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‣ Csm4, in Collaboration with Ndj1, Mediates Telomere-Led Chromosome Dynamics and Recombination during Yeast Meiosis

Wanat, Jennifer J.; Koszul, Romain; Zanders, Sarah; Alani, Eric; Kim, Keun Pill; Weiner, Beth Meryl; Kleckner, Nancy Elizabeth; Lichten, Michael Nicholas
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Chromosome movements are a general feature of mid-prophase of meiosis. In budding yeast, meiotic chromosomes exhibit dynamic movements, led by nuclear envelope (NE)-associated telomeres, throughout the zygotene and pachytene stages. Zygotene motion underlies the global tendency for colocalization of NE-associated chromosome ends in a “bouquet.” In this study, we identify Csm4 as a new molecular participant in these processes and show that, unlike the two previously identified components, Ndj1 and Mps3, Csm4 is not required for meiosis-specific telomere/NE association. Instead, it acts to couple telomere/NE ensembles to a force generation mechanism. Mutants lacking Csm4 and/or Ndj1 display the following closely related phenotypes: (i) elevated crossover (CO) frequencies and decreased CO interference without abrogation of normal pathways; (ii) delayed progression of recombination, and recombination-coupled chromosome morphogenesis, with resulting delays in the MI division; and (iii) nondisjunction of homologs at the MI division for some reason other than absence of (the obligatory) CO(s). The recombination effects are discussed in the context of a model where the underlying defect is chromosome movement, the absence of which results in persistence of inappropriate chromosome relationships that...

‣ Spatial and Temporal Organization of Chromosome Duplication and Segregation in the Cyanobacterium Synechococcus elongatus PCC 7942

Chen, Anna Hang; Afonso, Bruno; Silver, Pamela A.; Savage, David F.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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The spatial and temporal control of chromosome duplication and segregation is crucial for proper cell division. While this process is well studied in eukaryotic and some prokaryotic organisms, relatively little is known about it in prokaryotic polyploids such as Synechococcus elongatus PCC 7942, which is known to possess one to eight copies of its single chromosome. Using a fluorescent repressor-operator system, S. elongatus chromosomes and chromosome replication forks were tagged and visualized. We found that chromosomal duplication is asynchronous and that the total number of chromosomes is correlated with cell length. Thus, replication is independent of cell cycle and coupled to cell growth. Replication events occur in a spatially random fashion. However, once assembled, replisomes move in a constrained manner. On the other hand, we found that segregation displays a striking spatial organization in some cells. Chromosomes transiently align along the major axis of the cell and timing of alignment was correlated to cell division. This mechanism likely contributes to the non-random segregation of chromosome copies to daughter cells.

‣ Enrichment of HP1a on Drosophila Chromosome 4 Genes Creates an Alternate Chromatin Structure Critical for Regulation in this Heterochromatic Domain

Riddle, Nicole C.; Jung, Youngsook; Gu, Tingting; Alekseyenko, Artyom A.; Asker, Dalal; Gui, Hongxing; Kharchenko, Peter Vasili; Minoda, Aki; Plachetka, Annette; Schwartz, Yuri B.; Tolstorukov, Michael Y.; Kuroda, Mitzi I.; Pirrotta, Vincenzo; Karpen, Gar
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS...

‣ Identification of the SlmA Active Site Responsible for Blocking Bacterial Cytokinetic Ring Assembly over the Chromosome

Cho, Hongbaek; Bernhardt, Thomas G.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Bacterial cells use chromosome-associated division inhibitors to help coordinate the processes of DNA replication and segregation with cytokinesis. SlmA from Escherichia coli, a member of the tetracycline repressor (TetR)–like protein family, is one example of this class of regulator. It blocks the assembly of the bacterial cytokinetic ring by interfering with the polymerization of the tubulin-like FtsZ protein in a manner that is dramatically stimulated upon specific DNA binding. Here we used a combination of molecular genetics and biochemistry to identify the active site of SlmA responsible for disrupting FtsZ polymerization. Interestingly, this site maps to a region of SlmA that in the published DNA–free structure is partially occluded by the DNA-binding domains. In this conformation, the SlmA structure resembles the drug/inducer-bound conformers of other TetR–like proteins, which in the absence of inducer require an inward rotation of their DNA-binding domains to bind successive major grooves on operator DNA. Our results are therefore consistent with a model in which DNA-binding activates SlmA by promoting a rotational movement of the DNA-binding domains that fully exposes the FtsZ-binding sites. SlmA may thus represent a special subclass of TetR–like proteins that have adapted conformational changes normally associated with inducer sensing in order to modulate an interaction with a partner protein. In this case...

‣ Mammalian E-type Cyclins Control Chromosome Pairing, Telomere Stability and CDK2 Localization in Male Meiosis

Martinerie, Laetitia; Manterola, Marcia; Chung, Sanny S. W.; Panigrahi, Sunil K.; Weisbach, Melissa; Vasileva, Ana; Geng, Yan; Sicinski, Peter; Wolgemuth, Debra J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.

‣ The role of Xist RNA in the maintenance of X chromosome inactivation; Role of Xist ribonucleic acid in the maintenance of X chromosome inactivation

Csankovszki, Györgyi, 1971-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 177, [2] leaves; 12319159 bytes; 12318910 bytes; application/pdf; application/pdf
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The role of Xist RNA in silencing the inactive X of female somatic cells was investigated by generating a conditional allele of the Xist gene. A system was set up in which reactivation of two X-linked genes, the endogenous Hprt gene and an X-linked GFP transgene, can be quantitatively assessed. Mouse embryonic fibroblasts derived from mice carrying the conditional Xist mutation were cultured and infected with an adenovirus vector carrying the gene for Cre recombinase. After Cre mediated deletion of Xist, the inactive X remained transcriptionally silent, late replicating, and hypoacetylated on histone H4, confirming that X-inactivation can be maintained in the absence of Xist RNA. However, the Xist mutant inactive X was no longer enriched in histone macroH2A 1. Furthermore, the reactivation rate of GFP and Hprt increased, indicating Xist RNA does contribute to gene repression on the inactive X. DNA methylation, histone hypoacetylation and Xist RNA were found to act synergistically in X chromosome silencing. To investigate whether Xist RNA can also silence the active X chromosome of male somatic cells, Xist expression on the active X was induced by demethylation. Demethylation was achieved by Cre mediated deletion of a conditional mutant allele of DNA methyltransferase-l (Dnmtl) in male fibroblasts. In these cells...

‣ Investigation of the role of chromosome missegregation in embryo development and in tumorigenesis

Burds, Aurora A. (Aurora Ann), 1973-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 171 leaves; 12361544 bytes; 12361301 bytes; application/pdf; application/pdf
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The separation and partitioning of sister chromatids prior to cytokinesis must be tightly regulated to insure that progeny cells receive the correct number of chromosomes. The spindle assembly checkpoint prevents chromosome missegregation by arresting the cell before sister chromatid separation if any one chromatid is not properly attached to the spindle microtubules. Defects in chromosome segregation lead to the creation of aneuploid cells that contain too much or too little genetic material. Some aneuploid cells have gained oncogenes or lost tumor suppressor genes, and these genetic alterations allow the cells to ignore signals that regulate growth and proliferation. A majority of tumors are aneuploid, but it is not yet known whether chromosome instability is a driving force or merely a secondary effect of tumor development. In Chapter 2, I will present work showing that the protein most often altered in colon cancers, Adenomatous Polyposis Coli (AdPC) (Kinzler and Vogelstein 1996), is modified by members of the spindle assembly checkpoint and that a truncating mutation of the AdPC protein leads directly to aneuploidy in mouse cells. Although genetic disruption of any member of the spindle assembly checkpoint signaling cascade has proven lethal for metazoans on both the cellular and organismal level (Basu et al. 1999; Kitagawa and Rose 1999; Dobles et al. 2000; Kalitsis et al. 2000)...

‣ A role for the Bacillus subtilis Structural Maintenance of Chromosomes (BsSMC) protein in chromosome organization and compaction; Role for the BsSMC protein in chromosome organization and compaction

Lindow, Janet C. (Janet Christine), 1974-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 130 leaves; 9477059 bytes; 9476813 bytes; application/pdf; application/pdf
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All cells must compact their chromosomes in order for the DNA to fit inside the cell or nucleus. In Bacillus subtilis, and other bacteria, replication occurs simultaneously with the organization, compaction and segregation of newly duplicated chromosomal regions. My work indicates that the B. subtilis Structural Maintenance of Chromosomes (BsSMC) protein is involved in compacting and organizing the chromosome. Increasing the amount of supercoiling of DNA is a means to compact the chromosome. This thesis describes a role for BsSMC in supercoiling. I determined that BsSMC can alter the DNA topology of plasmids in vivo. There is also genetic evidence that BsSMC is involved in supercoiling. An smc null mutant is hypersensitive to inhibitors of DNA gyrase, which reduce the level of negative supercoiling in the cell. Conversely, depletion of Topoisomerase I, which increases the amount of negative supercoiling of the chromosome, partially suppresses the phenotype of an smc null mutant. These data are consistent with the model that BsSMC affects chromosome compaction by constraining positive supercoils. Interestingly, SMC-containing complexes in eukaryotes are able to constrain positive supercoils in vitro and affect chromosome architecture suggesting that there is a conserved function for SMC proteins in chromosome structure. I also determined the subcellular localization of BsSMC. I found that BsSMC is a moderately abundant protein that can bind to many regions of the chromosome. A portion of BsSMC localizes in a pattern similar to the replication machinery.; (cont.) Simultaneous localization of BsSMC and a component of the replisome revealed that they are usually in the same region of the cell but are not always colocalized. Finally...

‣ Studies on X chromosome inactivation and the X-linked disease Rett syndrome

Luikenhuis, Sandra, 1972-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 158 leaves; 7777941 bytes; 7799766 bytes; application/pdf; application/pdf
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(cont.) the RTT phenotype.; Deletion of the Xist gene results in skewed X-inactivation. To distinguish primary non-random choice from post-choice selection, we analyzed X-inactivation in early embryonic development in the presence of two different Xist deletions. We found that Xist is an important choice element, and that in the absence of an intact Xist gene, the X chromosome will never be chosen as the active X. To understand the molecular mechanisms that affect choice we analyzed the role of replication timing prior to X-inactivation. The X chromosomes replicated asynchronously before X-inactivation but analysis of cell-lines with skewed X-inactivation showed no preference for one of the two Xist alleles to replicate early, indicating that asynchronous replication timing prior to X-inactivation does not play a role in skewing of X-inactivation. Expression of the Xist is negatively regulated by its antisense gene, Tsix. In order to determine the role of transcription in Tsix function, we modulated Tsix transcription with minimal disturbance of the genomic sequence. Loss of Tsix transcription lead to non-random inactivation of the targeted chromosome, whereas induction of Tsix expression caused the targeted chromosome always to be chosen as the active X. These results for the first time establish a function for antisense transcription in the regulation of Xist expression. The X-linked disease Rett syndrome (RTT)...

‣ The spindle checkpoint : Bubs, Mads, and chromosome-microtubule attachment in budding yeast

Gillett, Emily S., 1976-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 145 leaves; 7400521 bytes; 7419921 bytes; application/pdf; application/pdf
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(cont.) DNA damage response, we hypothesize that the perinuclear pool of Mad proteins may be required for spindle checkpoint-independent functions such as invoking metaphase arrest following DNA damage.; The high fidelity of chromosome transmission in eukaryotes is achieved, in part, by the activity of the spindle checkpoint. This checkpoint monitors the status of chromosome-microtubule attachments and delays the onset of anaphase until all kinetochores have formed stable bipolar connections to the mitotic spindle. We have characterized the localization of the Bub and Mad spindle checkpoint proteins in Saccharomyces cerevisiae. In metazoan cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas S. cerevisiae Bublp and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Madlp and Mad2p are kinetochore-bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. We propose that differences in the behavior of spindle checkpoint proteins in metazoan cells and budding yeast are due primarily to evolutionary divergence in spindle assembly pathways. The spindle checkpoint proteins Madlp and Mad2p exhibit perinuclear localization in both budding yeast and metazoans. We find that the perinuclear localization of Madlp is dependent on Myosin-like proteins Mlplp and Mlp2p...

‣ Mitotic regulators and their effects on Drosophila : chromosome structure during development

Wallace, Julie Ann, 1977-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 2 v. (294 leaves); 14947522 bytes; 14987542 bytes; application/pdf; application/pdf
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Variants of the canonical cell cycle are frequently used in nature to accomplish specific developmental goals. In one such variant, the endocycle, synthesis phase alternates with a gap phase without an intervening mitosis, producing cells that have multiple copies of the genome. These cells show diversity in their chromosome structure; at one extreme, the sister chromatids are separate (polyploid) and at the other extreme, the sisters are held together (polytene). The endocycle itself can be modified and these variations are speculated to correlate with the observed differences in chromosome structure. In this thesis, we have analyzed the contribution of mitotic regulators to the endocycle and polytene chromosome structure in Drosophila. We show that morula, a gene required for the transition from polytene to polyploid chromosome structure in Drosophila nurse cells, is a subunit of the anaphase-promoting complex/cyclosome. Increasing levels of cyclin B, a known mitotic target of the APC/C, does not alter the timing of the transition, indicating that CYCLIN B is not the only APC/C target at the polyteny-polyploidy transition. In mitosis, activity of APC/C and POLO lead to the loss of sister-chromatid cohesion and we find that mutants in polo are unable to progress through the polyteny-polyploidy transition. Finally...

‣ Effects of the conserved proteins, Soj (ParA) and SpoOJ (ParB) on chromosome partitioning in Bacillus subtilis

Lee, Philina S. (Philina Sharlene), 1976-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 171 leaves; 6293206 bytes; 6290440 bytes; application/pdf; application/pdf
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The Par system contributes to stable inheritance of plasmids and chromosomes in diverse bacterial species. Faithful chromosome partitioning is important to dividing cells because anucleate, or chromosomeless, cells are not viable. Since the discovery of the Par system in low-copy number plasmids over 20 years ago, research has focused on characterizing its components: ParA, a DNA-binding ATPase, ParB, a DNA-binding protein that interacts with ParA, and parS, the site bound by ParB. How these proteins function to stabilize parS-containing plasmids or chromosomes remains to be elucidated. Less is known about the chromosomally-encoded Par systems. Intriguingly, in several organisms including Bacillus subtilis, chromosomally-encoded Par systems play important roles in cellular processes besides chromosome partitioning. This thesis focuses on characterizing the roles of Soj (ParA) and SpoOJ (ParB) of B. subtilis in chromosome partitioning. These proteins were first identified by their effects on sporulation. My results indicate that Soj and SpoOJ contribute to timely separation of replicated chromosomal origins, and argue against the model that SpoOJ anchors replicated origins to putative receptors in opposite halves of the cell. Additionally...

‣ Regulation of meiosis I chromosome segregation by Spol3 and Cdc5 in Saccharomyces cerevisiae

Lee, Brian H. (Brian Han), 1976-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 320 p.; 10215371 bytes; 10210891 bytes; application/pdf; application/pdf
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Meiosis is a specialized cell cycle that generates gametes for the purpose of disseminating genetic material to the next generation. The reduction of chromosome number by half is brought about two chromosome segregation phases following a single DNA replication phase. In the first division, homologs segregate away from each other and in the second division the sister chromatids separate. These two consecutive meiotic divisions necessitate innovations in chromosome dynamics and hence the involvement of both meiosis-specific modulators and regulators of the mitotic cell cycle. The work described herein characterizes the roles of two essential meiosis I regulators, a mitotic protein kinase, Cdc5 and a meiosis-specific gene, Spol 3. The conserved polo-like kinase Cdc5 regulates many essential aspects of meiosis I including the removal of cohesion between sister chromatids for homolog segregation, sister-kinetochore co-orientation and exit from meiosis I. Spol3 likely cooperates with Cdc5 to regulate some of these processes. SpoI3 controls kinetochore co-orientation and the retention of centromeric cohesion which is essential for accurate sister chromatid segregation in meiosis II. In sum, this work elucidated the roles of two important regulators of the meiotic cell cycle and defines a component of the complex regulatory circuit necessary for the specialized meiotic divisions.; by Brian H. Lee.; Thesis (Ph. D.)--Massachusetts Institute of Technology...

‣ Palindromes on the human X chromosome : testis-biased transcription, gene conversion and evolution

Saionz, Jennifer R., 1976-
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 134 leaves; 5802155 bytes; 5819548 bytes; application/pdf; application/pdf
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Recent genomic studies of the Y chromosome revealed massive, testis-specific palindromes that span 30% of the chromosome and are subject to gene conversion. We conducted studies to determine whether similar palindromes exist on the human X chromosome and, if they exist, to what degree they share the features of the Y chromosome palindromes. We performed an electronic search for palindromes on the human X chromosome resulting in the identification of 24 palindromes comprising 1.8% of the chromosome. The palindromes consist of sequences 9.5 to more than 140 kilobases long duplicated in inverted orientation separated by a 0.2 to 164 kilobase spacer. The paired palindrome arms display greater than 99 percent nucleotide identity. We determined the palindrome associated gene content and experimentally evaluated their transcription range. All the genes residing in palindrome arms and spacers are transcribed in the testis, with almost two thirds predominantly testis-transcribed. To determine if the testis-transcription bias is due to a chromosome-wide enrichment for testis-transcribed genes, we used publicly available expression data to compare the ratio of palindrome-associated X-linked testis genes with non-palindrome-associated X-linked testis genes. We confirmed that the proportion of testis genes in palindromes is significantly different than that of testis genes on the entire X chromosome. We pursued a comparative sequencing strategy to trace the evolution of the X chromosome palindromes. We sequenced bacterial artificial chromosomes (BACs) from chimpanzee...

‣ The role of the chromosomal passenger complex and condensin complex in meiotic chromosome cohesion and segregation

Resnick, Tamar Deborah
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 187 leaves
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The canonical mitotic cell cycle is modified in metazoans to achieve a variety of developmental goals. Among these, meiotic divisions reduce the DNA content of the cell, haploid gametes fuse and reenter mitotic cycling, and mitoses of early embryogenesis, in many animals, employ a rapid cell cycle that lacks gap phases. Much less is understood about regulation of these developmentally regulated cell cycles, than about canonical mitosis, because they cannot be studied in tissue culture systems and because mutations that specifically affect these processes are rare. This thesis describes a screen to characterize mutants that display defects in these programs in Drosophila melanogaster. This model system allowed use of powerful tools for genetic and cell biological analyses of these mutants. Nineteen mutants with defects in this developmental window were screened and placed them into five phenotypic classes, including mutants that displayed defects in fertilization, pronuclear fusion, and mitosis and DNA synthesis in the early divisions of embryogenesis. Among these mutations, one was mapped and molecularly characterized as an allele of the passenger complex component incenp.; (cont.) This allele was used to investigate roles of the passenger complex in meiosis in both male and female Drosophila. incenp mutant males displayed missegregation of sex chromosomes in both meiosis I and meiosis II...

‣ Autosomal random asynchronous replication is analogous to X-chromosome inactivation

Ensminger, Alexander Wilson
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 221 leaves
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A number of mammalian genes are expressed from only one of two alleles in either an imprinted or random manner. Those belonging to the random class include X-linked genes subject to X inactivation, as well as a number of autosomal genes, including odorant receptors, immunoglobulins, T-cell receptors, interleukins, natural killer-cell receptors, and pheromone receptors. Random asynchronous replication of DNA in S-phase represents an epigenetic mark that often parallels monoallelic expression. All randomly monoallelically expressed genes discovered to date replicate asynchronously in S-phase, though not all of the genes contained within asynchronous domains are monoallelically expressed. The focus of my work has been on understanding this random choice that cells make between two sequence-identical alleles. Using two-color fluorescent in situ hybridization (FISH) analyses, the random asynchronous replication of a large number of human and mouse genes appears to be coordinated at the level of entire chromosomes. This regulatory scheme is reminiscent of random X-chromosome inactivation, the dosage compensation machinery in mammals. We have shown that autosomal coordination responds to trisomy in a fashion similar to X inactivation, with one copy of the trisomic chromosome marked for early replication and the other two rendered late replicating.; (cont.) These observations raise the intriguing possibility that the mechanistic underpinnings of X inactivation and autosomal coordination may also be similar. Furthermore...

‣ Insights into Shugoshin localization and function in S. cerevisiae chromosome segregation; Insights into Sgo1 localization and function in S. cerevisiae chromosome segregation

Kiburz, Brendan Meredith
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 266 leaves
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Faithful chromosome segregation is crucial to the well being of organisms. In mitosis, the partitioning of chromosomes to daughter cells is monitored at many levels to ensure that identical genomic information is received. Meiosis is a specialized form of cell division during which two division phases follow replication of the genome. Several steps are taken in meiosis to allow for a unique pattern of chromosome segregation. Homologous chromosomes segregate during the first division and sister chromatids segregate during the second. Cohesins, protein complexes that associate along the length of chromosomes during DNA replication, are lost in a step-wise manner in meiosis. During the first division, cohesins are lost only along chromosome arms. Cohesins remain at centromeres during meiosis II and are required to establish proper orientation of sister chromatid kinetochores. Shugoshin (Sgo1) is a protein related to D. melanogaster Mei-S332 that is crucial for the protection of centromeric cohesins from cleavage until anaphase II of meiosis. Here we show that in S. cerevisiae cohesins are protected from cleavage during meiosis I in a 50 kb region of chromosomes and Sgo1 localizes to cohesin-associated regions within this region. Interestingly...

‣ Diverse outcomes of homologous recombination in the human Y chromosome

Lange, Julian H. (Julian Hendrik)
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 201 p.
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Mammalian sex chromosomes began diverging from an ordinary pair of autosomes roughly 300 million years ago. Inversions in the evolving Y chromosome sequentially suppressed recombination with the X chromosome. While pseudoautosomal regions in the human Y chromosome still participate regularly in allelic homologous recombination, the male-specific region of the Y (MSY) - the only haploid portion of the nuclear genome - does not. It does, however, engage in non-allelic homologous recombination. In this thesis, I examine modes and outcomes of non-allelic homologous recombination in the MSY. The predictions presented here are based on the double-strand break repair model of recombination between homologous chromosomes, in which a double-strand break (DSB) is the common precursor to crossing over and gene conversion. First, I show that massive MSY-specific palindromes, which maintain arm-to-arm sequence identity via gene conversion, are also the targets of crossing over. Crossover events in palindromes can lead to isochromosome formation and diverse reproductive disorders including sex reversal, male infertility, and Turner syndrome. Second, I demonstrate that a region of the MSY - thought to be recombinationally suppressed with the X chromosome - does undergo extensive X-Y gene conversion. This region encompasses hotspots of ectopic crossover events that lead to X-Y translocations associated with sex reversal syndromes. Although sequences in the MSY engage in productive recombination via gene conversion...

‣ Chromosome dynamics of the early meiotic cell cycle in S. cerevisiae

Blitzblau, Hannah G
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 186 p.
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In every cell cycle the genetic material must be duplicated and transmitted to the daughter cells. Meiosis is a developmental program that allows a diploid cell to produce haploid progeny. The reduction in chromosome number obtained during meiosis requires specialized mechanisms that are absent during the canonical mitotic cell cycle. Although previous studies found strong similarities between pre-mitotic and pre-meiotic DNA replication, pre-meiotic S phase is longer than pre-mitotic S phase, suggesting that meiosis-specific events regulate the rate of DNA replication. Additionally, after DNA replication, homologous recombination is initiated by the introduction of hundreds DNA double-strand breaks (DSBs) into the genome to produce physical DNA exchanges, or crossovers, between homologous chromosomes. To investigate the chromosome dynamics of the early meiotic cell cycle, I performed comprehensive analysis of pre-meiotic DNA replication and DSB formation in budding yeast. Genome-wide studies of pre-meiotic DNA replication confirmed that the same replication origins are selected and activated in pre-meiotic and pre-mitotic cells, although replication was delayed at a large number of origins. These results indicate that the regulation of DNA replication is similar in the meiotic and mitotic cell cycles...

‣ Sequence of the mouse Y chromosome

Alföldi, Jessica E
Fonte: Massachusetts Institute of Technology Publicador: Massachusetts Institute of Technology
Tipo: Tese de Doutorado Formato: 93 leaves
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The mouse Y chromosome has been studied for over 50 years, from the early sex determination and immunological phenotypes attributed to it in the 1950s, to the several mouse Y permatogenic phenotypes and the sex ratio distortion phenotype that are still being studied today. However, even though the draft sequence of the mouse genome was published in 2000, only 750 kb of the 95 Mb mouse Y chromosome was sequenced at that time. To fill that void, we are sequencing the male-specific portion of the mouse Y, and present the analysis of 72 Mb of the mouse Y chromosome here. We found that the X-degenerate portion of the mouse Y chromosome, the portion that descends from the autosomal progenitors of the mammalian X and Y chromosomes, is highly degenerate, even more so than the human Y's X-degenerate sequence. But the ampliconic portion of the mouse Y has expanded to an incredible degree, taking up 95% of the chromosome. Almost all of the ampliconic portion of the mouse Y chromosome is made up of a single 92 Mb segmental duplication that we have named the Huge Repeat array. The Huge Repeat array is a collection of 150-200 515 kb repeat units. The repeat units are internally repetitive, gene-containing, and have a repeat unit to repeat unit similarity ranging from 99% to 99.999%. This array has no homology to the human Y chromosome. The functions of the Huge Repeat are still unclear...