Embryonic chromatin diminution, the selective excision of large amounts of heterochromatic DNA from presomatic cell lineages, provides an example of an unusually large augmentation of the germline genome and raises questions regarding the source of the increased amount of DNA and its relevance to the biology of the organism. DNA levels in adult germ cell nuclei of the copepod Cyclops kolensis were determined by DNA-Feulgen cytophotometry and compared with those of somatic nuclei of adults and both pre- and postdiminuted embryos from the same mothers. Almost 75 pg DNA/nucleus is excised by diminution, resulting in the return of each generation to the approximately 1 pg DNA/nucleus level found for adult soma. To account for the increase in DNA levels of germ cells observed here, we propose alternative hypotheses to the original model of chromatin diminution: (1) repetitive endocycles or (2) proliferation of genetic elements. Specific tests for these hypotheses using next-generation sequencing and quantitative cytophotometry, as well as the functional significance of germ cell DNA augmentation to the copepod, are discussed.

Durante o desenvolvimento deste projeto adotou-se como estratégia o sequenciamento de ESTs, com a finalidade de encontrar mensagens relacionadas com desenvolvimento, metabolismo e principalmente amplificação/politenização em glândulas salivares de Rhynchosciara americana, um díptero (Sciarídeo) que apresenta cromossomos politênicos e amplificação gênica rigidamente regulada ao longo do desenvolvimento larval, tanto neste tecido quanto em outros. Um total de 8193 ESTs foi gerado, estas foram anotadas e categorizadas segundo os termos do Gene Ontology Consortium, proporcionando uma visão geral do status metabólico, como em um Northern eletrônico, de um ponto importante no desenvolvimento desta espécie, quando surgem amplificações gênicas específicas e a glândula salivar necessita secretar as proteínas do casulo. Outros frutos deste seqüenciamento foram a determinação de 91 polimorfismos e a criação de uma tabela de códon usage. Diversos ESTs foram identificados com potencial envolvimento com os endociclos observados neste tecido, destes, RaDup e RaMCM5 foram selecionados para estudo. Suas regiões genômicas foram isoladas e suas localizações cromossômicas foram identificadas, em relação a RaDup, toda a porção codificante de seu mensageiro e 12kb de DNA genômico contendo seu gene foram seqüenciados, revelando sua estrutura gênica. Anticorpos foram produzidos para detectar esta proteína, gerando evidências de sua participação tanto na replicação mitótica como nos endociclos presentes nas glândulas salivares. A localização cromossômica de RaDup é um dado muito interessante, pois pela primeira vez um pufe amplificado é relacionado com um gene regulatório. / In this work EST sequencing was used as strategy to find messages related to development, metabolism and polyteny/amplification in salivary glands of Rhynchosciara americana, a dipteran (Sciaridae) that shows in this tissue giant polytene chromosomes and gene amplification tightly regulated throughout development of the larvae. A total of 8193 EST sequences were generated, annotated and categorized using Gene Ontology Consortium terms, providing a general view of the metabolic status, like an electronic Northern, of an important point in development of the larvae, that shows where specific genes are amplified and the salivary gland needs to secrete the proteins to form the cocoon. Other data include determination of 91 SNPs and a statistic of codon usage. Several ESTs were identified with potential connection to endocicles, from these RaDup and RaMCM5 were selected for further studies. Both chromosomal loci were identified and genomic regions isolated, for RaDup the coding region of its mRNA and 12kb of genomic region were completely sequenced, revealing its gene structure, and antibodies were raised against this protein, making evident data about its involvement in replication in mitotic cells and in endocicles in salivary glands. About the chromosomal locus of RaDup, it becomes very interesting, because for the first time one amplified puff can be related to a regulatory gene.

Durante o desenvolvimento deste projeto adotou-se como estratégia o sequenciamento de ESTs, com a finalidade de encontrar mensagens relacionadas com desenvolvimento, metabolismo e principalmente amplificação/politenização em glândulas salivares de Rhynchosciara americana, um díptero (Sciarídeo) que apresenta cromossomos politênicos e amplificação gênica rigidamente regulada ao longo do desenvolvimento larval, tanto neste tecido quanto em outros. Um total de 8193 ESTs foi gerado, estas foram anotadas e categorizadas segundo os termos do Gene Ontology Consortium, proporcionando uma visão geral do status metabólico, como em um Northern eletrônico, de um ponto importante no desenvolvimento desta espécie, quando surgem amplificações gênicas específicas e a glândula salivar necessita secretar as proteínas do casulo. Outros frutos deste seqüenciamento foram a determinação de 91 polimorfismos e a criação de uma tabela de códon usage. Diversos ESTs foram identificados com potencial envolvimento com os endociclos observados neste tecido, destes, RaDup e RaMCM5 foram selecionados para estudo. Suas regiões genômicas foram isoladas e suas localizações cromossômicas foram identificadas, em relação a RaDup, toda a porção codificante de seu mensageiro e 12kb de DNA genômico contendo seu gene foram seqüenciados, revelando sua estrutura gênica. Anticorpos foram produzidos para detectar esta proteína, gerando evidências de sua participação tanto na replicação mitótica como nos endociclos presentes nas glândulas salivares. A localização cromossômica de RaDup é um dado muito interessante, pois pela primeira vez um pufe amplificado é relacionado com um gene regulatório. / In this work EST sequencing was used as strategy to find messages related to development, metabolism and polyteny/amplification in salivary glands of Rhynchosciara americana, a dipteran (Sciaridae) that shows in this tissue giant polytene chromosomes and gene amplification tightly regulated throughout development of the larvae. A total of 8193 EST sequences were generated, annotated and categorized using Gene Ontology Consortium terms, providing a general view of the metabolic status, like an electronic Northern, of an important point in development of the larvae, that shows where specific genes are amplified and the salivary gland needs to secrete the proteins to form the cocoon. Other data include determination of 91 SNPs and a statistic of codon usage. Several ESTs were identified with potential connection to endocicles, from these RaDup and RaMCM5 were selected for further studies. Both chromosomal loci were identified and genomic regions isolated, for RaDup the coding region of its mRNA and 12kb of genomic region were completely sequenced, revealing its gene structure, and antibodies were raised against this protein, making evident data about its involvement in replication in mitotic cells and in endocicles in salivary glands. About the chromosomal locus of RaDup, it becomes very interesting, because for the first time one amplified puff can be related to a regulatory gene.

Au sein de l’unité mixte de recherche 619 de l’Institut National de Recherche Agronomique, le groupe « Organogénèse du Fruit et Endoréduplication » étudie les acteurs moléculaires prenant part au contrôle du cycle cellulaire dans le fruit de tomate. L’objet de la présente thèse est l’étude de l’inhibiteur du cycle cellulaire Kip-Related Protein, et son rôle durant le développement du fruit. Identification de motifs protéiques fonctionnels chez l’Inhibiteur de Kinase Cycline-Dependent SlKRP1 chez Solanum lycopersicum : Leur rôle dans les interactions avec des partenaires du cycle cellulaire Les Kip-related proteins (KRPs) jouent un rôle majeur dans la régulation du cycle cellulaire. Il a été montré qu’ils inhibent les complexes CDK/Cyclin et ainsi bloquent la progression du cycle cellulaire. Malgré leur manque d’homologie avec leurs homologues animaux au delà de leur motif de liaison CDK/Cyclin, localisé à l’extrémité C-terminal de la protéine dans les séquences de plante, des études antérieurs ont montré la présence de motifs conservés spécifiques aux plantes chez certaines KRPs. Nous n’avons cependant que peu d’information concernant leur fonction. Nous montrons ici que les KRPs sont distribués en deux sous groupes phylogénétiques, et que chaque sous-groupe dispose de courts motifs spécifiques conservés. Les KRPs du sous-groupe 1 disposent ainsi de six motifs conservés entre eux. Utilisant SlKRP1, qui appartient au sous-groupe 1, nous avons identifié des motifs responsables de la localisation de la protéine et de ses interactions protéine-protéine. Nous montrons que le motif 2 est responsable de l’interaction avec CSN5, une sous-unité du complexe signalosome, et que le motif 5 a un effet redondant avec le motif 3 pour ce qui est de la localisation sub-cellulaire de la protéine. Nous montrons de plus que SlKRP1 est capable de guider SlCDKA1 et SlCycD3;1 vers le noyau, et ce même en l’absence du motif de liaison CDK/Cycline précédemment référencé. Ce nouveau site d’interaction est probablement localisé dans la partie centrale de la séquence de SlKRP1. Ces résultats apportent de nouveaux indices quant au rôle de la partie encore méconnue de cette protéine. La surexpression de SlKRP1 dans le mésocarpe de tomate détruit la proportionnalité entre endoréduplication et taille cellulaire Le fruit est un organe spécialisé résultant du développement de l’ovaire après pollinisation et fertilisation, et qui offre un environnement adéquat pour la maturation des graines et leur dispersion. De part leur importance en nutrition humaine et leur importance économique, les espèces à fruit charnu ont été le sujet d’étude développementales principalement orientée vers la formation de l’ovaire, la mise à fruit et la maturation du fruit. La phase de croissance du fruit a été beaucoup moins étudiée, bien que la division cellulaire et la croissance cellulaire prenant place durant cette période soient cruciales à la détermination de la taille finale du fruit, ainsi que de sa masse et sa forme. Le développement du mésocarpe du fruit de tomate se déroule par la succession d’une phase de division cellulaire suivie d’une phase d’expansion cellulaire associée à l’endoréduplication, menant à la formation de cellules géantes (jusqu’à 0,5mm) avec des niveaux de ploïdie pouvant atteindre 256C. Bien qu’une relation évidente entre endoréduplication et croissance cellulaire ait été montrée par de nombreux exemples chez les plantes, le rôle exact de l’endoréduplication n’a toujours pas été élucidé, étant donné que la plupart des expériences induisant une modification du niveau d’endoréduplication dans la plante affectaient aussi la division cellulaire. Nous avons étudié la cinétique du dévelopement du mésocarpe de tomate au niveau morphologique et cytologique et avons étudié l’effet de la diminution du niveau d’endoréduplication sur le dévelopement du fruit en sur-exprimant l’inhibiteur du cycle cellulaire Kip-Related Protein 1 (SlKRP1) spécifiquement dans les cellules en croissance du mésocarpe de tomate. Nous montrons une proportionnalité directe entre endoréduplication et taille cellulaire durant le développement normal du fruit, ce qui nous a permis de construire un modèle de développement du mésocarpe définissant l’épaisseur du péricarpe en ne prenant en compte que le nombre de divisions cellulaires et le nombre de tours d’endoréduplication. De façon surprenante, les mésocarpes de tomate affectés dans leur niveau d’endoréduplication par la sur-expression de SlKRP1 ne sont pas affectés au niveau de la taille des cellules ou du fruit, ni dans leur contenu métabolique. Nos résultats démontrent pour la première fois qu’alors que le niveau de ploïdie est étroitement lié avec la taille des cellules et du fruit, l’endoréduplication n’est pas responsable de la croissance cellulaire du mésocarpe de tomate. / Within the Joint Research Unit 619 of the National Institute of Agronomic Research (INRA), the group "Organogenesis of the Fruit and endoreduplication" examines the molecular players involved in cell cycle control in tomato fruit. The purpose of this thesis is the study of the cell cycle inhibitor Kip-Related Protein and its role during fruit development. Identification of protein motifs in the functional inhibitor of Cyclin-Dependent Kinase in Solanum lycopersicum SlKRP1: Their role in interactions with partners in the cell cycle The Kip-related proteins (KRPs) play a major role in the regulation of cell cycle. It has been shown to inhibit the CDK / Cyclin and thus block cell cycle progression. Despite their lack of homology with their counterparts in animals beyond their binding motif CDK / Cyclin, located at the C-terminal protein sequences in the plant, previous studies have shown the presence of conserved motifs plant specific in some KRPs, but there is little information about their function. We show here that the KRPs are distributed into two phylogenetic groups, and that each subgroup has specific short conserved motifs. The KRPs from subgroup 1 have six conserved motifs. Using SlKRP1, which belongs to subgroup 1, we have identified the motifs responsible for the localization of the protein and protein-protein interactions. We demonstrate that the pattern 2 is responsible for the interaction with CSN5, a subunit of the signalosome complex, and that the motif 5 is redundant with motif 3 with respect to the sub-cellular localization of the protein. We also show that SlKRP1 is capable of guiding SlCDKA1 and SlCycD3; 1 to the nucleus, even in the absence of CDK / cyclin binding motif previously referenced. This new site of interaction is probably located in the central part of the sequence of SlKRP1. These results provide new clues about the role of the little-known part of this protein. Overexpression of SlKRP1 in tomato mesocarp disrupts the proportionality between endoreduplication and cell size The fruit is a specialized organ which results from the ovary after pollination and fertilization, and provides a suitable environment for seed maturation and dispersal. Because of their importance in human nutrition and economic importance, fleshy fruit species have been the subject of study mainly focused on the developmental formation of the ovary, fruit set and fruit ripening. The stage of fruit growth has been much less studied, although cell division and cell growth taking place during this period are crucial to determining the final size of the fruit, as well as its mass and shape. The development of tomato fruit mesocarp occurs by the estate of a phase of cell division followed by a phase of cell expansion associated with endoreduplication, leading to the formation of giant cells (up to 0.5 mm) with ploidy levels of up to 256C. Although a clear relationship between endoreduplication and cell growth has been shown by many examples in plants, the exact role of endoreduplication has still not been elucidated, since most of the experiments leading to a change in the level of endoreduplication in plants also affected cell division. We studied the kinetics of the development of tomato mesocarp morphologically and cytologically and studied the effect of the reduced level of endoreduplication in the development of the fruit over-expressing the cell cycle inhibitor Kip-Related Protein 1 (SlKRP1) specifically in the growing cells of the tomato mesocarp. We show a direct proportionality between endoreduplication and cell size during normal development of the fruit, which allowed us to build a model for development of mesocarp defining the thickness of the pericarp by taking into account the number of cell divisions and the number of rounds of endoreduplication. Surprisingly, the tomato mesocarps affected in their level of endoreduplication by over-expression of SlKRP1 are not affected in terms of cell size and fruit, or on their metabolic content. Our results demonstrate for the first time that while the level of ploidy is closely linked with cell size and fruit, endoreduplication is not responsible for the cell growth of tomato mesocarp.