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Studium transkripční inaktivace pohlavních chromozomů během myší spermatogeneze / Meiotic sex chromosome inactivation within mouse spermatogenesisHomolka, David January 2012 (has links)
Meiotic sex chromosome inactivation (MSCI) is an essential epigenetic process, which transcriptionally silences X and Y chromosomes during spermatogenesis. It is accompanied by substantial chromatin remodeling resulting in a formation of so called sex or XY body, which is a characteristic of male pachytene spermatocytes. In spite of MSCI indispensability for male fertility, its biological role and molecular nature still remain rather unclear. However, the described link between chromosomal asynapsis and transcriptional silencing demonstrated that MSCI is tightly associated with the asynapsis of largely non-homologous sex chromosomes and is a specific form of more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC). The essential role of MSCI was demonstrated using mouse models, such as carriers of X- autosome translocations, where anomalous synapsis of sex chromosomes leads to impairment of MSCI and male sterility. Intriguingly, the exclusive spermatogenic arrest is a hallmark of not only X-autosome translocations but even various autosomal rearrangements, including autosomal translocations, inversions, or other structural mutations. Because the rearranged autosomes often intimately associate with the sex body, it...
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XGef functions independently of exchange factor activity to influence RINGO/CDK1 signaling and CPEB activation during Xenopus oocyte maturationKuo, Peiwen January 2009 (has links)
Thesis advisor: Laura E. Hake / Metazoan development depends on cytoplasmic polyadenylation, a key mechanism that controls the translation of maternally deposited mRNAs. In Xenopus laevis oocytes, CPEB regulates the translation of several developmentally important mRNAs, which drive meiotic progression and the production of fertilizable eggs. Most of our current knowledge of this process, also referred to as oocyte maturation, has been acquired from experiments conducted in Xenopus laevis oocytes. Despite over 30 years of research devoted to the exploration of progesterone signaling during maturation, the very early events that occur from progesterone receptor engagement to CPEB activation are not well understood. XGef, a putative Rho family guanine nucleotide exchange factor (GEF), interacts with CPEB and facilitates CPEB activation and timely meiotic progression. To further our understanding of XGef function during meiotic progression, the requirement for exchange factor activity and the activities of several Rho GTPases during maturation were examined. Despite previous reports of XGef activation of Cdc42 in mammalian cell culture, XGef does not stimulate the activation of Cdc42 in maturing Xenopus oocytes. Further, Cdc42 activity does not affect CPEB phosphorylation and overexpression of a dominant negative Cdc42 mutant does not affect maturation. Inhibition of Toxin B sensitive Rho GTPases, including Cdc42, Rac1 and Rho A-C, also fails to affect CPEB activation or meiotic progression. Lastly, the overexpression of XGef exchange deficient point mutants did not affect maturation compared to oocytes overexpressing wildtype XGef. Together, these results suggest that as a facilitator of CPEB activation and meiotic progression, XGef functions independently of exchange factor activity and Rho GTPase activation. Additionally, we found that XGef activity influences the function of RINGO/CDK1, a novel component of the progesterone signaling pathway. XGef inhibition depresses RINGO-induced GVBD, whereas XGef overexpression enhances this process. XGef interacts with RINGO in oocyte extracts and the interaction is direct in vitro. Our protein interaction data, in total, suggest that a XGef/RINGO/MAPK/CPEB complex forms in ovo to facilitate CPEB activation. Lastly, inhibition of RINGO activity directly compromises CPEB phosphorylation during early maturation, which suggests that RINGO/CDK1 directly mediates CPEB-activation. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
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Parameter estimation of a probabilistic automata model of DNA meiosisWu, Kuan Chan January 2010 (has links)
Digitized by Kansas Correctional Industries
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Identifying natural modifiers of meiotic crossover frequency in Arabidopsis thalianaLawrence, Emma Jane January 2019 (has links)
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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The regulation of cohesin cleavage during meiosis in Saccharomyces cerevisiaeGalander, Stefan January 2017 (has links)
Meiosis is a specialized form of cell division where homologous chromosomes are segregated in meiosis I before sister chromatids are segregated in meiosis II. To establish this pattern, a number of changes to the mitotic chromosome segregation machinery are put in place. Firstly, sister kinetochores orient towards the same pole in meiosis I (mono-orientation). Secondly, homologue recombination creates chiasmata, which link homologues together. And thirdly, cohesin, the molecule that holds sister chromatids together, is cleaved in a step-wise manner. This is achieved because the Shugoshin (Sgo1) protein recruits protein phosphatase 2A (PP2A) to centromeres to counteract cohesin phosphorylation, which is required for its cleavage. The work presented here has investigated two critical aspects of cohesin protection: firstly, how cohesin protection is deactivated in meiosis II and, secondly, how a meiosis-specific protein called Spo13 helps to set up cohesin protection in meiosis I. Previously, our lab had shown that Sgo1 is removed from chromosomes when sister chromatids come under tension during mitosis. I therefore sought to investigate whether sister kinetochore mono-orientation allows Sgo1 to stay on centromeres during meiosis I and carry out its protective function. To this end, I modified meiosis I chromosomes to lack both chiasmata and mono-oriented kinetochores. Under these conditions, where sister chromatids are forced to be under tension in metaphase I, Sgo1 is undetectable on chromosomes. As a consequence, centromeric cohesin is largely lost in anaphase I leading to the premature separation of sister chromatids in a fraction of cells. Since mono-orientation of sister kinetochores is exclusive to meiosis I, these findings suggest that Sgo1 localisation is influenced by sister kinetochore tension in both mitosis and meiosis. Therefore, our findings suggest a mechanism that could contribute to the deprotection of cohesin in meiosis II. However, loss of cohesin protection upon bi-orientation is not complete, suggesting that other factors are involved in the efficient protection and deprotection of cohesin. One such factor is the meiosis-specific protein Spo13, which had previously been shown to be required for cohesin protection as well as kinetochore monoorientation. Although it had been suggested that Spo13 regulates Sgo1 recruitment to centromeres, I could not find any evidence to support a loss of Sgo1, or PP2A, in spo13Δ cells. Additionally, even when Sgo1 is stabilised and clearly visible in anaphase I of spo13Δ mutants, pericentromeric cohesion is still defective. Therefore, I investigated the effect that polo kinase Cdc5, an interactor of Spo13, has on Sgo1. While cellular Sgo1 levels are increased in response to Cdc5 loss, this effect seems to be independent of Spo13. However, Spo13 is required for proper levels of Cdc5 at centromeres and the centromeric recruitment of Cdc5 by Spo13 is likely to be functionally important because tethering of Cdc5 to kinetochores rescued the mono-orientation phenotype of spo13Δ cells. In contrast, I found no evidence that the Spo13-Cdc5 interaction is required for cohesin protection. Meiotic overexpression of SPO13 enhances cohesin protection in meiosis I, apparently independent of its robust interaction with Cdc5, and causes increased Sgo1 enrichment at centromeres. This suggested that Spo13 might recruit Sgo1 to cohesin itself to facilitate its protection. Although I could not detect a loss of Sgo1-cohesin interaction in spo13Δ cells, tethering of Sgo1 to cohesin restores pericentromeric Rec8 to spo13Δ mutants in anaphase I. Surprisingly, sister chromatids still segregate in this case, suggesting that pericentromeric cohesion is defective, despite maintenance of Rec8. Furthermore, inhibition of either one of the cohesin kinases, DDK and Hrr25, restores sister chromatid cohesion to spo13Δ cells. Therefore, the findings in this study suggest that Spo13 is at the centre of a complex regulatory network that coordinates cohesin protection and sister chromatid cohesion in meiosis I.
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Meiotic spindle organization and chromosome condensation in Drosophila oocytesNikalayevich, Elvira January 2014 (has links)
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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Molecular evolution of meiosis genes in fungiSavelkoul, Elizabeth Jennings 01 December 2013 (has links)
Meiosis as a general process is prevalent across the eukaryotes, as are the orthologs of many genes encoding proteins known to function in meiosis. However, many organisms have experienced derived losses of otherwise well-conserved meiosis genes without losing meiosis and sexual reproduction. Although this general conservation of meiosis genes and precedent for derived meiosis gene losses has been previously established, questions remain about the frequency of and evolutionary forces contributing to these trends. This work sought (i) to characterize the phylogenetic distribution of 15 meiosis genes (most of which are known to function only in meiosis) in the exemplar eukaryotic kingdom Fungi and (ii) to use this dataset to investigate evolutionary processes contributing to the loss and retention of these genes.
Orthologs of 15 meiosis genes (Rad51, Rad21, Spo11, Rec8, Dmc1, Hop2, Mnd1, Sae3/Swi5, Mei5/Sfr1, Pch2, Hop1, Msh4, Msh5, Mer3, Zip3) were identified by BLAST-based techniques and phylogenetically validated in most of the 109 publicly available sequenced fungal genomes investigated, but numerous putative derived losses were also detected. Rad51, Rad21, Rec8, and Spo11 were nearly universally conserved; the remaining genes were each undetectable or independently pseudogenized multiple times within fungi, particularly often for Pch2. Genes with previously known functional interactions tended to show parallel presence, absence, or pseudogenization patterns. Although this work primarily established the conserved presence of meiosis gene orthologs at the DNA level, examination of expressed sequence tags (ESTs) showed that many species--including some not previously known to undergo sexual reproduction--were competent to transcribe (and often splice) mRNA from the identified meiosis genes.
Factors potentially influencing derived meiosis gene losses were investigated in two ways. First, degenerate PCR was used to amplify loci expected to contain orthologs of Msh4, Msh5, Pch2, and Zip3 in various Aspergillus species closely related to Aspergillus nidulans (a species with undetected or pseudogenized orthologs of these four genes.) The loss of Pch2 substantially predated the pseudogenization of Msh4, Msh5, and Zip3. Evolutionary rate analyses using the Ka/Ks ratio found no change in nonsynonymous substitution patterns in Msh4 and Msh5 in species that had lost Pch2 compared to those retaining Pch2. Elevated Zip3 Ka/Ks values were found in species with pseudogenized Msh4 and Msh5, suggesting possible obligate functional interactions of Zip3 with Msh4 and Msh5. Second, phylogenetically independent contrasts (PIC) analyses were performed on species from the 109-taxon inventory with published chromosome number and chromosome size estimates to investigate whether changes in either parameter were consistently associated with changes in the presence or absence of meiosis genes. Many analyses had low statistical power, neither detecting nor being able to exclude an association between gene loss and the tested variables. However, several comparisons did detect significant or nearly significant trends: for example, fungi that had lost genes related to crossover interference (Msh4, Msh5, or Pch2) tended to have fewer and/or larger chromosomes than their closest relatives without gene loss.
A final objective was to determine the distribution of meiosis genes in lichenized fungi and green algae to see whether this form of symbiosis was associated with differences in the presence or molecular evolution of meiosis genes. Rad51, Dmc1, and Mnd1 were each amplified by degenerate PCR from multiple lichenized fungi that lacked sequenced genomes, and no systematic difference in evolutionary rate was found between examined lichenized fungi compared to other examined classes in phylum Ascomycota. Bioinformatic analyses of meiosis gene distribution in green algae revealed not only no obvious increased tendency for derived gene losses in examined lichenized green algae but also very few derived meiosis gene losses in green algae in general. This suggests that lichenization may not be associated with consistent differences in the evolution of meiosis genes in either fungal or green algal symbionts. The green algal results also illustrate the need to investigate the extent to which eukaryotes as a whole exhibit the same trends of meiosis gene evolution described here for fungi: frequent derived losses of meiosis genes, genes encoding proteins with function interactions showing similar distributions, likely roles for post-transcriptional regulation of meiosis gene transcripts, and loss of crossover distribution-related genes potentially being associated with constraints on chromosome size and/or haploid chromosome number.
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The evolution and expression of Drosophila meiosis genesBeekman, Danielle Jeanine 01 December 2013 (has links)
Drosophila melanogaster is unique amongst model organisms in that males utilize achiasmatic meiosis, where formation of the synaptonemal complex (SC) and recombination are absent. Most organisms require the SC and chiasmata for the successful completion of meiosis and production of viable gametes, making D. melanogaster an ideal system for the study of meiotic variation. The goal of my research was to examine in detail the origin and evolution of male achiasmatic meiosis in Diptera. This was done in three parts: 1) assessing the presence and absence of meiosis genes across dipteran species, 2) analyzing the rate of evolution of Drosophila achiasmatic meiosis genes, and 3) evaluating differences in expression and splicing of meiosis genes between D. melanogaster males and females. I queried genome and transcriptome data from eleven dipteran species for both canonical and achiasmatic meiosis genes. Surprisingly, I found that a set of meiosis-specific genes was lost prior to the gain of Drosophila male achiasmy genes, suggesting that the latter were a later addition to an already non-canonical meiotic process. To assess the evolution of fourteen Drosophila achiasmatic meiosis genes, I performed phylogenetic, rate, selection and co-evolution analyses. My results show that, although these genes appear to be evolving under purifying selection, they are all evolving rapidly compared to their paralogs and paralogous genes throughout the Drosophila genome. Some groups of these genes are also co-evolving, supporting their potential for encoding members of protein complexes. These results suggest that male achiasmy is globally influencing the rapid evolution of these genes, even though their functions within meiosis vary greatly. Lastly, I investigated the expression and splicing of meiosis genes between male and female D. melanogaster. As expected, many meiosis genes with sex-limited roles showed biased expression for the sex that utilized them. However, some genes were expressed equally in both sexes or higher in the opposite sex. I also found evidence that sex-biased splicing may have a role in regulating protein production for some meiosis genes. These results indicate that the regulation of meiotic gene expression is more complex than originally thought and that multiple mechanisms, including alternative splicing, are utilized to control protein production. The combination of results from all parts of this work highlight some of the major events that occurred prior to and during the evolution of Drosophila male achiasmy and lay groundwork for future studies examining the details of this unusual evolutionary path.
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Analysis of meiotic recombination initiation in Saccharomyces cerevisiaeKoehn, Demelza Rae 01 July 2009 (has links)
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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Evolution of meiosis genes in sexual vs. asexual Potamopyrgus antipodarumRice, Christopher Steven 01 May 2015 (has links)
How asexual reproduction affects genome evolution, and how organisms that are ancestrally sexual alter their reproductive machinery upon becoming asexual are both central unanswered questions in evolutionary biology. While these questions have been addressed to some extent in organisms such as asexual clams, rotifers, ostracods, arthropods, and fungi, the most powerful and direct tests of how sex and its absence influence evolution requires direct comparisons between closely related and otherwise similar sexual and asexual taxa. Here, I quantify the rates and patterns of molecular evolution in the meiosis-specific genes Msh4, Msh5, and Spo11 in multiple sexual and asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail. Because asexual P. antipodarum reproduce apomictically (without recombination), genes used only for meiosis should be under relaxed selection relative to meiosis-specific genes in sexual P. antipodarum, allowing me to directly study how asexuality affects the evolution of meiosis-specific genes. Contrary to expectations under relaxed selection, I found no evidence that these meiosis-specific genes are degrading in asexual P. antipodarum; instead they display molecular patterns consistent with purifying selection. The presence of intact meiosis-specific genes in asexual P. antipodarum hints that the asexuals may maintain the ability to perform meiosis despite reproducing apomictically. Asexual meiotic capability suggests that some meiotic components may persist or acquire a new role in these asexuals.
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