Les crossing-overs (CO) sont indispensables dans la création variétale pour permettre l'introgression de régions d'intérêt dans les variétés agronomiques d'espèces cultivées telles que le blé tendre (Triticum aesstivum L.). Afin d'évaluer l'impact des facteurs qui influencent leur formation, nous avons entrepris une caractérisation fine de leur distribution sur le plus grand chromosome ( chromosome 3B, 995Mb) du blé tendre en s'appuyant sur la carte physique récemment développée et le séquençage de quelques régions de plusieurs mégabases. La comparaison entre une carte génétique dense (102 marqueurs) et une carte physique de délétion montre que 77% des CO sont présents dans les régions distales couvrant seuleument 25% du chromosome. La comparaison de différentes cartes génétiques montre de plus que cette distribution est conservée entre populations avec cependant des différences de taux de CO locaux entre populations mais également entre méiose mâle et femelle. Cette distribution est influencée par une interférence positive forte à des distances inférieures à 10 cM. Cependant, les faibles fréquences de CO observées au sein des régions proximales restent inexpliquées. En effet, la faible augmentation du taux de CO observée au sein des régions proximales placées en position distale suggère que la position proximale de ces régions sur le chromosome ne semble pas responsable de leur faible fréquence de CO. De plus, nous avons montré que ces faibles fréquences ne seraient pas non plus dues à une divergence de séquence entre chromosomes homologues au sein des régions proximales, la fréquence de CO étant toujours aussi faible au sein de celles-ci entre deux chromosomes homozygotes. En revanche, l'analyse à l'échelle d'une région séquencée de 3.1 Mb indique que les fréquences de CO importantes sont fortement corrélées avec la présence de gènes. L'inhibition de la formation des CO au sein des régions proximales pourrait ainsi s'expliquer par la présence de gènes en quantité moins importante dans ces régions comparées aux régions distales.

<p>Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured. </p><p>In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time. </p><p>DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background. </p><p> In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 130-133).

Resumo: A variabilidade entre progênies é criada pela segregação cromossômica independente dos genes e pela recombinação genética intracromossomal durante a meiose. O objetivo deste estudo foi analisar a variabilidade derivada de crossing-overs em cruzamentos biparentais (G2 e J2), quádruplos (G4 e J4) e óctuplos (G8 e J8), avaliados em populações segregantes derivadas de parentais contrastantes para resistência ao nematóide de cisto da soja (raça 3) - NCS - e ao oídio - O. A análise foi realizada em populações F2, através de marcadores SSR (single sequence repeat) concentrados em uma região de 55 cM ao redor do gene rmd (resistência ao oídio) e rhg1 (resistência ao NCS). Após o teste dos marcadores, quanto ao polimorfismo, apenas marcadores polimórficos foram utilizados para detectar crossing-over. Todos os marcadores analisados foram não significativos pelo teste de qui-quadrado (P > 0,05), indicando que os valores observados se ajustam à proporção genotípica esperada em F2 (1:2:1). As maiores médias de crossing-over por genótipo foram obtidas para G4 (4,00), no grupo G, e J8 (2,91), no grupo J. Por outro lado, as maiores médias de crossing-over considerando o número de gerações para formar cada população, foram para G2 (2,02) e J8 (0,97). A recombinação entre alelos ocorreu em algumas populações, entretanto para G4 e J8 em 1,89% dos genótipos não ocorreram. Em geral, nos cruzamentos com maior número de parentais envolvidos a ocorrência de crossingover foi maior, sendo satisfatórios na criação de variabilidade. O progresso no melhoramento de soja tem sido alcançado em partes pela criação de novas combinações alélicas dentro dos cromossomos. / Abstract: The variability among the progenies is created by chromosome segregation, independent assortment of genes, and intra-chromosomal genetic recombination during meiosis. The objective of this study was to analyze the variability derived from crossovers in soybean biparental (G2 and J2), quadruple (G4 and J4) and octuple (G8 and J8) crosses, measured in segregant population derived from contrasting parental regarding their resistance to cist nematode (race 3) - SCN and powdery mildew - PM. The analyses were made in F2 population through SSR (single sequence repeat) markers located in a 55CM region around Rmd (powdery mildew) and Rhg1 (cist nematode) resistance genes. After screening makers for their polymorphism, only polymorphic markers were used to detect crossovers. All markers were not significant by chi-square test (P > 0.05), showing that observed values corroborates to genotypic inheritance ratio expected in F2 population (1:2:1). Thus, the higher average of crossovers for some populations were observed for G4 (4.00), at linkage group G and J8 (2.91), at linkage group J. On the other hand, the higher average of crossovers considering the generation number to form each population, was found for G2 (2.02) and J8 (0.97). The recombination between alleles occurred in some populations, however, to G4 and J8, in 1.89% of the genotypes not showing crossover. In general, the crosses with larger numbers of parents showed higher number of crossovers, being very satisfactory for the creation of genetic variability. Soybean breeding progress has been accomplished in part by creating on new within_chromossome allele combinations. / Orientador: Antonio Orlando Di Mauro / Coorientador: Todd Pfeiffer / Banca: Natal Antonio Vello / Banca: Osvaldo Toshiyuki Hamawak / Banca: José Roberto Môro / Banca: Janete Apparecida Desidério Sena / Doutor

Plant gene families organized as linked clusters are capable of evolving by a process of unequal crossing-over. This results in the formation of chimeric genes that may impart a novel function. However, the frequency and functional consequences of these unequal cross-over events are poorly characterized. Plant disease resistance genes (R genes) genes are frequently organized as gene clusters. In this study, I constructed an elaborately designed reconfigurable synthetic RPP1 (for resistance to Paranospora parasitica) gene cluster (synthRPP1) to model R gene evolution by meiotic recombination. This experimental design utilizes gain-of-luciferase phenotype (luc+) to identify and isolate recombinant R genes and uses two alternatively marked alleles to distinguish and measure different types of meiotic recombination (intra- vs. inter-chromosomal). Two putative single copy transgenic plants containing the synthRPP1 gene cluster were generated. These synthRPP1 gene clusters were reconfigured in vivo by two kinds of site-specific recombination systems (CRE/Lox, FLP/FRT) to generate two alternative versions of the synthRPP1 gene clusters in vivo. These lines, as well as others being developed, will be used in future genetic crosses to identify and characterize plants expressing chimeric RPP1 genes. My second area of research was to use a previously developed synthetic RBCSB gene cluster (synthRBCSB) gene cluster to investigate the relative frequency of meiotic unequal crossing over between paralogous genes located on either homologous chromosomes (homozygous lines) or sister chromatids (hemizygous lines). In contrast to published somatic recombination frequencies using a different reporter gene system, no statistically significant difference of meiotic unequal crossing over was observed between homo- and hemi-zygous synthRBCSB lines. This result suggests that meiotic unequal crossing-over between paralogs located on homologous chromosomes occurs at about the same frequency as paralogs located on sister chromatids. To investigate the rate of somatic recombination in synthRBCSB lines, a QRT-PCR method was developed to estimate the frequency of somatic recombination. Preliminary results suggest that the somatic recombination frequency was about 10,000 fold higher than meiotic recombination in the same generation. Moreover, two of five cloned chimeric genes that formed by somatic recombination indicated a different distribution of resolution sites than those observed in meiotic recombination. This finding suggests there are significant differences in both the frequency and character of somatic versus meiotic unequal crossing-over between paralogous genes in Arabidopsis. / Ph. D.

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

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-09-28Bitstream added on 2014-06-13T18:43:25Z : No. of bitstreams: 1 muniz_frs_dr_jabo.pdf: 1182648 bytes, checksum: 94ba816afe15c07503cde37414bc5a99 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A variabilidade entre progênies é criada pela segregação cromossômica independente dos genes e pela recombinação genética intracromossomal durante a meiose. O objetivo deste estudo foi analisar a variabilidade derivada de crossing-overs em cruzamentos biparentais (G2 e J2), quádruplos (G4 e J4) e óctuplos (G8 e J8), avaliados em populações segregantes derivadas de parentais contrastantes para resistência ao nematóide de cisto da soja (raça 3) – NCS – e ao oídio - O. A análise foi realizada em populações F2, através de marcadores SSR (single sequence repeat) concentrados em uma região de 55 cM ao redor do gene rmd (resistência ao oídio) e rhg1 (resistência ao NCS). Após o teste dos marcadores, quanto ao polimorfismo, apenas marcadores polimórficos foram utilizados para detectar crossing-over. Todos os marcadores analisados foram não significativos pelo teste de qui-quadrado (P > 0,05), indicando que os valores observados se ajustam à proporção genotípica esperada em F2 (1:2:1). As maiores médias de crossing-over por genótipo foram obtidas para G4 (4,00), no grupo G, e J8 (2,91), no grupo J. Por outro lado, as maiores médias de crossing-over considerando o número de gerações para formar cada população, foram para G2 (2,02) e J8 (0,97). A recombinação entre alelos ocorreu em algumas populações, entretanto para G4 e J8 em 1,89% dos genótipos não ocorreram. Em geral, nos cruzamentos com maior número de parentais envolvidos a ocorrência de crossingover foi maior, sendo satisfatórios na criação de variabilidade. O progresso no melhoramento de soja tem sido alcançado em partes pela criação de novas combinações alélicas dentro dos cromossomos. / The variability among the progenies is created by chromosome segregation, independent assortment of genes, and intra-chromosomal genetic recombination during meiosis. The objective of this study was to analyze the variability derived from crossovers in soybean biparental (G2 and J2), quadruple (G4 and J4) and octuple (G8 and J8) crosses, measured in segregant population derived from contrasting parental regarding their resistance to cist nematode (race 3) – SCN and powdery mildew – PM. The analyses were made in F2 population through SSR (single sequence repeat) markers located in a 55CM region around Rmd (powdery mildew) and Rhg1 (cist nematode) resistance genes. After screening makers for their polymorphism, only polymorphic markers were used to detect crossovers. All markers were not significant by chi-square test (P > 0.05), showing that observed values corroborates to genotypic inheritance ratio expected in F2 population (1:2:1). Thus, the higher average of crossovers for some populations were observed for G4 (4.00), at linkage group G and J8 (2.91), at linkage group J. On the other hand, the higher average of crossovers considering the generation number to form each population, was found for G2 (2.02) and J8 (0.97). The recombination between alleles occurred in some populations, however, to G4 and J8, in 1.89% of the genotypes not showing crossover. In general, the crosses with larger numbers of parents showed higher number of crossovers, being very satisfactory for the creation of genetic variability. Soybean breeding progress has been accomplished in part by creating on new within_chromossome allele combinations.

La recombinaison méiotique via les Crossing-Overs (COs) est le principal mécanisme permettant le brassage de la diversité génétique. Cependant, le nombre et la position des COs entre paires de chromosomes homologues sont strictement régulés, limitant la séparation des loci en sélection variétale. Dans le cas du colza B. napus, l’utilisation d’allotriploïdes (AAC, 2n=3x=29), issus du croisement entre le colza (AACC, 2n=4x=38) et l’un de ses progéniteurs B. rapa (AA, 2n=2x=20), permet d’augmenter considérablement le nombre de COs entre chromosomes homologues A. L’objectif de cette étude était de déterminer les conséquences d’une telle variation sur la distribution des COs le long des chromosomes ainsi que d’identifier des facteurs régulant ce phénomène. Suite à la production et à la caractérisation cytogénétiques d’hybrides F1 présentant différents caryotypes, la recombinaison homologue a été évaluée par des analyses génétiques via des marqueurs SNPs physiquement ancrés sur l’ensemble duNous avons montré que l’addition du génome C chez les allotriploïdes conduit toujours à (1) la formation de COs surnuméraires, dont le nombre varie fonction des méioses mâle/femelle et du fond génétique, (2) une modification des profils de recombinaison, notamment au voisinage des centromères, et (3) une réduction de l’intensité d’interférence. De plus, nous avons révélé que le contrôle génétique de ces variations est imputé à des chromosomes C spécifiques et aurait divergé dans un contexte polyploïde. Nous avons donc identifié un levier permettant d’optimiser le brassage de la diversité gén / Meiotic recombination via crossovers (COs) is the main mechanism responsible for mixing genetic diversity. However, the number and position of COs between the pairs of homologous chromosomes are strictly regulated, limiting the loci separation in plant breeding. In the case of the rapeseed B. napus, the use of allotriploids (AAC, 2n=3x=29), resulting from the cross between rapeseed (AACC, 2n=4x=38) and one of its progenitors B. rapa (AA, 2n=2x=20), allows a substantial increase of the number of COs between homologous A chromosomes. The objective of this study was to determine the consequences of such a variation on the distribution of COs along the chromosomes and to identify factors regulating this phenomenon. Following the production and cytogenetic characterization of F1 hybrids with different karyotypes, homologous recombination was assessed by genetic analyzes via SNPs markers physically anchored on the whole A genome.We showed that the additional C genome in allotriploids always leads to (1) the formation of extra COs, for which the number depends on the male/female meiosis and the genetic background, (2) the modification of the recombination landscapes, especially in the vicinity of centromeres, and (3) the decrease of CO interference. In addition, we revealed that the genetic control of these variations is assigned to specific C chromosomes and could have evolved in a polyploid context. We have therefore identified a way to optimize the shuffling of genetic diversity in rapeseed breeding.

Mieux connaitre les facteurs qui gouvernent l’apparition des évènements de recombinaison (crossing-overs ; CO) chez le blé tendre (Triticum aestivum L.) est primordial car ce processus est l’outil principal du sélectionneur pour permettre le brassage de la diversité génétique et l’introgression de régions d’intérêt dans des variétés agronomiques. L’utilisation de techniques de cytogénétique développées sur l’orge a permis de comparer la mise en place de la synapse lors de la méiose chez des lignées de blé tendre délétées de tout ou partie du bras long du chromosome 3B et qui avaient été préalablement montrées comme présentant un nombre de chiasmas réduit par rapport à la variété euploïde. Les analyses cytogénétiques couplées à des études bioinformatiques de la séquence montrent que le synapsis a lieu quasiment normalement chez les mutants et que la délétion de certains gènes connus comme impactant le déroulement de la méiose pourrait expliquer le phénotype observé. De plus, le développement de ressources génomiques (SNP, séquence) à destination des sélectionneurs a permis la réalisation de cartes génétiques haute densité des 21 chromosomes ancrées sur la séquence du génome. Tous les chromosomes montrent le même profil de recombinaison avec un accroissement dans les parties distales et une réduction drastique dans les parties centromériques et péri-centromériques. L’exploitation de plus de 250 CO localisés dans des fenêtres de moins de 25 kb sur le chromosome 3B utilisé comme modèle pour l’étude de l’impact de la séquence sur la recombinaison, montre que les profils de recombinaison ancestrale et actuelle sont conservés et que les CO ont lieu préférentiellement dans les parties promotrices des gènes exprimés en méiose ce qui suggère que la conformation chromatinienne influence la recombinaison. Finalement, ces données ont aussi été l’opportunité de détecter des motifs liés à la recombinaison qui présentent des similarités avec celui ciblé par la protéine PRDM9 qui conduit à la recombinaison chez l’humain. Cela suggère que les mécanismes de contrôle de la recombinaison sont conservés chez les eucaryotes. / Better knowledge of the factors that drive recombination (crossovers; COs) in bread wheat (Triticum aestivum L.) is of main interest since this process is the main tool allowing breeders to admix the genetic diversity and to introgress regions of interest in agronomic varieties. We used cytogenetic techniques previously developed on barley to compare the establishment of synapsis during meiosis in deletion lines missing part or whole of the long arm of chromosome 3B of bread wheat and which were previously shown as having a reduced chiasmata number compared to euploid varieties. Cytogenetic analysis combined with bioinformatics studies showed that the synapsis occurs almost normally in mutants and that deletion of some genes known to impact meiosis behavior may explain the observed phenotype. In addition, development of genomic resources (SNPs, sequence) for wheat breeders allowed simultaneous elaboration of high density genetic maps for the 21 chromosomes anchored on genome sequence. All chromosomes present the same recombination pattern with an increase of recombination in the distal parts and reduction in centromeric/pericentromeric regions of the chromosomes. Analysis of more than 250 COs mapped in windows lower than 25kb located on chromosome 3B used as model to study the impact of sequence features on recombination showed that current and ancestral recombination patterns are conserved and that COs preferentially occur in the promoter part of gene expressed in meiosis suggesting that chromatin conformation impacts recombination. Finally, these data were the opportunity to detect recombination-associated motif which presents similarities with the motif targeted by the PRDM9 protein driving recombination in human. This suggests that the control of recombination mechanisms is conserved among eukaryotes.

Repair of DNA double-strand breaks via homologous recombination is an essential pathway for vertebrate cell development and maintenance of genome integrity throughout the organism’s lifetime. The Rad51 enzyme provides the central catalytic function of homologous recombination while many other proteins are involved in regulation and assistance of Rad51 activity, including a group of five proteins referred to as Rad51 paralogs (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3). At the start of my work, cellular studies of human Rad51 (HsRad51) had shown only that it forms distinct nuclear foci in response to DNA damage. Additionally, no information regarding the cellular localization, potential DNA damage-induced redistribution or cellular functions for any of the Rad51 paralog proteins was available. Therefore, the goals of this work were to (1) present a more complete description of the cellular localization and DNA damage-induced redistribution of Rad51 and the two paralog proteins known to specifically associate with Rad51, Rad51C and Xrcc3, and (2) to define specific functional roles for Rad51C and Xrcc3 in mediating Rad51 activity. I focused on the use of cellular, RNAi and immunofluorescence methods to study endogenous Rad51, Rad51C and Xrcc3 in human cells. In my initial studies we showed for the first time that Xrcc3 forms distinct foci in both the nucleus and cytoplasm independent of DNA damage, that the distribution of these foci did not change significantly throughout the time course of DNA damage and repair, and that Xrcc3 focus formation is independent of Rad51. Additionally, and unlike most previously published images of nuclear Rad51, we found that the majority of DNA damage-induced nuclear Rad51 foci do not colocalize with gamma H2AX, a histone marker used to indicate the occurrence of DNA double strand breaks. As a consequence of these initial outcomes, a significant amount of effort was devoted to developing and optimizing immunofluorescence methods. Importantly, we developed a purification method for commercially available monoclonal antibodies against Rad51C and Xrcc3 that significantly improved their reactivity and specificity. My next study concentrated on Rad51C. Similar to Xrcc3, we found for the first time that Rad51C forms distinct nuclear and cytoplasmic foci independent of DNA damage and Rad51. An additional and surprising outcome was our discovery that Rad51C plays an important role in regulating the ubiquitination and proteasome-mediated degradation of Rad51. While biochemical functions for Rad51 paralog proteins had been suggested in the literature, this was the first demonstration of a specific biochemical function for Rad51C that contributes directly to the Rad51 activity in the homologous recombination pathway. Our improved immunofluorescence methods allowed us to see the accumulation of Rad51, Rad51C and Xrcc3 at the nuclear periphery early in response to DNA damage, suggesting the existence of a DNA damage-dependent trafficking mechanism that promoted movement of these proteins from the cytoplasm to the nucleus. This led to further studies in which we show distinct co-localization of cytoplasmic Rad51 with actin as well as alpha and beta tubulin. Using both immunofluorescence and sub-cellular fractionation methods our recent results strongly suggest that trafficking of Rad51 to the nucleus in response to DNA damage is regulated at least in part by its association with cytoskeletal proteins, and involves movement of both existing pools of Rad51 and newly synthesized protein. In a particularly exciting development, in collaboration with Leica Microsystems and Dr. Joerg Bewersdorf at The Jackson Laboratory, Bar Harbor, ME., I have been able to exploit a new technology, 4Pi microscopy, to provide the first images of endogenous nuclear proteins using this method. Results presented in this thesis have added significantly to a more complete understanding of cellular localization Rad51, Rad51C and Xrcc3, and have provided important insights into possible mechanisms of cellular trafficking of Rad51 in response to response to DNA damage. Additionally, we have defined a specific function for Rad51C in its regulation of Rad51 ubiquitination. These findings open several new avenues of investigation for furthering our understanding of the cellular and molecular functions of proteins with critical roles in the maintenance of genome integrity in human cells.