desaparece, sugiriendo algún tipo de degradación. Además, los estudios anteriores no consideraron el papel de los miembros de la familia de Bcl-2 en la apoptosis inducida por estrés osmótico. Aquí mostramos que pJNK1-2 es proteolizada en Asp385 por caspasa-3, y que la proteína resultante acelera la liberación del citocromo c y la activación de caspasa-3, creando así un bucle de retroalimentación positiva. También mostramos que la sobreexpresión de Bcl-xL en oocitos de Xenopus protege a estos de la apoptosis inducida por shock hiperosmótico. Los oocitos que expresan Bid en combinación con Bcl-xL, presentan tres formas distintas de Bid: no ubiquitinada, mono- y biubiquitinada. Todas las formas de Bid se localizan en el citosol y la mitocondria. El shock hiperosmótico incrementa ligeramente, y de forma rápida, las formas mono- y biubiquitinadas de Bid en la mitocondria. Posteriormente, una pequeña parte de Bid es proteolizada en Asp52, probablemente por una caspasa iniciadora, generando un fragmento N-terminal (nBid) y un fragmento C-terminal altamente pro-apoptótico (tBid). Cuando el citocromo c es liberado y se activa la caspasa-3 se produce una proteólisis masiva de Bid (formas no ubiquitinada y monoubiquitinada) en Asp52, mediada por caspasa-3, creando así otro bucle de retroalimentación positiva. Aunque algunos experimentos sugieren que la forma no ubiquitinada de Bid se proteoliza más rápido y es más pro-apoptótica que las otras formas, los efectos funcionales de la ubiquitinación en la regulación de la apoptosis no son tan claros. Sin embargo, la función pro-apoptótica de Bid se atenúa notablemente en el mutante Bid-D52N, que no puede ser proteolizado por caspasas. En conclusión, la activación de caspasa-3 inducida por shock hiperosmótico pone en marcha dos bucles de retroalimentación positiva mediante la proteólisis de JNK1-2 y Bid, promoviendo así la muerte irreversible de los oocitos.In the model organism Xenopus laevis, oocytes at stage VI are standing in prophase (G2/M) of meiosis I indefinitely until proper hormone stimulation. A positive regulation network around the Mos/MEK/ERK cascade ensures the rapid maturation (GVBD) of the oocytes upon stimulation with progesterone. However, for the stress associated MAPK families, JNK and p38, their involvement in meiotic resumption is not so clear. Here we analyze a protein of 42 kDa detected by pJNK antibodies (XpJNK-p42) that appears around GVBD in progesterone treated oocytes. Ectopic expression of a constitutively active MEKK1 accelerates oocyte maturation through activation of the p38 and ERK signaling pathways, but not the JNK cascade. Moreover, four dormant JNK3 transcripts are described in Xenopus oocytes and none of them are activated during progesterone-induced oocyte maturation. Protein mass spectrometry analysis indicates that XpJNK-p42 is actually phosphorylated ERK2. Intriguingly, the pJNK antibody only recognizes pERK2 in mature oocytes but not in oocytes exposed to hyperosmotic shock, suggesting that a posttranslational modification of pERK2 occurs during meiotic progression. Importantly, neither ERK2 overexpression nor JNK inhibitor SP600125 affects c-Jun phosphorylation detected in mature oocytes extracts. In conclusion, JNK proteins are not involved in Xenopus oocyte maturation, and the phosphorylation of c-Jun detected in mature oocytes is independent of JNK and ERK2. We previously reported that hyperosmotic shock induces apoptosis in Xenopus oocytes through activation of four independent pathways: p38, JNK, calpains and Smac/DIABLO release. We also reported that activation of p38β, JNK1-1, and JNK1-2 is clearly pro-apoptotic. However, several hours after hyperosmotic shock the JNK1-2 isoform disappears, suggesting some type of degradation during cell death. In addition, our previous studies did not address the role of the Bcl-2 family members in the regulation of cytochrome c release. Here we show that Xenopus pJNK1-2 is proteolyzed at Asp385 by caspase-3, and the resulting cleaved protein accelerates cytochrome c release and caspase-3 activation, thus creating a positive feedback loop. We also show that overexpression of Bcl-xL in Xenopus oocytes protect from osmostress-induced apoptosis. In oocytes expressing Bid in combination with Bcl-xL, three different types of Bid are detected: non-ubiquitinated Bid, mono- and bi-ubiquitinated Bid. All Bid types reside both in the cytosol and the mitochondria. Hyperosmotic shock rapidly induces a slight increase of mono- and bi-ubiquitinated Bid in the mitochondria. Subsequently, Bid is cleaved at Asp52 at very low levels, probably by an initiator caspase, generating an N-terminal fragment (nBid) and a highly pro-apoptotic C-terminal fragment (tBid). When cytochrome c is released and caspase-3 is activated a massive proteolysis of non-ubiquitinated and mono-ubiquitinated Bid occurs at Asp52, mediated by caspase-3, thus creating another positive feedback loop. Although some experiments suggest that non-ubiquitinated Bid is proteolyzed faster and is more pro-apoptotic than wild type Bid, the functional effects of Bid ubiquitination are not so clear. However, the pro-apoptotic function of Bid is markedly attenuated in mutant Bid-D52N that is not cleaved by caspases, indicating that Bid proteolysis regulates osmostress-induced apoptosis. In conclusion, caspase-3 activation induced by hyperosmotic shock engages two positive feedback loops through the cleavage of JNK1-2 and Bid, thus promoting an irreversible death of the oocytes.
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Pightling, Arthur William. "The evolutionary history of meiotic genes: early origins by duplication and subsequent losses." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/2960.
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Meiosis is necessary for sexual reproduction in eukaryotes. Genetic recombination between non-sister homologous chromosomes is needed in most organisms for successful completion of the first meiotic division. Proteins that function during meiotic recombination have been studied extensively in model organisms. However, less is known about the evolution of these proteins, especially among protists. We searched the genomes of diverse eukaryotes, representing all currently recognized supergroups, for 26 genes encoding proteins important for different stages of interhomolog recombination. We also performed phylogenetic analyses to determine the evolutionary relationships of gene homologs. At least 23 of the genes tested (nine that are known to function only during meiosis in model organisms) are likely to have been present in the Last Eukaryotic Common Ancestor (LECA). These genes encode products that function during: i) synaptonemal complex formation; ii) interhomolog DNA strand exchange; iii) Holliday junction resolution; and iv) sister-chromatid cohesion. These data strongly suggest that the LECA was capable of these distinct and important functions during meiosis. We also determined that several genes whose products function during both mitosis and meiosis are paralogs of genes whose products are known to function only during meiosis. Therefore, these meiotic genes likely arose by duplication events that occurred prior to the LECA. The Rad51 protein catalyzes DNA strand exchange during both mitosis and meiosis, while Dmc1 catalyzes interhomolog DNA strand exchange only during meiosis. To study the evolution of these important proteins, we performed degenerate PCR and extensive nucleotide and protein sequence database searches to obtain data from representatives of all available eukaryotic supergroups. We also performed phylogenetic analyses on the Rad51 and Dmc1 protein sequence data obtained to evaluate their utility as phylogenetic markers. We determined that evolutionary relationships of five of the six currently recognized eukaryotic supergroups are supported with Bayesian phylogenetic analyses. Using this dataset, we also identified ten amino acid residues that are highly conserved among Rad51 and Dmc1 protein sequences and, therefore, are likely to confer protein-specific functions. Due to the distributions of these residues, they are likely to have been present in the Rad51 and Dmc1 proteins of the LECA.
To address an important issue with the gene inventory method of scientific inquiry, we developed a heuristic metric for determining whether apparent gene absences are due to limitations of the sequence search regimen or represent true losses of genes from genomes. We collected RNA polymerase I (Pol I), Replication Protein A (RPA), and DNA strand exchange (SE) sequence data from 47 diverse eukaryotes. We then compared the numbers of apparent absences to a single measure of protein sequence length and sequence conservation (Smith-Waterman pairwise alignment (S-W) scores) obtained by comparing yeast and human protein sequence data. Using Poisson correlation regression to analyze the Pol I and RPA subunit datasets, we confirmed that S-W scores and apparent gene absences are correlated. We also determined that genes encoding products that are critical for interhomolog SE in model organisms (Rad52, Rad51, Dmc1, Rad54, and Rdh54) have been lost frequently during eukaryotic evolution. Saccharomyces cerevisiae null rad52, dmc1, rad54, and rdh54 mutant phenotypes are suppressed by rad51 overexpression or mutation. If rad51 overexpression or mutation affects other eukaryotes in a similar fashion, this phenomenon may account for frequent losses of genes whose products are critical for the completion of meiosis in model organisms. Finally, we place this work into greater context with a review of hypotheses for the selective forces and mechanisms that resulted in the origin of meiosis. The review and the data presented in this thesis provide the basis for a model of the origin of meiotic genes in which meiosis arose from mitosis by large-scale gene duplication, following a preadaptation that served to reduce increased numbers of chromosomes (from diploid to haploid) caused by erroneous eukaryotic cell-cell fusions.
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Hartl, Tom A. "CONDENSIN II CHROMOSOME INDIVIDUALIZATION IS NECESSARY FOR MEIOTIC SEGREGATION AND ANTAGONIZES INTERPHASE CHROMOSOME ALIGNMENT." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/195995.
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Maintenance of an intact genome and proper regulation of the genes within are crucial aspects for life. The work of this dissertation has implicated the Drosophila condensin II complex in both processes. Condensin II's ability to reconfigure chromosomes into spatially separated and discrete units is necessary to ensure proper meiotic segregation. When this "individualization" activity fails in a condensin II mutant, chromosomes remain entangled, and either cosegregate or become lost during cell division. This leads to the creation of aneuploid sperm. We have also implicated condensin II as a factor necessary to individualize interphase somatic chromosomes from one another. This is relevant in Drosophila because the association of homologous chromosomes is thought to facilitate gene regulation activity in trans. We speculate that condensin II individualization spatially distances aligned chromosomes from one another and prevents this trans-communication between allelic loci. This is supported first by an increase of homologous chromosome pairing in a condensin II mutant background. Secondly, loss of condensin II leads to elevated production from alleles that are known to depend on pairing for transcriptional activation. These meiotic and interphase condensin II roles support its necessity to Drosophila genome integrity and transcriptional regulation. Given the conservation of condensin from bacteria to humans, it is likely that equivalent or related roles exist in a variety of species.
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Altemose, Nicolas Frank. "Novel genetic and molecular properties of meiotic recombination protein PRDM9." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:1afe17c3-5f75-4166-8697-7da1471a5230.
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Meiotic recombination is a fundamental biological process in sexually reproducing organisms, enabling offspring to inherit novel combinations of mutations, and ensuring even segregation of chromosomes into gametes. Recombination is initiated by programmed Double Strand Breaks (DSBs), the genomic locations of which are determined in most mammals by PRDM9, a rapidly evolving DNA-binding protein. In crosses between different mouse subspecies, certain Prdm9 alleles cause infertility in hybrid males, implying a critical role in fertility and speciation. Upon binding to DNA, PRDM9 deposits a histone modification (H3K4me3) typically found in the promoters of expressed genes, suggesting that binding might alter the expression of nearby genes. Many other questions have remained about how PRDM9 initiates recombination, how it causes speciation, and why it evolves so rapidly. This body of work investigates these questions using complementary experimental and analytical methodologies. By generating a map of human PRDM9 binding sites and applying novel sequence analysis methods, I uncovered new DNA-binding modalities of PRDM9 and identified sequence-independent factors that predict binding and recombination outcomes. I also confirmed that PRDM9 can affect gene expression by binding to promoters, identifying candidate regulatory targets in meiosis. Furthermore, I showed that PRDM9âÃôs DNA-binding domain also mediates strong protein-protein interactions that produce PRDM9 multimers, which may play an important functional role. Finally, by generating high-resolution maps of PRDM9 binding in hybrid mice, I provide evidence for a mechanism to explain PRDM9-mediated speciation as a consequence of the joint evolution of PRDM9 and its binding targets. This work reveals that PRDM9 binding on one chromosome strongly impacts DSB formation and/or repair on the homologue, suggesting a novel role for PRDM9 in promoting efficient homology search and DSB repair, both critical for meiotic progression and fertility. One consequence is that PRDM9 may play a wider role in mammalian speciation.
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James, Rosalina Dee. "Cohesin proteins SMC1 and SMC3 : roles in aneuploidy and in meiotic chromosome dynamics /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/6333.
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Galland, Lanie Maria. "Investigation of chromosome size effect on the rate of crossovers in the meiotic yeast Saccharomyces cerevisiae." DigitalCommons@CalPoly, 2014. https://digitalcommons.calpoly.edu/theses/1193.
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Meiosis is a specialized type of cell division characterized by a single round of DNA replication and two rounds of chromosome segregation, ultimately resulting in four haploid cells. During meiosis I, chromosomes align and reciprocal recombination results in the formation of a crossover, creating the tension required to properly segregate homologs during the first round of meiosis.
Two mechanisms involved in regulating the occurrence of crossing over are assurance and interference. Crossover assurance describes the phenomenon that at least one crossover will form between each pair of homologous chromosomes during prophase I. Crossover interference, on the other hand, describes the nonrandom placement of crossovers between homologs, increasing the probability that a second crossover will occur at a discrete distance away from the first one.
In addition to assurance and interference, chromosome size may play a role in the rate of meiotic recombination during prophase I. As a result of crossover assurance, small chromosomes receive a minimum of one crossover, the obligate crossover. Assuming chromosome size does not influence the rate of recombination, pairs of large chromosomes should experience the same number of crossovers per base pair as small chromosomes. Previous studies have been inconsistent: Kaback et al. (1999) saw decreased rates of crossing over between large chromosomes relative to small ones, suggesting that crossover interference acts across a larger distance on large chromosomes. Turney et al. (2004), however, saw no such effect, suggesting that these findings may be site- or sequence-specific.
The current study used the Cre-loxP system to create translocated chromosomes, decreasing the size of chromosome VIII from 562 kb to 125 kb. The rate of crossing over was evaluated using nutrient marker genes that were inserted on the left arm of chromosome VIII to facilitate phenotypic detection of crossing over between homologous translocated chromosomes in comparison to crossing over between homologous nontranslocated chromosomes.
Translocated strains were attempted, though further testing suggests that the translocation itself may be lethal. In the future, we plan to further investigate the potential lethal nature of the translocation.
We also experienced difficulty in curing yeast cells of the Cre expression plasmid: as pSH47 was removed, translocated chromosomes reverted to nontranslocated chromosomes. In addition, crossing over in nontranslocated yeast, along with subsequent molecular analysis, revealed that one of the marker genes presumed to be on the left arm of chromosome VIII is, in fact, located on a different chromosome, preventing analysis of crossing over in this region. As a result, we were unable to proceed with current experimentation.
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Brockway, Heather Marie. "A role for the CSN/COP9 signalosome in synaptonemal complex assembly and meiotic progression." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/1296.
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Defects in meiotic prophase I events, resulting in aneuploidy, are a leading cause of birth defects in humans; however, these are difficult to study in mammalian systems due to their occurrence very early in development. The nematode, Caenorhabditis elegans, is an excellent model for prophase I studies as its gonad is temporally and spatially organized around these meiotic events. Homolog pairing, synapsis, meiotic recombination and crossover formation are essential to the proper segregation of chromosomes into the respective gametes, either the egg or sperm. Disturbances in these events leads to missegregation of chromosomes in the gametes in the meiotic divisions. Synapsis is especially critical in meiosis as it precedes and is required for meiotic recombination in C. elegans. The formation of the synaptonemal complex (SC) is fundamental to chromosomal synapsis, yet the molecular mechanisms of synaptonemal complex morphogenesis are largely unknown. The investigations described in this thesis were undertaken to better understand the molecular contributions to synaptonemal complex morphogenesis. Chapter One reviews knowledge of morphogenesis and its relationship to the events of meiotic prophase I. Recent studies in our laboratory have implicated AKIRIN, a nuclear protein with multiple biological functions, as having a role in synaptonemal complex disassembly, specifically preventing the aggregation of synaptonemal proteins (Clemons et al., 2013). As a result of our efforts to discern the mechanism by which AKIRIN regulates disassembly, we found that the highly conserved CSN/COP9 signalosome has a role in SC assembly, leading to defects in prophase I events and in MAPK signaling , leading to the arrest of nuclei in the later stages of meiosis. While the CSN/COP9 signalosome has been implicated in general fertility in C. elegans (Pintard et al., 2003), no role had been defined in earlier meiotic stages until this study. Chapter Two describes an RNAi enhancer/suppressor screen undertaken in the akir-1 mutant background. Several RNAi clones were selected for future study based on a reduction in brood size; one of which, csn-5/, is the focus of the analysis presented in Chapter 3.
Chapter Three describes the phenotypic characterization of two CSN/COP9 signalosome subunits, csn-2 and csn-5. Alleles of both genes display synaptonemal complex protein aggregation and defects in mitotic cell proliferation, homologous chromosome pairing, meiotic recombination and crossover formation, leading to an increase in apoptosis. Oocyte maturation is also disrupted by a lack of MAPK signaling, resulting in a lack of viable oocytes, which renders the csnmutant homozygotes sterile. These findings support a model suggesting the CSN/COP9 signalosome has an essential role in regulating meiotic prophase I events and oocyte maturation.
Chapter 4 describes the methodology used in this study.
Chapter 5 provides a summary of the thesis findings and examines the future directions to extend this work.
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Bouftas, Nora. "Control of meiotic divisions in oocytes : a novel role for cyclin B3." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS176.
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La méiose est un processus très réglementé composé de deux divisions successives, la méiose I et II, qui doivent être complétées dans l’ordre pour obtenir des gamètes haploïdes avec le nombre correct de chromosomes. La méiose chez les femelles est un processus sujet aux erreurs, où les erreurs de ségrégation créent des gamètes aneuploïdes. De plus, l'incidence d'aneuploïdie augmente avec l'âge. Comprendre la régulation de la méiose chez les femelles mammifères est donc essentiel. Les divisions méiotiques sont régulées par les cyclines associées à leurs partenaires catalytiques, les Cdks. J'ai étudié le rôle d'une cycline unique, la cycline B3, grâce à l'utilisation de souris cycline B3 KO. J'ai trouvé que l’absence d'activité de cycline B3-Cdk1 dans les ovocytes KO affecte l'activité de l'APC/C et induit un arrêt en métaphase I en raison des taux élevés de cycline B1, de l'activité de la Cdk1 et de la séparase inactive. Étonnamment, la cycline B3 d’autres espèces a pu sauver le phénotype des ovocytes de cycline B3 KO. J'ai aussi pu montrer que la cycline B3 était capable d'inhiber l'arrêt CSF. Les données récentes suggèrent que les ovocytes KO entraînent un arrêt précoce en CSF conduisant à l’arrêt en métaphase I observé. Mon travail de thèse a donc montré que la cycline B3 est essentielle pour la méiose I chez les femelles et pour empêcher un arrêt CSF précoce en méiose IMeiosis is a tightly regulated process made up of two successive divisions, meiosis I and II. They must be completed in an orderly manner to obtain haploid gametes with the correct number of chromosomes. Female mammalian meiosis is an error-prone process where errors in segregation create aneuploid gametes. In addition, incidence of aneuploidy increases in correlation with age. Understanding the regulation of female mammalian meiosis is therefore essential. Meiotic cell divisions are regulated by cyclins associated to their binding catalytic partners Cdks. I investigated the role of a unique cyclin, cyclin B3, through the use of cyclin B3 KO female mice. I found that lack of cyclin B3-Cdk1 activity in KO oocytes affects APC/C activity and induces an arrest at metaphase I due to high cyclin B1 levels, high Cdk1 activity, and inactive separase. Surprisingly, cyclin B3 from other species was able to rescue mouse cyclin B3 KO oocytes. I was also able to show that cyclin B3 is able to inhibit CSF arrest. My recent data suggests that cyclin B3 KO oocytes put in place a precocious CSF arrest, leading to the metaphase I arrest observed. Hence, my PhD work has shown that cyclin B3 is essential for female meiosis I and to prevent precocious CSF arrest in meiosis I instead of meiosis II
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Lee, Chih-ying. "Bouquet formation, rapid prophase movements and homologous pairing during meiotic prophase in Saccharomyces cerevisiae." Oklahoma City : [s.n.], 2009.
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Cromer, Laurence. "Etude de deux régulateurs de l’APC/C et de leurs rôles dans le contrôle du cycle cellulaire et de la cohésion lors de la méiose chez Arabidopsis thaliana." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112046/document.
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La méiose est la division cellulaire qui aboutit à la production de gamètes haploïdes. Lors de la méiose, un unique évènement de réplication est suivi de deux divisions afin de réduire la ploïdie. Lors de ces deux divisions, la cohésion entre chromatides sœurs est éliminée de façon séquentielle pour permettre la succession de deux ségrégations de chromosomes équilibrées. La progression du ‘’cycle méiotique’’ est contrôlée par des régulateurs communs à la mitose et à la méiose mais également par des mécanismes nécessitant des protéines spécifiques à la méiose. L’objectif de de mon travail de thèse était de décrypter les mécanismes moléculaires permettant l’enchainement de deux divisions équilibrées pour la production de gamètes haploïdes. Nous avons pu montrer que la protéine OSD1 inhibait l’APC/C pour permettre la progression méiotique. Nous avons également mis en évidence un réseau fonctionnel, comprenant OSD1, CYCA1;2/TAM et TDM, indispensable à trois étapes clés de la progression méiotique chez Arabidopsis ; la transition prophase-méiose I, la transition méiose I-méiose II et la sortie de méiose. Ces travaux ont également permis de caractériser chez Arabidopsis les deux paralogues de Shugoshin, qui sont des protéines conservées et impliquées dans la protection de la cohésion centromérique. Nous avons également identifié Patronus comme un nouveau protecteur de la cohésion centromérique en méiose. Les résultats obtenus suggèrent que Patronus est un régulateur de l’APC/C qui permet d’empêcher l’élimination de la cohésion centromérique en interkinèse méiotiqueMeiosis is a specialized type of cell division that generates haploid gametes. At meiosis, two divisions follow a single DNA replication event leading to ploidy halving. A stepwise sister chromatids cohesion release also occurs to allow the two successive balanced rounds of chromosome segregation. In addition to general cell-cycle regulators, meiosis requires specific proteins. The aim of this thesis was to understand the molecular mechanisms leading to two successive balanced chromosome segregations. We show that OSD1 promotes meiotic progression through APC/C inhibition and we identified a functional network between OSD1, CYCA1;2/TAM and TDM in Arabidopsis. This functional network controls three key steps of meiotic progression; the prophase-meiosis I transition, the meiosis I-meiosis II transition and the meiosis exit. In addition, we characterized the two Arabidopsis thaliana Shugoshin paralogs, which are conserved proteins involved in sister chromatid cohesion protection. We also identified Patronus, an uncharacterized protein, as a novel protector of meiotic centromeric cohesion. We suggest that Patronus is a novel APC/C regulator that prevents cohesins release during meiotic interkinesis. This work identified two APC/C regulators essential for meiosis in Arabidopsis thaliana
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Nore, Alexandre. "Biochemical properties and regulation of the TopoVI-like complex responsible for the initiation of meiotic recombination." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT062.
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Afin de transmettre leurs informations génétiques d'une génération à l'autre, les organismes à reproduction sexuée doivent réduire de moitié leur contenu chromosomique pour former des gamètes haploïdes. Cette réduction se produit lors d'une division cellulaire appelée méiose, durant laquelle une étape de réplication est suivie de deux divisions successives, la méiose I et II. Au cours de la méiose I, les chromosomes homologues se séparent et leur bonne ségrégation dépend de la création entre eux d’un lien physique. En méiose c’est le processus de réparation appelé recombinaison homologue, qui à la suite de l’induction dans le génome de centaine de cassures double brin par la protéine Spo11, permet d’établir ce lien. Spo11 est l'orthologue méiotique de la sous-unité catalytique de la topoisomérase VI, TopoVIA. Comme TopoVI est composée de deux sous-unités, TopoVIA et TopoVIB, l’existence d’un orthologue méiotique de TopoVIB était une question posée depuis l'identification de Spo11. Au cours de ma thèse, j'ai contribué à identifier une nouvelle famille de protéine, que l’on a nommé TopoVIB-like, orthologue à TopoVIB et nécessaire à la formation des cassures double-brin d'ADN méiotiques(Robert et al, 2016). Ces protéines ont des domaines similaires à ceux de TopoVIB, à savoir un GHKL (impliqué dans la liaison et l'hydrolyse de l'ATP), un domaine transducteur et un domaine CTD. Nous avons démontré que chez la souris, SPO11 forme un complexe avec TOPOVIBL. De plus, nous avons démontré que cette protéine est nécessaire à la formation des CDB. Ces résultats suggèrent que chez la souris, les CDB méiotiques sont catalysées par un complexe TopoVI-like. Chez S. cerevisiae, il n'y a pas d'orthologue clair de TopoVIB, mais nous avons trouvé que la protéine Rec102, connue pour être nécessaire à la formation des CDB méiotiques, présente une homologie partielle avec le domaine transducteur des TopoVIB-like. Rec102 forme un complexe avec Rec104, une protéine également requise pour la formation des CDB. Ainsi, nous avons émis l'hypothèse que le complexe Rec102 / Rec104 était l'orthologue méiotique de TopoVIB chez la levure, interagissant avec Spo11 pour former un complexe de type TopoVI-like. Malgré l'importance de Spo11, son mode d'action est mal connu. Cette absence de données biochimiques est due à l’insolubilité de la protéine. Le but de ma thèse était de caractériser le mode d'action et la régulation du complexe TopoVI-like dans la formation des CDB méiotiques. Tout d'abord, biochimiquement, en purifiant in vitro une forme soluble du complexe TopoVI-like de levure composé de Spo11 / Rec102 / Rec104 / Ski8 (un partenaire direct de Spo11) en co-exprimant ces protéines dans deux systèmes d'expression, E. coli et S. cerevisiae. En utilisant E. coli, j'ai réussi à purifier un complexe soluble formé par Spo11 / Rec102 / Rec104 / Ski8 et en utilisant S. cerevisiae, j'ai purifié deux complexes différents, l'un formé par les quatre protéines, et un formé uniquement par Spo11 et Rec102. Néanmoins, les tests d'activité sur différents substrats d'ADN n'ont révélé aucune activité de coupure de l’ADN. Le deuxième objectif de ma thèse était d'étudier comment, chez la souris, TOPOVIBL régule l'activité de SPO11 en interagissant avec d'autres protéines nécessaires à la formation des CDB. En double hybride, j'ai prouvé que, comme chez la levure, l'orthologue méiotique de TopoVIB chez la souris interagissait avec REC114, une autre protéine nécessaire à la formation des CDB. La cartographie de cette interaction à l'échelle de l’acide aminé a conduit à l'identification d'un résidu sur TOPOVIBL essentiel pour l'interaction entre TOPOVIBL et REC114. Afin d'étudier in vivo le rôle de l'interaction entre TOPOVIBL et REC114, une souris mutante pour le résidu identifié de TOPOVIBL a été générée à l'aide de CRISPER-Cas9 et son phénotype a été analyséTo properly transmit their genetic information from one generation to another, sexually reproductive organisms need to halve their genome to form haploid gametes. This reduction occurs during a special cell division called meiosis, which proceeds through one round of DNA replication followed by two successive divisions called meiosis I and II. During meiosis I homologous chromosomes segregate, and their proper segregation depends on the homologous recombination pathway that establishes a physical link between the homologues. During meiosis, homologous recombination events are triggered by the formation of DNA double strand break (DSB) catalyzed by the evolutionarily conserved Spo11 protein. Spo11 is the meiotic ortholog of the catalytic subunit of the TopoVI topoisomerase, TopoVIA. As TopoVI is composed of two subunits, TopoVIA and TopoVIB, the requirement for meiotic DSB formation of a B subunit was under investigation since the identification of Spo11. During my PhD, I contributed to the identification of a new family of protein, the TopoVIB-like family, ortholog to the Topoisomerase VI B subunit (TopoVIB) and required for meiotic DNA double strand break formation (Robert et al, 2016). These proteins share domains in part similar to the canonical TopoVIB which are a GHKL domain (involved in ATP binding and hydrolysis), a transducer domain and a CTD domain. We demonstrated that in mice, SPO11 forms a complex with TOPOVIBL. Biochemical characterization of this complex showed a structure compatible with an A2B2 organization. Furthermore, we demonstrated that this protein is required for meiotic DSB formation. These results suggest the existence, in mice, of a TopoVI-like complex that catalyzes the formation of meiotic DSB. In S. cerevisiae, there is no clear TopoVIB-like ortholog, but we found that the Rec102 protein, which is known to be required for the formation of meiotic DSB, shows a partial homology with the transducer domain of the TopoVIB-like proteins. Rec102 forms a complex with Rec104, a protein also essential for DSB formation. Thus, we hypothesized that the Rec102/Rec104 complex is the yeast meiotic ortholog of TopoVIB, interacting with Spo11 to form a meiotic TopoVI-like complex. Despite the importance of Spo11 little is known about its mode of action. This absence of biochemical data is due to the lack of solubility of the protein. The aim of my PhD was to characterize the mode of action and regulation of the TopoVI-like complex for meiotic DSB formation. First, biochemically, by purifying in vitro a soluble form of the yeast TopoVI-like complex composed by Spo11/Rec102/Rec104/Ski8. To achieve this objective, I co-expressed these proteins in two different expression systems, E. coli and meiotic culture of S. cerevisiae. Using E. coli I managed to purify a soluble complex formed by Spo11/Rec102/Rec104/Ski8, and using meiotic culture of S. cerevisiae, I purified two different complexes, one formed, by the four proteins, and one formed only by Spo11 and Rec102. Nevertheless, in vitro activity essays on different DNA substrates did not reveal any DNA cleavage activity. The second goal of my PhD was to study how in mouse, the activity of TOPOVIBL / SPO11 may be regulated by other proteins known to be required for DSB formation. Using Y2H experiment I was able to prove that, as in yeast, mouse TOPOVIBL interacts with REC114, a protein required for DSB formation. The mapping of this interaction at the amino-acid scale, leads to the identification of one residue on TOPOVIBL essential for the interaction between TOPOVIBL and REC114. In order to investigate in vivo the role of the interaction between TOPOVIBL and REC114, a mutant mouse carrying a mutation in the identified residue of TOPOVIBL was generated using CRISPER-Cas9, and its phenotype analyzed
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Zielinska, Agata Pamela. "Studies in oocytes from three mammalian species demonstrate that meiotic kinetochores are composed of previously unidentified subdomains and reveal two novel mechanisms behind the maternal-age effect in humans." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/285004.
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Poor egg quality is the leading cause of pregnancy loss and Down's syndrome. While even eggs in young women frequently contain an incorrect number of chromosomes and are therefore unlikely to give rise to a viable pregnancy, the incidence of chromosomally abnormal eggs increases strikingly with advancing maternal age. Why egg quality declines dramatically as women approach their forties remains one of the outstanding questions in developmental biology. This PhD thesis demonstrates how unforeseen features of kinetochore organization that are unique to meiosis render this cell division process in mammals particularly prone to errors. Firstly, my results uncovered an unexpected multi-subunit organization of the meiotic kinetochore, which is widely conserved across mammals and biases eggs towards errors. Secondly, I identified two independent mechanisms that predispose eggs from older women to aneuploidy. The first mechanism affects the fidelity of meiosis I. My analysis revealed that human oocytes challenge the paradigm that sister kinetochores are fully fused. Instead, I demonstrated that sister kinetochores disjoin as women get older, which promoted erroneous kinetochore-microtubule attachments. This in turn allowed chromosomes to rotate on the spindle and provided a mechanistic explanation for reverse segregation - a recently discovered meiotic error that is unique to humans. Secondly, I pioneered the use of super-resolution microscopy to study chromosome architecture in human eggs and discovered that individual kinetochores during meiosis II in mammals are composed of previously unidentified subdomains. In young females, these subdomains are joined together by cohesin complexes. With age, kinetochores fragment into two pieces. Fragmented kinetochores frequently attach merotelically to spindle microtubules, which predisposes aged eggs to errors. What severely hinders our progress in identifying causes of human infertility is that numerous features of human meiosis are not represented in mice. To overcome this challenge, I developed an experimental platform to mimic the age-related changes that occur in humans in oocytes from young mice. I achieved this by extending the applications of Trim-Away, a novel method to degrade endogenous proteins even in primary cells, to partially deplete proteins. Furthermore, I established a new experimental model system to study human-like aspects of meiosis in live non-rodent cells in real time: pig oocytes. Together, these results set foundations for new therapeutic approaches to extend reproductive lifespan by counteracting the age-related loss in kinetochore integrity that this study identified. Furthermore, partial Trim-Away and studying meiosis in pigs opens new directions for meiotic research.
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Bolte, Melanie. "Regulation of the anaphase promoting complex (APC-C) in the mitotic and meiotic cell cycle of Saccharomyces cerevisiae." Doctoral thesis, [S.l.] : [s.n.], 2004. http://webdoc.sub.gwdg.de/diss/2004/bolte/bolte.pdf.
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Martínez, Marchal Ana. "Regulation of the oocyte pool in mammals." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667797.
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Durant la oogènesi dels mamífers, les oogònies proliferen forman els anomenats cists. Les oogònies entren en meiosis progressant en la profase I i els cists es trenquen al mateix temps que es produeix una mort massiva perinatal dels oòcits. En la profase I, s’indueixen trencaments de doble cadena (DSBs) per tot el genoma, que son reparats per recombinació homòloga per a promoure la sinapsi dels cromosomes homòlegs. Existeixen diferents mecanismes que s’activen en resposta a errors en aquests processos i que aturen el cicle cel·lular i produeixen l’apoptosi de les cèl·lules danyades. La resposta al dany al DNA (DDR) es activada en presència d’oòcits i d’espermatòcits amb errors de recombinació en l’anomenat checkpoint de recombinació. Per l’altre banda, errors en la sinapsi activen el checkpoint de sinapsi. El nostre objectiu era caracteritzar les funcions de la DDR i del checkpoint de sinapsi durant l’oogènesi en mamífers. Contràriament al que succeeix en espermatòcits, els oòcits presenten un alt número de DSBs no reparats a l’estadi de paquitè en el moment en que es produeix la mort oocitària massiva i el trencament del cists. Per tal d’esbrinar si el checkpoint de recombinació participa en la regulació del número d’oòcits en mamífers, hem analitzat el número de DSBs, el número d’oòcits en femelles perinatals i adultes, el trencament dels cists, la formació de fol·licles i la vida reproductiva de femelles de ratolí control i mutants per a la quinasa efectora de la via de la DDR, la proteïna CHK2. Les nostres dades han revelat la implicació de CHK2 en la regulació del número d’oòcits, però només en ovaris fetals, obrint la possibilitat de l’existència d’una via alternativa regulant el número d’oòcits després del naixement. Els nostres estudis utilitzant ovaris cultivats in vitro en presència d’inhibidors, suggereixen que CHK1 podria compensar l’absència de CHK2 in vivo. Per tant, la via de la DDR controlaria el número d’oòcits en mamífers. A més, hem trobat un augment del número d’oòcits en adultes velles mutants per CHK2 suggerint que la DDR controla la llargada de la vida reproductiva en mamífers. Finalment, hem estudiat el possible paper de TRIP13 en el checkpoint de sinapsi. La proteïna TRIP13 es necessària per a la recombinació, però també per a la sinapsi dels cromosomes sexuals i per a la formació de la vesícula sexual, suggerint un possible rol al checkpoint de sinapsi. Hem analitzat el número d’oòcits en ovaris Spo11-/- Trip13mod/mod i Dmc1-/- Chk2-/- Trip13mod/mod per a esbrinar si TRIP13 es necessària per a activar el checkpoint de sinapsi en femelles. Les nostres dades han revelat un rescat en el número d’oòcits en el triple mutant, però no en el doble. Aquest resultats obren la possibilitat de que TRIP13 participi en el checkpoint de sinapsis, però com a alternativa, proposem que aquesta participació podria ser compatible amb una possible regulació per part de TRIP13 de la elecció de la via de reparació dels DSBs.During mammalian oogenesis, oogonia proliferate forming the so-called cysts. The oogonia enter meiosis progressing through prophase I and the cysts break down concomitantly to massive perinatal oocyte death. During meiotic prophase I, double strand breaks (DSBs) are induced throughout the genome and repaired by homologous recombination to promote the synapsis of the homologous chromosomes. In response to errors in these processes, different response pathways are activated triggering cell cycle arrest or even apoptosis. The DNA damage response (DDR) is activated in response of meiocytes with recombination failure in the recombination checkpoint; while errors in synapsis trigger the synapsis checkpoint. We aimed to characterize the roles of the DDR and synapsis checkpoint in mammalian oogenesis. Contrary to what occurs in spermatocytes, oocytes present high numbers of unrepaired DSBs at pachynema, at the time of the massive oocyte death and cyst breakdown. In order to know if the recombination checkpoint participates in the regulation of the oocyte number in mammals, we analyzed the presence of DSBs, the oocyte number in both perinatal and adult females, the cyst breakdown, the formation of follicles and the reproductive lifespan using control and mutant mice for the effector kinase of the DNA damage response pathway, CHK2. Our data revealed the involvement of CHK2 in the regulation of the oocyte number but only in fetal ovaries prior to birth, raising the question of a possible alternative regulator acting just after birth. Our studies using in vitro ovarian cultures using inhibitors, suggest that CHK1 may compensate the loss of CHK2 perinatally in vivo. Thus, revealing that the DDR pathway controls the oocyte number in mammals. Furthermore, we found an increased number of oocytes in elder Chk2 mutant females suggesting that the DDR controls the reproductive lifespan extension in mammals. Finally, we studied the possible involvement of TRIP13 in the synapsis checkpoint. The protein TRIP13 is required for recombination, but it is also needed for the synapsis of sex chromosomes and the sex body formation. Thus, suggesting a possible role in the synapsis checkpoint. We analyzed the oocyte number in females from Spo11-/- Trip13mod/mod and Dmc1-/- Chk2-/- Trip13mod/mod ovaries in order to infer if TRIP13 is required to implement the synapsis checkpoint in females. Our data revealed a rescue in the number of oocytes in the triple mutant, but not in the double mutant. These results leave open the possibility of a participation of TRIP13 in the synapsis checkpoint, but as an alternative, they could be compatible with a possible role of TRIP13 regulating the DSB repair pathway choice.
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Phizicky, David V. (David Vincent). "Mechanisms preventing DNA replication between Meiosis I and Meiosis II." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/117786.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged student-submitted from PDF version of thesis.
Includes bibliographical references.
The vast majority of multicellular organisms reproduce using sexual reproduction, which requires the production of haploid gametes. These gametes are produced by meiosis, a specialized cell division during which one round of DNA replication is followed by two rounds of chromosome segregation, Meiosis I (MI) and Meiosis II (MII). This imbalance between rounds of DNA replication and chromosome segregation causes diploid cells to produce haploid gametes. In contrast, mitotically-dividing cells maintain ploidy by alternating between rounds of replication and segregation. It is unclear how meiosis accomplishes two sequential chromosome segregation events without an intervening round of DNA replication. In mitotic cells, both DNA replication and chromosome segregation are regulated by oscillations of cyclin-dependent kinase (CDK) activity. Both events initiate during G1 due to the associated low CDK-activity state, and both events are completed later in the cell cycle due to increased CDK activity. During meiosis, uncoupling replication and segregation presents a unique problem. After completion of MI, CDK activity decreases and then increases to drive MII chromosome segregation. However, DNA replication must remain inhibited between MI and MII. Given that an oscillation of CDK activity is sufficient for genome re-duplication in mitotic cells, I sought to understand how meiotic cells prevent DNA replication while resetting the chromosome segregation program. In this thesis, I show that meiotic cells inhibit two distinct steps of DNA replication: (1) loading of the replicative helicase onto replication origins, and (2) activation of the replicative helicase. CDK and the meiosis-specific kinase Ime2 cooperatively inhibit helicase loading during the meiotic divisions, and their simultaneous inhibition causes inappropriate helicase reloading. Further studies of Ime2 revealed two mechanisms by which it inhibits this process. First, I showed that Ime2-phosphorylation of the helicase directly inhibits its loading onto origins. Second, Ime2 cooperated with CDK to transcriptionally and proteolytically repress Cdc6, an essential helicase-loading protein. In addition, I found that meiotic cells use CDK and the polo-like kinase Cdc5 to promote degradation of Sld2, an essential helicase-activation protein. Together, these data demonstrate that multiple kinases inhibit both helicase loading and activation between MI and MII, thereby ensuring a reduction in ploidy.
by David V. Phizicky.
Ph. D.
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Wells, Jennifer. "Schizosaccharomyces pombe meiotic linear elements." Thesis, Bangor University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432058.
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Testori, Sarah. "Cohesin dynamics during meiotic prophase." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/29857.
Full textAbstract:
For faithful segregation during meiosis, chromosomes must be physically linked by both sister chromatid cohesion (SCC), provided by cohesin, and at least one crossover (CO). In mitosis, cohesin is dynamically associated with chromatin and this has been shown to be crucial for the repair of DSBs. Although DSBs are purposely made to start meiotic recombination, it is unknown if meiotic cohesin is dynamically associated with chromatin. However, cohesin loss or degradation is thought to be involved in the high incidence of aneuploidy observed in human eggs. In Caenorhabditis elegans (C. elegans), the cohesin loader SCC-2 remains associated with the axial element of meiotic chromosomes following the completion of S-phase, hinting that cohesin may be reloaded during meiotic prophase. To confirm this, I investigated if depleting SCC-2 by RNAi after entrance into meiotic prophase had an effect on cohesin association with chromosomes. This revealed loss of the cohesin subunit REC-8 from late prophase nuclei, suggesting that without reloading cohesin is removed from chromatin. Furthermore, scc-2 RNAi also resulted in the impairment of chiasmata, raising the possibility that cohesin reloading plays a role in CO formation or in chiasma maintenance. Two key mediators of cohesin removal are known to operate during the G2 phase of the mitotic cell cycle: the presence of DSBs and the cohesion anti-establishment factor Wapl1. Here I show for the first time that WAPL-1 modulates the cohesiveness of complexes containing the meiosis-specific kleisins COH-3 and COH-4. Furthermore, cohesin complexes containing different kleisins are differentially modulated by DSBs, and only REC-8-containing cohesin complexes can undertake the repair of DNA damage. Finally, I have developed several genetic tools to allow the visualization of cohesin turnover during meiosis. These findings show the exceptional complexity of cohesin dynamics during meiotic prophase, as well as demonstrating roles for cohesin outside of the provision of SCC.
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Marcet, Ortega Marina. "Surveillance mechanisms in mammalian meiosis." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/387429.
Full textAbstract:
Per tal de protegir les cèl·lules germinals de sofrir inestabilitat genòmica, diversos mecanismes de control s’encarreguen de que la progressió de la meiosis sigui correcte. En mamífers, els espermatòcits que presenten defectes de recombinació o de la formació de la vesícula sexual pateixen un bloqueig a l’estadi de paquitè. Estudis previs del nostre laboratori descriuen que la via complex MRE11-ATM-CHK2 activa l’arrest dependent de recombinació en presència de trencaments de doble cadena (DSBs) no reparats. L’objectiu d’aquest treball ha estat identificar si els membres de la família p53, els quals són possibles substrats de ATM i CHK2, participen en l’activació del arrest depenent de recombinació. En una aproximació genètica, hem obtingut ratolins doble mutants portadors d’una mutació de un membre de la família p53 (p53, Tap63 o p73) en un fons defectiu per Trip13. La mutació de Trip13 causa defectes de recombinació, el qual activa l’arrest depenent de recombinació en els espermatòcits a l’estadi de paquitè. Per tant, hem estudiat com l’absència d’algun membre de la família p53 afectava aquest fenotip d’arrest el espermatòcits Trip13mod/mod. Els nostres resultats demostren que tant la deficiència de p53 com Tap63, però no p73, permeten que els espermatòcits progressin més enllà i arribin a l’estadi de paquitè tardà tot i acumular nombrosos DSBs no reparats. Addicionalment, l’absència de p53 o Tap63 resulta en una disminució del nombre d’espermatòcits apoptòtics a l’estadi de paquitè primerenc. Així, els nostres resultats indiquen que p53 i TAp63 són responsables d’activar l’arrest dependent de recombinació en els espermatòcits de ratolí. Tot i així, els espermatòcits doble mutants encara presenten un bloqueig a l’estadi de paquitè. Per tal d’estudiar si els espermatòcits doble mutants arresten a causa de l’activació de l’arrest depenent de la correcta formació de la vesícula sexual, hem analitzat la funcionalitat del MSCI en els mutants Trip13. Per tant, el fet de saltar-se l’arrest dependent de recombinació ens ha permès elucidar el paper de TRIP13 en el silenciament meiòtic, de manera que al fallar la vesícula sexual es desencadena l’apoptosi i bloqueig dels mutants Trip13. Aquests resultats infereixen que el bloqueig depenent de recombinació i el depenent de la correcta formació de la vesícula sexual, són mecanismes que s’activen per mecanismes genèticament separats. A partir de l’observació que TRIP13 és necessari per implementar el silenciament del MSCI, he dut a terme un anàlisis exhaustiu de la transcripció en els mutants de Trip13. Els nostres resultats de marcatge de RNA amb EU i activació de la RNA polimerasa II fosforilada (S2) suggereixen que la expressió de RNA en els espermatòcits mutants per Trip13 es troba incrementada en els estadis inicials de la meiosis. Addicionalment, la seqüenciació del RNA ha permès observar que els gens dels cromosomes sexuals i gens pre-meiòtics es troben sobre expressats en els mutants de Trip13, suggerint que TRIP13 és necessari per mantenir l’expressió d’aquests gens a nivells baixos. En conjunt, els resultats presentats en aquest treball contribueixen a entendre com els mecanismes de control regulen diversos passes crucials de la progressió de la profase meiòtica en els espermatòcits de mamífer.In order to protect germinal cells from genomic instability, surveillance mechanisms ensure that meiosis occurs properly. In mammals, spermatocytes that display recombination or sex body defects experience an arrest at pachytene stage. Previous studies from our lab described that the MRE11 complex-ATM-CHK2 pathway activates the recombination-dependent arrest in the presence of unrepaired double strand breaks (DSBs). In this work we aimed to identify if p53 family members, which are putative targets of ATM and CHK2, participate in the activation of the recombination-dependent arrest. As a genetic approach, we bred double mutant mice carrying a mutation of a member of the p53 family (p53, TAp63, p73) in a Trip13 defective background. Trip13 mutation causes recombination defects, which activate the recombination-dependent arrest in pachytene-stage spermatocytes. Thus, we studied how the absence of p53 family members affected the arrest phenotype of Trip13mod/mod spermatocytes. Our data showed that p53 and TAp63 deficiency, but not p73, allowed spermatocytes to progress further into late pachynema, despite accumulating numerous unrepaired DBSs. In addition, lack of p53 or TAp63 resulted in a decrease of apoptotic spermatocytes at early pachytene stage. Therefore, our results indicate that p53 and TAp63 are responsible to activate the recombination-dependent arrest in mouse spermatocytes. Even though, double mutant spermatocytes still arrested at pachytene stage. To study if double mutant spermatocytes were arresting due to the activation of the sex body deficient arrest we analyzed MSCI functionality in Trip13 mutants. Thus, by bypassing the recombination-dependent arrest has allowed us to elucidate a role for TRIP13 protein in meiotic silencing, which consequently triggers apoptosis in double mutants at late pachytene stage due to sex body impairment. These results infer that the recombination-dependent and the sex-body deficient arrest are activated by two genetically separated mechanisms. From the observation that TRIP13 is required to implement MSCI silencing, we performed an exhaustive analysis of transcription in Trip13 mutants. Our results suggested that RNA expression in Trip13 mutants was increased in early meiotic stage spermatocytes, assessed by EU-labeling RNA and phosphorylated(S2)-RNA polymerase II. Moreover, RNA sequencing data highlighted the observation that sex chromosome genes and pre-meiotic genes are overexpressed in Trip13 mutants, suggesting that TRIP13 is required to maintain the expression of these genes at low levels. Overall, the data presented in this work contributes to the understanding on how surveillance mechanisms control several crucial steps of meiotic prophase progression in mammalian spermatocytes.