Orientador: Luciana Bolsoni Lourenço Morandini / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T15:06:51Z (GMT). No. of bitstreams: 1 Targueta_CintiaPelegrineti_D.pdf: 14058240 bytes, checksum: 609267a5b0419113c3e1035f998113c6 (MD5) Previous issue date: 2013 / Resumo: Dentre as espécies do gênero Engystomops, somente as três espécies do clado Edentulus (E. freibergi, E. petersi e E. pustulosus) e duas das seis espécies do clado Duovox (E. pustulatus e E. puyango) tinham o número diploide descrito, e a localização cariotípica das NORs (regiões organizadores de nucléolo) e das regiões heterocromáticas era conhecida apenas para E. freibergi, E. petersi e E. puyango. Os dados disponíveis mostravam que as espécies do clado Duovox já cariotipadas apresentavam 2n=20, enquanto o número cromossômico diploide de todas as espécies do clado Edentulus era 2n=22, assim como o das espécies de Physalaemus e Edalorhina, gêneros mais proximamente relacionados a Engystomops. Dessa forma, uma redução do número diploide de 2n=22 para 2n=20 era considerada na história de Engystomops, mas não era possível inferir se essa era uma sinapomorfia de E. puyango e E. pustulatus ou de todo o clado Duovox. Em relação ao clado Edentulus, algumas dúvidas também persistiam. Recentes estudos propunham a presença de um complexo de espécies amazônicas sendo confundidas com E. petersi. Citogeneticamente puderam ser reconhecidos três grupos nesse complexo, que corresponderam a populações de Puyo (Equador), Yasuní (Equador) e La Selva (Equador), diferentes ainda daquele de E. freibergi (Brasil). Nem sempre é possível a identificação de homeologias cromossômicas entre esses grupos, o que dificulta o reconhecimento dos possíveis rearranjos cromossômicos envolvidos na divergência cariotípica no gênero em questão. Também permanecia pouco explorada a diferenciação entre os cromossomos sexuais heteromórficos reconhecidos em populações de E. petersi e em E. freibergi. Os objetivos do presente trabalho foram estudar citogeneticamente as espécies de Engystomops do clado Duovox, melhor caracterizar os cromossomos sexuais de E. freibergi por meio de pintura cromossômica e buscar novos marcadores citogenéticos para auxiliar na identificação de homeologias cromossômicas nesse gênero. A análise cariotípica mostrou que as espécies E. randi, E. guayaco, E. montubio, E. pustulatus e E. coloradorum apresentaram 2n=20, assim como as espécies E. puyango e E. pustulatus, previamente cariotipadas, corroborando a hipótese de essa característica provavelmente seja uma sinapomorfia do grupo Duovox. Em E. coloradorum, foi encontrada uma fêmea triploide, que consistiu no primeiro relato de poliploidia para Engystomops. Em todas e somente nas fêmeas dessa espécie foi encontrado um heteromorfismo referente à presença de NOR dentre os cromossomos 10 homólogos o que sugere que esses sejam cromossomos sexuais e que o sistema de determinação do sexo em E. coloradorum seja ZZ/ZW. Quanto à análise dos cromossomos sexuais de E. freibergi, foi possível isolar por microdissecção os cromossomos X e Y e produzir sondas a partir da amplificação de segmentos desses cromossomos. A utilização da técnica de pintura cromossômica com tais sondas permitiu inferir que as regiões proximais do cromossomo X e do cromossomo Y de E. freibergi apresentam similaridade molecular, o que sugere a possibilidade de se tratarem de regiões pseudo-autossômicas. As sondas dos cromossomos sexuais de E. freibergi não detectaram os cromossomos X e Y de E. petersi de Puyo nem os cromossomos do par 11 homomórfico de E. petersi de Yasuní, não evidenciando, portanto, grande similaridade entre eles. O isolamento, a caracterização e a localização cariotípica do DNAr 5S das espécies pertencentes ao grupo Duovox permitiram inferir a possível homeologia do par cromossômico 6 dessas espécies entre si e com o par 6 das espécies do grupo Edentulus. Ainda, o uso de sondas PcP190EcoRI, referentes a uma família de DNA repetitivo derivada de DNAr 5S, permitiu identificar os cromossomos 5 de E. randi, E. guayaco e E. coloradorum e diferenciá-los dos cromossomos 6 dessas espécies, portadores do sítio de DNAr 5S do tipo II. Além disso, essa mesma sonda identificou a região pericentromérica do braço curto do cromossomo 3 (e do cromossomo 5 de E. petersi de Yasuní) de todas as espécies do gênero, com excessão de E. coloradorum, sugerindo outra possível homeologia entre esses cromossomos. Dessa forma, com o emprego de técnicas citogenéticas clássicas e moleculares foi possível fornecer importante contribuição para o estudo da evolução do gênero Engystomops / Abstract: Of the species in the genus Engystomops, only three species of the Edentulus clade (E. freibergi, E. petersi and E. pustulosus) and two species of the Duovox clade (E. pustulatus and E. puyango) have had their diploid number described, while the NOR (nucleolus organizer region) and the heterochromatic sites were known only for E. freibergi, E. petersi and E. puyango karyotypes. The species of Duovox clade had 2n=20, while the diploid chromosome number of all species of Edentulus clade was 2n=22, which is the same diploid number of species of Physalaemus and Edalohina, genera closely related to Engystomops. Thus, the diploid number reduction from 2n=22 to 2n=20 was supposed, but it was not possible to infer if this was a synapomorphy of E. puyango and E. pustulatus or a synapomorphy of the entire Duovox clade. With regards to the Edentulus clade, some doubts also persisted. Recent studies proposed a complex of Amazonian species misidentified as E. petersi and it was possible to cytogenetically recognize three groups in this complex that corresponded to populations from Puyo (Ecuador), Yasuní (Ecuador) and La Selva (Ecuador). All of these karyotypes also differed from the E. freibergi (Brazil) karyotype. Chromosome homologies are not easily recognized between these groups, a fact that makes difficult the identification of chromosome rearrangements involved in karyotypic divergence in this genus. Additionally, the differentiation of the heteromorphic sex chromosomes found in the E. petersi and E. freibergi karyotypes remained unexplored. The goals of the present work are (1) to cytogenetically study the species of Duovox clade, (2) to better characterize the sex chromosomes of E. freibergi by means of chromosome painting and (3) to find new cytogenetic markers to identify potential chromosome homologies in this genus. The karyotypic analysis showed that the species E. randi, E. guayaco, E. montubio, E. pustulatus and E. coloradorum had 2n=20, as previously described in E. pustulatus and E. puyango. This corroborates the hypothesis that the diploid number 2n=20 is likely a synapomorphy of the Duovox clade. Among the specimens of E. coloradorum, a triploid female was found, which is the first report of polyploidy for the genus Engystomops. In all the females of this species, there was a NOR heteromorphism in the homologous chromosomes 10, suggesting that these may be ZZ/ZW sex chromosomes. Regarding the sex chromosomes of E. freibergi, it was possible to isolate the X and Y chromosomes by microdissection and to produce probes by amplifying segments of the microdissected chromosomes. The hybridization of these probes in E. freibergi metaphases showed that the proximal regions of the X and Y chromosomes of this species had molecular similarity, thus suggesting that they may be pseudoautosomal regions. The sex chromosome probes from E. freibergi detected neither the X nor the Y chromosome of E. petersi from Puyo nor the chromosomes of homomorphic pair 11 of E. petersi from Yasuní. This implies no significant similarity between them and the E. freibergi sex chromosomes. The isolation, characterization and karyotypic localization of the rDNA 5S from the species of the Duovox clade made it possible to infer the homeology of chromosome pair 6 of those species, as well as their homeology with chromosome pair 6 of the species of the Edentulus clade. Further, the probes PcP190EcoRI, which are related to a family of satellite DNA derived from rDNA 5S, made it possible to identify chromosome 5 of E. randi, E. guayaco and E. coloradorum and to differentiate them from chromosome 6, which carry type II rDNA 5S. In conclusion, both classical and molecular cytogenetic techniques provided important contributions for the study of the evolution of the genus Engystomops / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

Orientador: Eliana Regina Forni Martins / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T15:34:53Z (GMT). No. of bitstreams: 1 Oliveira_VanessaMancusode_M.pdf: 1586139 bytes, checksum: 790cc37633d3cc0d647e5e8e47cbb71c (MD5) Previous issue date: 2005 / Resumo: Foram estudadas, através da análise mitótica (técnica de Giemsa), 14 espécies do gênero Vernonia sensu Baker (Asteraceae, Vernonieae), pertencentes à seção Lepidaploa, correspondentes às subseções Axilliflorae, Macrocephalae, Oligocephalae, Paniculatae e Scorpioideae, objetivando subsidiar as propostas de seu desmembramento em gêneros menores (sensu Robinson) ou da manutenção de sua integridade (sensu Baker). As espécies foram coletadas em áreas de cerrado e campo rupestre, nos Estados de São Paulo, Minas Gerais e Goiás. Foram realizadas contagens cromossômicas, que variaram de 2n=20 a 2n=ca.80 e, elaborados cariótipos, verificando-se o predomínio de cromossomos metacêntricos, e alguns submetacêntricos. Foram observados cromossomos B em uma das populações analisadas de V. geminata. O tamanho dos cromossomos variou de 0,9 a 4,9µm, o tamanho total de cromatina (CTC) de 29,7 a 50,7 µm e, o índice de assimetria TF% de 41,2 a 46,9. O índice de assimetria intracromossômica (A1) variou de 0,13 a 0,29, enquanto o índice de assimetria intercromossômica (A2) de 0,14 a 0,21. A população 1 da espécie V. geminata foi a que mostrou ter cariótipo mais assimétrico. Foram observadas diferenças cariotípicas entre populações de V. remotiflora e V. polyanthes. Foram aplicados em V. geminata bandamentos C, NOR, CMA/DA/DAPI e a técnica de hibridação de DNA in situ para a seqüência de 45S de rDNA. A espécie apresentou dois pares de bandas C, sendo duas bandas terminais e duas centroméricas; um par de bandas CMA+ terminais; dois pares de bandas NOR, sendo duas bandas terminais e duas centroméricas. A hibridação in situ evidenciou dois pares de sítios de rDNA 45S, sendo dois sítios terminais e dois centroméricos. Houve coincidência de localização entre bandas C, CMA, NOR e sítios de rDNA 45S. Não foi possível comparar os resultados dos bandamentos e sítios de hibridação in situ com outras espécies de Vernonia, por não existir dados disponíveis para o gênero na literatura. Embora a representatividade da amostra seja pequena, os dados cariotípicos obtidos, no presente trabalho e em literatura, ainda não permitiram apoiar conclusivamente qualquer das propostas taxonômicas vigentes para Vernonia, devido à inexistência de um padrão cariotípico característico/distintivo para cada grupo taxonômico, ou seja, seções e subseções (sensu BAKER 1873) ou novos gêneros (sensu ROBINSON 1999a). No entanto, até o momento, parece existir, uma tênue relação com a conceituação de ROBINSON (1999a) para os gêneros Lessingianthus, Vernonanthura, e Chrysolaena / Abstract: We studied, from the mitotic analyses (Giemsa technique), 14 species of Vernonia sensu BAKER (Asteraceae, Vernonieae), belonging to Lepidaploa section, purposing to assistant the proposal of its separate in little genus (sensu ROBINSON) or maintenance of its complete (sensu BAKER). We colleted species in ¿cerrado¿ and ¿campo rupestre¿ areas, in São Paulo, Minas Gerais and Goiás states. Chromosome numbers (2n=20 to ca.80) and karyotypes are analyzed, with predominance of metacentric and some submetacentric chromosomes. We observed B chromosomes in a population of V. geminata analyzed. Chromosomes size varied 0,9 to 4,9µm, total size of chromatin 29,7 to 50,7 µm and, asymmetry index TF% 41,2 to 46,9. The intrachromosomal asymmetry index (A1) varied 0,13 to 0,29 and, the interchromosomal asymmetry index (A2) varied 0,14 to 0,21. The population 1 of V. geminata showed the most asymmetric karyotype. Some differences of karyotypes are observed in V. remotiflora and V. polyanthes populations. We applied banding in V. geminata neither C, NOR, CCD and in situ hybridization technique for 45S rDNA sequences. It showed two pairs of bands C, it are two terminal bands and two centromeric; one pair of CMA+ terminal bands; two neither pairs of NOR band, its are two terminal and Two centromeric. The in situ hybridization showed two pairs of rDNA 45S sites, two terminal and two centromeric bands. There are coincidence of localization among C, CMA, NOR bands and rDNA 45S sites. We can not compare the results of the banding and in situ hybridization sites with others Vernonia species, because there are not datas for the genus in literature. The karyotype datas obtained here do not permitted support conclusively the taxonomic proposes to Vernonia, because the inexistence of a characteristic/distinctive karyotype pattern for each taxonomic group, ou seja, sections and subsections (sensu BAKER 1873) or new genus (sensu ROBINSON 1999a). Além disso, the representative of the samples is little. However, while, look exist, a little relationship between the chromosomes number obtained here and in the literature with RobiNSON¿s propose (1999a) for the genus Lessingianthus, Vernonanthura e Chrysolaena / Mestrado / Mestre em Biologia Vegetal

Oligoryzomys é o gênero mais especioso da tribo Oryzomyini e está amplamente distribuído na região Neotropical. O objetivo deste trabalho é contribuir para a citotaxonomia e investigar a evolução cromossômica no gênero. Foram analisados 117 exemplares pertencentes às espécies: O. flavescens (2n=64-66, NF=66), O. fornesi (2n=62, NF=64), O. microtis (2n=64, NF=64), O. moojeni (2n=70, NF=72), O. nigripes (2n=62, NF=78-82), O. stramineus (2n=52, NF=68) e Oligoryzomys sp. A (2n=70, NF=72). As seis primeiras possuem cariótipos espécie-específicos e, dessa forma, reiteramos a importância da informação citogenética para a citotaxonomia. A pintura cromossômica comparativa (Zoo-FISH) com sondas de O. moojeni revelou hibridação em 29 segmentos autossômicos em O. fornesi; 30 em O. microtis; 31 em O. nigripes; e 32 em O. rupestris e Oligoryzomys sp. 2. Os resultados mostraram uma extensa reorganização genômica na evolução cromossômica do gênero, decorrente de fissões, fusões em tandem, rearranjos Robertsonianos e perda/inativação, surgimento ou reposicionamento de centrômero. / Oligoryzomys is the most specious genus within the tribe Oryzomyini and it is distributed throughout Neotropical region. This work aims to contribute to citotaxonomy and to investigate the chromosomal evolution of the genus. A total of 117 individuals were cytogenetically analysed, and they belong to the species: O. flavescens (2n=64-66, FN=66), O. fornesi (2n=62, FN=64), O. microtis (2n=64, FN=64), O. moojeni (2n=70, FN=72), O. nigripes (2n=62, FN=78-82), O. stramineus (2n=52, FN=68), and Oligoryzomys sp. A (2n=70, FN=72). The first six species possess species-specific karyotypes, and therefore we emphasize the importance of cytogenetic studies for citotaxonomy. Comparative chromosome painting (Zoo-FISH) with O. moojeni probes hybridized to 29 segments on metaphases of O. fornesi, 30 on O. microtis, 31 on O. nigripes, and 32 on O. rupestris and Oligoryzomys sp. 2. The results showed an extensive genomic reshuffling, due to fissions, tandem and Robertsonian fusions, loss/inactivation or repositioning of centromeres.

La mitose permet la multiplication des cellules, contribuant ainsi à générer de nouveaux organismes unicellulaires, ou à construire des organismes multicellulaires. Pendant la mitose, le génome répliqué de la cellule mère est réparti entre les deux cellules filles. Les erreurs survenant lors de la répartition peuvent mener à l’aneuploïdie, une caractéristique de certaines maladies développementales dont les cancers. La fidélité de la répartition des chromatides sœurs dépend du fuseau mitotique, un réseau bipolaire de microtubules qui dirigent les chromosomes via leurs interactions avec les kinétochores assembles sur les chromatides sœurs. Ces interactions mènent à l’alignement des chromosomes, et à leur biorientation. Les chromatides sœurs sont alors attachés à des microtubules .manant des pôles opposés du fuseau. La ségrégation des chromatides sœurs a alors lieu en anaphase, et simultanément le fuseau central est assemblé entre les deux jeux de chromosomes. Cette structure composée de microtubules contribue à la ségrégation des chromatides sœurs en spécifiant la localisation et en favorisant l’ingression du sillon de division cellulaire. Pendant ma thèse, j’ai étudié les fonctions d’un ensemble de protéines du kinétochore, BUB-1, HCP-1/2CENP-F et CLS-2CLASP, lors de la mitose dans les zygotes de C. elegans. En combinant des approches de génétique et de vidéo-microscopie, j’ai montré que ces protéines participent à l’alignement et à la ségrégation des chromosomes. En particulier, BUB-1 contribue à l’alignement des chromosomes en accélérant l’attachement des microtubules aux kinétochores, tout en contrôlant la conformation et la maturation de ces attachements. Ces activités dépendent du recrutement de HCP-1/2 et CLS-2 par BUB-1, mais aussi du complexe RZZ et de la dynéine, ainsi que d’une activité de BUB-1 inhibant le recrutement du complexe SKA aux kinétochores. De plus, j’ai montré que BUB-1, HCP-1/2 and CLS-2 contribuent à l’assemblage des microtubules du fuseau central via l’activité polymérase de CLS-2. Cette fonction dépend du pré-recrutement de ces protéines aux kinétochores en métaphase, en aval de KNL-1, révélant une nouvelle fonction pour les kinétochores dans l’assemblage du fuseau central. Ce travail identifie donc des fonctions versatiles pour ces protéines, les plaçant comme des gardiennes majeures de l’intégrité génétique / Mitosis is a process by which cells multiply, contributing to the generation of new unicellular organisms, or the construction of multicellular organisms. During mitosis, the daughter cells inherit an identical copy of the mother cell’s replicated genome. Errors in genetic material distribution can lead to aneuploidy, a hallmark of developmental diseases including cancer. The accurate segregation of sister chromatids relies on the mitotic spindle, a bipolar network of microtubules that governs chromosome movements by interacting with the kinetochores assembled on sister chromatids. This drives chromosome alignment at the spindle equator, and chromosome bi-orientation meaning that sister kinetochores are connected to opposite spindle poles, laying the ground for sister chromatid segregation during anaphase. Once segregation has initiated, the microtubule-based central spindle is assembled between the two sets of chromosomes. This structure contributes to sister chromatid segregation, by specifying the location and favoring the ingression of the cytokinesis furrow. During my thesis, I have studied the functions of a subset of conserved kinetochore proteins called BUB-1, HCP-1/2CENP-F and CLS-2CLASP, during mitosis in C. elegans zygotes. By combining genetics and live imaging, I have shown that these proteins are involved both in chromosome alignment and segregation. In particular, I have shown that BUB-1 contributes to chromosome alignment by accelerating the establishment of end-on kinetochore-microtubule attachments, while controlling the conformation and maturation of these attachments. These activities rely on BUB-1’s downstream partners HCP-1/2CENP-F and CLS-2CLASP, but also on the RZZ complex and dynein, as well as an activity for BUB-1 in inhibiting the recruitment of the SKA complex. Additionally, I have shown that BUB-1, HCP-1/2CENP-F and CLS-2CLASP contribute to central spindle microtubule assembly, via CLS-2CLASP’s polymerase activity. This function relies on the prior kinetochore recruitment of these proteins during metaphase by the kinetochore scaffold protein KNL-1, revealing a new function for the kinetochore in central spindle assembly. Together, this work identifies versatile functions for this subset of conserved kinetochore proteins, making them major safe-keepers of genomic integrity

L'endoribonucléase RNase L est essentiellement connu comme étant un acteur critique de l'immunité innée pour enrayer la progression d'une infection virale en clivant les ARN cellulaires. Son activité est régulée par de nombreux facteurs tels que la 2-5A et son inhibiteur, la RLI. Au cours de cette étude, nous avons démontré une implication de l'activité de la RNase L dans la régulation de la structure du domaine centromérique. Nous présentons dans ce manuscrit, les perturbations majeures engendrées par une augmentation ou une inhibition de l'activité de la RNase L représentées par une délocalisation de HP1-alpha et de CENP-C causant une déstructuration générale des chromosomes. Ces délocalisations de protéines centrales de la structure chromatinienne seraient causées par un défaut de la maturation des transcrits majeures péricentromériques lors d'une modulation de l'activité de la RNase L. Pour terminer, nous avons également identifié un potentiel trafic cyto-nucléaire empreinté par la RNase L. Nous proposons ainsi une fonction nucléaire inattendue de la RNase L par son implication dans la régulation des transcrits péricentromériques assurant l'intégrité structurale de la chromatine. / The endoribonuclease Latente (RNase L) is mostly known as a critical factor in the innate immunity during the cell's defence against a viral infection. The antiviral activity of RNase L which is characterize by it capacity of cleavage of viral RNA, is regulated by several factors like it activator the oligoadénylates 2-5A and his inhibitor RLI. In this manuscript, we have studied the role of the activity of RNase L in the regulation of the structure of centromeric domains. Our results show a general destructuration of chromosomes observed in cells over-expressing RNase L or RLI. These major aberrations are demonstrated by a delocalization of essentials proteins for the structure of chromatin: HP1-alpha and CENP-C. The mislocalization of these proteins could be provoked by a default in the maturation of major transcripts due to a modulation of the activity of RNase L. moreover, in this study, we have identified a mechanism regulating the cyto-nuclear shuttling of RNase L. therefore, we propose that a new nuclear function of RNase L: it's implication in the regulation of pericentromeric transcripts needed to stabilize the integrity of the structure of chromatin.

Evolution of sex chromosomes and karyotypes in iguanas (Squamata: Pleurodonta) PhD Thesis Marie Altmanová Abstract This PhD thesis is composed of five published articles and one manuscript, and is focused on the evolution of the sex chromosomes and karyotype of the iguanas (Pleurodonta). Based on our primary research of available data, only male heterogamety (XX/XY) with ancestral karyotype 2n = 36 chromosomes was recorded in iguanas. However, in many species sex chromosomes have not been uncovered by classical cytogenetics, probably due to their homomorphy. The partially-known X chromosome content of Anolis carolinensis allowed us to compare the relative gene doses of X-specific genes between male and female of representatives of all iguana families, and to reveal homologous and well-differentiated sex chromosomes across all iguanas, with the exception of basilisks. Thus, due to the comparable age with sex chromosomes of mammals and birds, the results put into question the importance of endothermy for the formation of stable sex chromosomes. The striking feature of the iguanas is the relatively frequent occurrence of multiple sex chromosomes in their karyotypes. Using the ancestral state analysis of the type of sex chromosomes, it has been found that these multiple sex chromosomes developed at least twelve...

Chromosomes are very dynamic structures that are constantly undergoing physical changes necessary for cell survival. Studies in yeast and metazoans have shown that chromosomal loci exhibit large-scale changes in mobility in response to DNA double-strand breaks (DSBs). If left unrepaired, DSBs can lead to disease and even cell death. One of the predominant cellular pathways utilized to repair DSBs is homologous recombination (HR). DSB repair via HR requires a homologous DNA template to recover the missing genetic information lost at the break site. Our lab proposes that increased chromosome mobility (ICM) facilitates recombination by helping a broken chromosome successfully find its homolog. In support of this view, ICM is under the genetic control of the HR machinery and requires activation of the DNA damage checkpoint response. However, there is currently no consensus on the precise functional role of ICM in HR. In Chapter 1, I describe in detail the known steps of DSB repair via the HR pathway, and discuss some of the important advancements made in the field of cell biology that has helped shape our understanding of HR. I highlight the use of in vivo cell imaging and fluorescently labeled DNA repair proteins during the study of HR. Additionally, I discuss some of the first studies that examined chromosome dynamics within the nucleus in live cells. Lastly, I describe the phenomenon of increased chromosome mobility and expand upon why it needs to be studied further. In Chapter 2, I present in detail our method for measuring the pairing of DNA loci during HR at a site-specific DSB in Saccharomyces cerevisiae. This method utilizes live cell imaging and a chromosome tagging system in diploid yeast to visualize homologous chromosomes during HR-mediated repair. Using this method, we demonstrate that in wild type (WT) cells, homologous chromosomes come together, repair and then move apart after repair is complete. Importantly, the kinetics we observe in the pairing of homologous chromosomes match the kinetics of site-specific DSB formation and the subsequent gene conversion of that site. In Chapter 3, I describe our study that elucidates the relationship between ICM and multiple HR steps. We find a tight temporal correlation between the recruitment of the recombination proteins, ICM, the physical pairing of homologous loci, and gene conversion. Importantly, we can shift the timing of ICM by altering the initiation of DNA end resection - an early step in the HR process. Our data highlight the importance of DNA end resection as a vital precursor to ICM and demonstrate a strong temporal linkage between ICM and HR. Taken together our data support the claim that ICM is essential to HR and mechanistically involved in the process of DNA repair. In Chapter 4, we explore chromosome mobility in response to different forms of DNA damage such as spontaneous DSBs, collapsed replication forks, and ionizing radiation (IR). We find that spontaneous DSBs and collapsed replication forks do not induce a change in chromosome mobility. However, exposure to ionizing radiation results in a robust increase in global chromosome mobility that is dependent on activation of the DNA damage checkpoint. Overall, these findings demonstrate how ICM is tightly regulated and highly dependent on the circumstances surrounding the formation of the DSB. Lastly, in Chapter 5, I summarize all of my findings and discuss how they relate to one another with respect to the linkage between ICM and HR. I also provide a perspective on future experiments needed to advance the field.

The unique chromosome biology of the fungus fly Sciara coprophila has fascinated investigators for over 80 years. Male meiosis exhibits a monopolar spindle, nonrandom segregation of imprinted chromosomes and nondisjunction of the X chromosome. The unusual mechanism of sex determination requires selective elimination of X chromosomes in embryogenesis. Super-numerary (L) chromosomes are also eliminated from the soma during early cleavage divisions. Distinctive DNA puffs on the larval salivary gland chromosomes are sites of DNA amplification. As a foundation for future genome studies to explore these many unusual phenomena, we have used DNA-Feulgen cytophotometry to determine genome size from hemocyte nuclei of male (X0) and female (XX) larvae and adults. The DNA content of the X chromosome is ∼0.05 pg DNA and the autosomal complement is ∼0.45 pg DNA. Measurements of DNA levels for individual sperm from adults showed that the DNA contribution of the germ line-limited (L) chromosomes constitutes as much as 35% of the DNA of the male gamete. A parallel study using Sciara ocellaris, a related species lacking L chromosomes, confirmed the presence of two X chromosomes in the sperm of this species.

A study was undertaken to investigate the application of tissue culture and histochemical banding techniques normally used in mammalian studies to fish cells and to examine the mechanisms of chromosome evolution and speciation in salmonids by comparing the chromosomes of the hybrid to those of the parental types. The application of banding techniques was generally unsuccessful in revealing bands, though quinacrine staining did reveal the presence of two fluorescent spots in the vicinity of the centromere. These were thought to be heterochromatic blocks, possibly related to the high incidence of centric fusion or fission in salmonids. Results of chromosome analysis showerd a higher incidence of acrocentric chromosomes in the hybrid than in either parental type which together with evidence for tetraploidization in salmonids is consistant with some of the requirements for species diversification. Conditions of divergence in chromosome evolution and changes which would have to occur in the hybrid for divergence from the parental types to take place are discussed. The results of a preliminary electrophoretic examination of LDH and esterase activity tended to confirm hybridization.

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strandbreaks (DSBs) into homology-directed repair (HDR) domains. Using genomic approaches, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. Chromosome conformation capture techniques (Hi-C) reveal multi-scale alterations in genome organization following damage, including changes in chromatin insulation and compartmentalization. Nuclear actin polymerization promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements as visualized by high-throughput translocation assays (HTGTS). Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.