Zooxanthellae are very important for the coral reef ecosystem. The diversity of coral hosts is high in the Indo-Pacific, but the diversity of zooxanthellae has not been broadly investigated. Southern Taiwan and Penghu Islands are coral reef and non-reefal communities, respectively. These localities were chosen as the sampling sites for this study to maximize the opportunity of surveying this region in the Indo-Pacific. Zooxanthellae diversity was investigated in 40 host species including 32 species of Scleractinia, 4 species of Actiniaria, 3 species of Milleporina and 1 species of Helioporacea using polymerase chain reaction (PCR) of the ssrRNA gene and restriction fragment length polymorphism (RFLP) patterns. The phylogenetic relationship of partial and complete sequences of the ssrRNA gene were also analysed. Aiptasia puchella harbors clade B; Oulastrea crispata only harbors clade E; while Acropora palifera and Montipora cactus harbor both clades C and E. Zooxanthellae isolated from all except the above 4 host species are identified as "clade C" sensu Rowan and Powers (1991a). Therefore, the clade C is the dominant type in the Indo-Pacific. Phylogenetic analyses based on partial and complete sequences obtained in this study and also from the GenBank data base demonstrate 4 clades (A, B, C and E) in the genus Symbiodinium. Clade E, classed as D3 RFLP type in previous studies, is a distinct clade differing from A, B and C by RFLP and sequencing data. Clade E has only been found in Scleractinia host species collected in shallow-water habitats in the Pacific. The composition of zooxanthellae clades and ecological pattern of polymorphic symbiosis is not consistent with the irradiance adaptation hypothesis in the Caribbean. A literature survey of zooxanthellae in Scleractinian hosts indicates a significant difference between the Caribbean and the Pacific. The documented biogeography of zooxanthellae clades and the ecological pattern of polymorphic symbiosis are also differ between the Caribbean and the Indo-Pacific.

Ribosomes are universally conserved macromolecular machines that translate cellular genetic information into proteins. All ribosomes are com- posed of two ribonucleoprotein subunits. In eukaryotes these are called 40S (small) and 60S (large) subunits. Biogenesis of both subunits begins from a common precursor ribosomal RNA (rRNA) transcript in the nucleolus. The 18S rRNA of the small subunit is encoded in the 5ʹ end of the precursor transcript. U3 snoRNA and about 70 accessory factors associate with the 50 end of the pre-rRNA, to form the SSU processome or 90S pre-ribosome, which can be observed as terminal knobs in electron micrographs. After the initial processing and folding, the pre-rRNA is cleaved at site A2 to release the pre--40S. This event is dependent on the formation of the central pseudoknot, a structure that maintains the integrity of 40S architecture. Bud23 is the methyltransferase responsible for modification of the base G1575 in the P-site of the small subunit. Work presented here demonstrates that the in vivo stability, and thus function, of Bud23 is dependent on the presence of Trm112, a novel ribosome biogenesis factor identified in this work. Analysis of rRNA processing and strong negative genetic interactions with RNaseMRP mutants, provide strong evidence for that BUD23 is required for A2 cleavage. Extragenic suppressors of bud23 [delta] were identified in UTP14, UTP2, IMP4 and ECM16, coding for SSU processome components. Bud23 and the RNA helicase Ecm16 interact physically as well as genetically. Most fascinatingly, using ecm16 enzymatic mutants, this work provides compelling evidence that Ecm16 facilitates removal of U3 snoRNA from pre-rRNA, a prerequisite for central pseudoknot formation and 90S to pre--40S transition. These findings suggest a model in which binding of Bud23 monitors the status of 40S assembly, triggering Ecm16 activity to promote release of the pre--40S from 90S only after the critical folding of the small subunit rRNA. / text

The ribosome is one of the most universal molecular machinery, synthesizing proteins in all living systems. The small ribosomal subunit plays a crucial role in decoding the messenger RNA during translation. We propose and validate a new architectural model of the ribosomal small subunit, with broad implications for function, biogenesis and evolution. We define an rRNA domain: compact and modular, stabilized by self-consistent molecular interactions, with ability to fold autonomously when it is isolated from surrounding RNA or protein. Each rRNA helix must be allocated uniquely to a single domain. These criteria identify a core domain of small subunit rRNA (domain A), which acts as a hub, linking to all other domains by A-form helical spokes. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2’OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with autonomous folding, and therefore classification as a domain. We show that the domain concept is applicable and useful for understanding the small ribosomal subunit. Our results support the utility of the concept of the domain as applied to at least some RNAs, the interdependence of the elements of domain A, and its ability to fold autonomously. Moreover, domain A, which exhibits elements of tRNA mimicry, is the essential core of the small ribosomal subunit. Understanding the structure and dynamics of domain A will provide valuable insight into the translational machinery.

The molecular characterization of twenty six Theileria spp. isolates and one C. felis isolate were done on the small subunit ribosomal RNA (SSU rRNA) gene, the 5.8S gene, and the two internal transcribed spacer regions using gDNA. The SSU rRNA gene is increasingly accepted as a widely used marker for characterization, taxonomic classification, and phylogenetic analysis and this gene has been sequenced from a variety of different organisms, resulting in a large database for sequence comparisons (Chae et al. 1998; Chae et al., 1999 a,b,c; Stockham et al., 2000; Cossio-Bayugar et al., 2002; Gubbels et al., 2000). The genomic region consists of the internal transcribed spacer 1 (ITS 1), the 5.8S gene, and internal transcribed spacer 2 (ITS 2) (ITS 1-5.8S-ITS 2 gene region) and separates the SSU rRNA gene from the large subunit ribosomal RNA gene. The 5.8S rRNA gene is highly conserved in size and nucleotide sequence, is relatively constant in molecular weight, and has an average chain length of approximately 160 nucleotides and has proven useful in dividing subgenera of Gyrodactylus ((Nazar, 1984; Zietara et al., 2002). Pairwise comparisons were done between the clones of an individual isolate and among the clones of the different isolates. Phylogenetic trees were made from the resulting sequences. This study shows that different SSU rRNA genes may be associated with ITS 1-5.8S-ITS 2 gene regions of distinct sequence in the same isolate. This study also demonstrates that considerable ITS 1-5.8S-ITS 2 gene region sequence variation may exist within a species. This may be useful for subspeciation designation, or may simply reflect considerable variation within the population. This study shows that the ITS 1-5.8S-ITS 2 gene region may be a useful molecular marker for the taxonomy of Theileria spp.

Ribosomes are the cellular structures responsible for the synthesis of protein in all branches of life. All ribosomes are made from a large and small subunit that in turn are composed of protein and RNA. The synthesis of eukaryotic ribosomes is a complex process involving more than 200 factors and spans three cellular compartments: the nucleolus, the nucloplasm, and the cytoplasm. The precise function of most of these ribosome biogenesis factors remains unknown. The RNA component of ribosomes is in part processed from a large RNA transcript that yields most of the RNA present in mature ribosomes. Part of the maturation process involves modification of this ribosomal RNA as processing is carried out. Recent work constructing protein interaction networks in Saccharomyces cerevisiae suggested the methyltransferase Bud23 was involved in ribosome biogenesis (1). This thesis describes my work to characterize Bud23 and place it within the ribosome biogenesis pathway. Bud23 is a SAM methyltransferase important for the proper biogenesis of the small ribosomal subunit. Here I will demonstrate that Bud23 methylates G1575 of the small subunit ribosomal RNA (SSU rRNA), and its absence delays export of the SSU rRNA from the nucleolas, and the nucleus, and results in the delayed maturation of the SSU rRNA. Finally, I will show that Bud23 function is connected to small subunit processome factor Utp14 through identification of a Utp14 mutant that suppresses the bud23[Delta] deletion phenotype. / text

No description available.

No description available.

Les résultats ont été obtenus avec le logiciel "Insight-2" de Accelris (San Diego, CA) / En 2000, les structures à hautes résolutions des deux sous-unités ribosomiques ont finalement été mises à la disposition du public. L'année suivante, la structure aux rayons X de l'ensemble du ribosome bactérien a été publiée. Ces grandes réalisations ont ouvert une nouvelle ère dans l'étude des mécanismes de la synthèse des protéines. Dès lors, il est devenu possible de relier différents aspects de la fonction du ribosome à des éléments particuliers de sa structure tertiaire. L'établissement de la relation structure-fonction peut toutefois être problématique en raison de l'immense complexité de la structure du ribosome. En d'autres termes, pour que les données cristallographiques sur la structure tertiaire du ribosome soient vraiment utiles à la compréhension du fonctionnement du ribosome, ces données devraient elles-mêmes faire l'objet d'une analyse approfondie. Le travail, présenté ici, peut être vu comme une tentative de ce genre. En appliquant l’analyse systématique des structure cristallographiques du ribosome disponibles, nous avons essayé de résoudre deux problèmes fondamentaux de la biologie ribosomale concernant (1) la nature des réarrangements du ribosome qui ont lieu à différentes étapes de son cycle de fonctionnement et (2) la possibilité de reconstitution de l'évolution du ribosome du monde-à-ARN jusqu’à nos jours. Dans le premier projet, nous avons systématiquement comparé les structures du ribosome disponibles et de sa sous-unité afin d'identifier les domaines rigides, qui ont toujours la même conformation, et les régions flexibles dont la conformation peut varier d'une structure de ribosome à une autre. Il y a deux types de réarrangements structuraux connus dont nous voulions comprendre les mécanismes: le « ratchet-like movement » et la «fermeture de domaines ». Le premier a lieu au cours de la translocation du ribosome et est plus ou moins perçu comme une rotation d'une sous-unité par rapport à l'autre. Le deuxième se produit dans la petite sous-unité et est associé à la reconnaissance codon-anticodon au site A. La comparaison des conformations ribosomales disponibles a révélé les mécanismes spécifiques des deux réarrangements. Bien que la sélection de l'aminoacyl-ARNt appropriée au site A et la translocation du ribosome n'ont jamais été considérés comme ayant quelque chose en commun, nous démontrons ici que les réarrangements de la structure des ribosomes associés au premier processus répète les réarrangements associés au deuxième mais dans l’ordre inverse. En d'autres termes, pendant le cycle d'élongation, la fermeture de domaine et le « ratchet » peuvent ii être considérés comme un mouvement de va-et-vient, qui renvoie finalement le ribosome à sa conformation initiale. Dans le second projet, nous avons fait une tentative de reconstitution de l'évolution de l'ARNr 23S, du monde-à-ARN jusqu`à nos jours. Ici nous nous sommes basés sur la supposition que l'évolution de cette molécule a procédé par des insertions aléatoires des régions relativement courtes dans différentes parties de la chaîne poly-nucléotidique. Pour cela, nous avons élaboré des critères de l'intégrité de la structure ribosomale et présumé que lors de l'évolution, la structure du ribosome s’est toujours adaptée à ces standards. Nous avons examiné l'interaction de type A-mineur, un arrangement fréquent dans la structure de l’ARN ribosomique, constitué d'un empilement d’adénosines non-appariées, attachées à une double hélice. Nous avons supposé que dans toutes les interactions A-mineurs existantes dans le ribosome, la double hélice est apparue avant ou au moins simultanément avec la pile d’adénosines correspondantes. L'application systématique de ce principe à la structure tertiaire de l’ARN 23S a permis d'élucider de manière progressive l'ordre dans lequel les parties différentes de l’ARN 23S ont rejoint la structure. Pris ensemble, les deux projets démontrent l'efficacité de l'analyse systématique in-silico de la structure tertiaire du ribosome et ouvrent la voie à de futures découvertes. / In the year 2000, the first high-resolution structures of the individual ribosomal subunits became available to the public. The following year, the X-ray structure of the complete bacterial ribosome was published. These major achievements opened a new era in studying the mechanisms of protein synthesis. From then on, it became possible to attribute different aspects of the ribosome function to particular elements of its tertiary structure. However, establishing the structure-function relationships is problematic due to the immense complexity of the ribosome structure. In other words, in order to make the crystallographic data on the ribosome tertiary structure really useful for understanding of how the ribosome functions, it must be thoroughly analyzed. Here, based on systematic analysis of the available X-ray conformations of the ribosome we have tried to resolve two fundamental problems of the ribosome biology: concerning (1) the nature of rearrangements in the ribosome that take place at different steps of its functional cycle, and (2) the reconstruction of the ribosome evolution from the RNA world to present time. In the first project, we systematically compared the available structures of the ribosome and its subunits to identify rigid domains, which always have the same conformation, and flexible regions, where the conformation can vary from one ribosome structure to another. There were two known types of structural rearrangements whose mechanisms we wanted to understand: the ratchet-like motion and the so-called domain closure. The ratchet-like motion takes place during the ribosomal translocation and is roughly seen as a rotation of one subunit with respect to the other. The domain closure occurs in the small subunit and is associated with the cognate codon-anticodon recognition in the A-site. Comparison of the available ribosome conformations revealed the detailed mechanisms of both rearrangements. Although the selection of the cognate amino-acyl-tRNA in the A-site and of the ribosomal translocation have never been thought to have anything in common, we demonstrate that the rearrangements in the ribosome structure associated with the first process repeat in reverse order the rearrangements associated with the second process. In other words, during the ribosome elongation cycle, the domain closure and the ratchet-like motion can be seen as a back-and-forth movement, which eventually returns the ribosome to the initial conformation. iv In the second project, we attempted to reconstruct the evolution of the 23S rRNA from the RNA world to present time based on the presumption that the evolutionary expansion of this molecule proceeded though random insertions of relatively short regions into different regions of the polynucleotide chain. We developed criteria for integrity of the ribosome structure and presumed that during the evolutionary expansion, the ribosome structure always matched to these standards. For this, we specifically considered the A-minor interaction, a frequent arrangement in the rRNA structure consisting of a stack of unpaired adenosines tightly attached to a double helix. We presumed that in all A-minor interactions present in the ribosome, the double helix emerged before or at least simultaneously with the corresponding adenosine stack. The systematic application of this principle to the known tertiary structure of the 23S rRNA allowed us to elucidate in a step-vise manner the order in which different part of the modern 23S rRNA joined the structure. Taken together, the two projects demonstrate the effectiveness of the systematic in-silico analysis of the ribosome tertiary structure and pave the way for future discoveries.

En 2000, les structures à hautes résolutions des deux sous-unités ribosomiques ont finalement été mises à la disposition du public. L'année suivante, la structure aux rayons X de l'ensemble du ribosome bactérien a été publiée. Ces grandes réalisations ont ouvert une nouvelle ère dans l'étude des mécanismes de la synthèse des protéines. Dès lors, il est devenu possible de relier différents aspects de la fonction du ribosome à des éléments particuliers de sa structure tertiaire. L'établissement de la relation structure-fonction peut toutefois être problématique en raison de l'immense complexité de la structure du ribosome. En d'autres termes, pour que les données cristallographiques sur la structure tertiaire du ribosome soient vraiment utiles à la compréhension du fonctionnement du ribosome, ces données devraient elles-mêmes faire l'objet d'une analyse approfondie. Le travail, présenté ici, peut être vu comme une tentative de ce genre. En appliquant l’analyse systématique des structure cristallographiques du ribosome disponibles, nous avons essayé de résoudre deux problèmes fondamentaux de la biologie ribosomale concernant (1) la nature des réarrangements du ribosome qui ont lieu à différentes étapes de son cycle de fonctionnement et (2) la possibilité de reconstitution de l'évolution du ribosome du monde-à-ARN jusqu’à nos jours. Dans le premier projet, nous avons systématiquement comparé les structures du ribosome disponibles et de sa sous-unité afin d'identifier les domaines rigides, qui ont toujours la même conformation, et les régions flexibles dont la conformation peut varier d'une structure de ribosome à une autre. Il y a deux types de réarrangements structuraux connus dont nous voulions comprendre les mécanismes: le « ratchet-like movement » et la «fermeture de domaines ». Le premier a lieu au cours de la translocation du ribosome et est plus ou moins perçu comme une rotation d'une sous-unité par rapport à l'autre. Le deuxième se produit dans la petite sous-unité et est associé à la reconnaissance codon-anticodon au site A. La comparaison des conformations ribosomales disponibles a révélé les mécanismes spécifiques des deux réarrangements. Bien que la sélection de l'aminoacyl-ARNt appropriée au site A et la translocation du ribosome n'ont jamais été considérés comme ayant quelque chose en commun, nous démontrons ici que les réarrangements de la structure des ribosomes associés au premier processus répète les réarrangements associés au deuxième mais dans l’ordre inverse. En d'autres termes, pendant le cycle d'élongation, la fermeture de domaine et le « ratchet » peuvent ii être considérés comme un mouvement de va-et-vient, qui renvoie finalement le ribosome à sa conformation initiale. Dans le second projet, nous avons fait une tentative de reconstitution de l'évolution de l'ARNr 23S, du monde-à-ARN jusqu`à nos jours. Ici nous nous sommes basés sur la supposition que l'évolution de cette molécule a procédé par des insertions aléatoires des régions relativement courtes dans différentes parties de la chaîne poly-nucléotidique. Pour cela, nous avons élaboré des critères de l'intégrité de la structure ribosomale et présumé que lors de l'évolution, la structure du ribosome s’est toujours adaptée à ces standards. Nous avons examiné l'interaction de type A-mineur, un arrangement fréquent dans la structure de l’ARN ribosomique, constitué d'un empilement d’adénosines non-appariées, attachées à une double hélice. Nous avons supposé que dans toutes les interactions A-mineurs existantes dans le ribosome, la double hélice est apparue avant ou au moins simultanément avec la pile d’adénosines correspondantes. L'application systématique de ce principe à la structure tertiaire de l’ARN 23S a permis d'élucider de manière progressive l'ordre dans lequel les parties différentes de l’ARN 23S ont rejoint la structure. Pris ensemble, les deux projets démontrent l'efficacité de l'analyse systématique in-silico de la structure tertiaire du ribosome et ouvrent la voie à de futures découvertes. / In the year 2000, the first high-resolution structures of the individual ribosomal subunits became available to the public. The following year, the X-ray structure of the complete bacterial ribosome was published. These major achievements opened a new era in studying the mechanisms of protein synthesis. From then on, it became possible to attribute different aspects of the ribosome function to particular elements of its tertiary structure. However, establishing the structure-function relationships is problematic due to the immense complexity of the ribosome structure. In other words, in order to make the crystallographic data on the ribosome tertiary structure really useful for understanding of how the ribosome functions, it must be thoroughly analyzed. Here, based on systematic analysis of the available X-ray conformations of the ribosome we have tried to resolve two fundamental problems of the ribosome biology: concerning (1) the nature of rearrangements in the ribosome that take place at different steps of its functional cycle, and (2) the reconstruction of the ribosome evolution from the RNA world to present time. In the first project, we systematically compared the available structures of the ribosome and its subunits to identify rigid domains, which always have the same conformation, and flexible regions, where the conformation can vary from one ribosome structure to another. There were two known types of structural rearrangements whose mechanisms we wanted to understand: the ratchet-like motion and the so-called domain closure. The ratchet-like motion takes place during the ribosomal translocation and is roughly seen as a rotation of one subunit with respect to the other. The domain closure occurs in the small subunit and is associated with the cognate codon-anticodon recognition in the A-site. Comparison of the available ribosome conformations revealed the detailed mechanisms of both rearrangements. Although the selection of the cognate amino-acyl-tRNA in the A-site and of the ribosomal translocation have never been thought to have anything in common, we demonstrate that the rearrangements in the ribosome structure associated with the first process repeat in reverse order the rearrangements associated with the second process. In other words, during the ribosome elongation cycle, the domain closure and the ratchet-like motion can be seen as a back-and-forth movement, which eventually returns the ribosome to the initial conformation. iv In the second project, we attempted to reconstruct the evolution of the 23S rRNA from the RNA world to present time based on the presumption that the evolutionary expansion of this molecule proceeded though random insertions of relatively short regions into different regions of the polynucleotide chain. We developed criteria for integrity of the ribosome structure and presumed that during the evolutionary expansion, the ribosome structure always matched to these standards. For this, we specifically considered the A-minor interaction, a frequent arrangement in the rRNA structure consisting of a stack of unpaired adenosines tightly attached to a double helix. We presumed that in all A-minor interactions present in the ribosome, the double helix emerged before or at least simultaneously with the corresponding adenosine stack. The systematic application of this principle to the known tertiary structure of the 23S rRNA allowed us to elucidate in a step-vise manner the order in which different part of the modern 23S rRNA joined the structure. Taken together, the two projects demonstrate the effectiveness of the systematic in-silico analysis of the ribosome tertiary structure and pave the way for future discoveries. / Les résultats ont été obtenus avec le logiciel "Insight-2" de Accelris (San Diego, CA)