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The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.

Dans la levure S. cerevisiae, la mitose nécessite le positionnement du fuseau mitotique le long de l’axe cellule mère-bourgeon (future cellule fille) afin d‘assurer une bonne ségrégation des chromosomes. Ce phénomène requiert le fonctionnement de deux mécanismes impliquant les protéines Kar9 et Dyn1. Durant la métaphase, Kar9 se positionne de manière asymétrique le long du fuseau mitotique, avec une accumulation notable sur les microtubules qui émanent de l’ancien « spindle pole body » (SPB; l’équivalent du centrosome dans les vertébrés), qui est normalement dirigé vers le bourgeon. Dans le cas d’un défaut d’alignement du fuseau mitotique, un mécanisme appelé « Spindle Position Checkpoint » (SPOC) inhibe la sortie de mitose et la cytokinèse, afin de permettre un réalignement correct du fuseau mitotique. La principale cible de ce checkpoint est une GTPase Tem1. Dans le cas d’alignement correct du fuseau mitotique, Tem1 active une voie de signalisation appelée le « Mitotic Exit Network » (MEN) qui permet de mener à la sortie de mitose et à la cytokinèse. Lors de la transition métaphase/anaphase Tem1 se positionne asymétriquement sur les SPBs jusqu’à se concentrer majoritairement sur l’ancien SPB. Des données récentes ont montré que des composants du MEN, Tem1 inclus, sont également impliqués dans la régulation de la localisation de la protéine Kar9 à l’SPB, et dans l’établissement d’une polarité correcte des SPBs durant la métaphase. En effet, Kar9 se positionne plus symétriquement dans le cas des mutants du MEN que dans le type sauvage, ce qui engendre des problèmes d’orientation du fuseau et de ségrégation des SPBs. Nous cherchons à élucider comment l’activité du MEN régule la localisation de Kar9 et l’orientation du fuseau mitotique en métaphase alors que les fonctions du MEN liées à la sortie de mitose restent bloquées jusqu’à la télophase. Nous avons émis l’hypothèse que les modifications post-traductionnelles de Tem1 pourraient jouer un rôle dans la régulation du MEN. Il a été montré que les résidus Y40 et Y45 sont phosphoryles in vivo. Afin de disséquer le rôle de ces résidus nous les avons mutés en phénylalanines. Ces mutations peuvent complémenter la létalité induite par la délétion de TEM1, suggérant que ce mutant conserve les fonctions essentielles de Tem1. Par ailleurs, la cinétique de progression du cycle cellulaire du mutant est la même que celle du type sauvage, signifiant que la perte de phosphorylation sur Tem1 ne semble pas agir sur la sortie de mitose. De plus, l’allèle mutant n’affecte pas la localisation aux SPBs de Tem1 ni celle de sa « GTPase-activating protein » Bub2/Bfa1 durant le cycle cellulaire. Bien que l’activité GTPasique de la protéine Tem1-Y40F,Y45F soit réduite in vitro, les mutations ne causent pas des défauts de SPOC in vivo et le mutant répond efficacement au mauvais alignement de fuseau mitotique en s’arrêtant en anaphase. Tous ces résultats nous suggèrent que la perte de phosphorylation de Tem1 n’affecte pas les fonctions de fin de mitose de cette GTPase. Par contre, nous avons découvert que la phosphorylation de Tem1 est requise pour la localisation asymétrique de Kar9 sur les SPBs, ainsi que pour l’alignement correct du fuseau mitotique durant la métaphase (la distribution de Kar9 est plus symétrique dans les cellules TEM1-Y40F,Y45F et que le fuseau mitotique n’est pas aligné correctement). Nous cherchons alors à trouver quelle kinase phosphoryle Tem1 et régule son activité. Les kinases potentielles sont la protéine Swe1 (la seule vraie kinase phosphorylant les tyrosines dans la levure) ainsi que la kinase Mps1 (kinase qui contrôle la duplication des SPBs). Nous développons actuellement des outils nous permettant de vérifier l’implication de ces deux candidats. Mots clés : Tem1, Kar9, cycle cellulaire, Mitotic Exit Network (MEN), Spindle Position Checkpoint (SPOC), phosphorylation on tyrosines. / In the budding yeast Saccharomyces cerevisiae a faithful mitosis requires positioning of the mitotic spindle along the mother-bud axis to ensure proper chromosome segregation. This is achieved by two distinct but functionally redundant mechanisms that require the APC (adenomatous polyposis coli)-like protein Kar9 and dynein (Dyn1), respectively. During metaphase, Kar9 localizes asymmetrically on the mitotic spindle, with a prominent accumulation on astral microtubules emanating from the old spindle pole body (SPB – i.e. the yeast equivalent of the centrosome) that is normally directed towards the bud. In case of spindle misalignment, a surveillance mechanism called Spindle Position Checkpoint (SPOC) inhibits mitotic exit and cytokinesis, thereby providing the time necessary to correct spindle alignment. The main target of the SPOC is the small GTPase Tem1, which activates a signal transduction cascade called Mitotic Exit Network (MEN) that drives cells out of mitosis and triggers cytokinesis. Tem1 is localized at SPBs, with an increasingly asymmetric pattern during the progression from metaphase to anaphase, when Tem1 is concentrated on bud-directed old SPB. Recent data have implicated MEN components also in the regulation of Kar9 localization at SPBs and in setting the right polarity of SPBs inheritance during metaphase. In particular, Kar9 localizes more symmetrically in MEN mutants than in wild type cells and this leads to spindle orientation and SPB inheritance defects (i.e. with the new SPB being oriented towards the bud). A key question emerging from these data is how MEN activity is regulated to promote proper Kar9 localization and spindle positioning in metaphase, while being restrained until telophase for what concerns its mitotic exit and cytokinetic functions. We hypothesised that Tem1 post-translational modifications might be relevant for this control and for this reason we have been focusing on the role of Tem1 phosphorylation. Tem1 was found in a wide phosphoproteomic study to be phosphorylated on two tyrosines (Y40 and Y45) located at its N-terminus. We constructed a non-phosphorylatable mutant, TEM1-Y40F,Y45F, where the two phosphorylated tyrosines were mutated to phenylalanine. This mutant allele was able to rescue the lethality caused by TEM1 deletion, suggesting that it retains all its the essential functions. The kinetics of cell cycle progression of TEM1-Y40F,Y45F cells was similar to that of wild type cells, suggesting that lack of Tem1 phosphorylation is unlikely to affect mitotic exit. In addition, the TEM1-Y40F,Y45F allele did not affect the SPB localization of Tem1 and its regulatory GTPase-activating protein Bub2/Bfa1 during the cell cycle. Moreover, although the Tem1-Y40F,Y45F mutant protein showed reduced GTPase activity in vitro, it did not cause SPOC defects in vivo and could efficiently respond to spindle mispositioning. Altogether, these results suggest that lack of Tem1 phosphorylation does not affect the late mitotic functions of the GTPase. In contrast, we found that Tem1 phosphorylation is required for Kar9 asymmetry at SPBs and proper spindle positioning during metaphase. Indeed, TEM1-Y40F,Y45F cells display a more symmetric pattern of Kar9 distribution at SPBs in this cell cycle stage, as well as spindle position and orientation defects. We are currently investigating if Tem1 phosphorylation also regulates the pattern of SPB inheritance. Finally, an important question that we are trying to answer is “what is the kinase that phosphorylates Tem1?” The best candidates are the wee1-like kinase Swe1, which is the only true tyrosine kinase of budding yeast, and Mps1, a dual-specificity protein kinase controlling SPB duplication. While we are developing specific tools to study Tem1 phosphorylation and ultimately identify its promoting kinase, we gained preliminary data suggesting that both kinases might be involved in spindle positioning.

All cellular RNAs contain numerous chemically modified nucleosides, but the largest number and the greatest variety are found in transfer RNA (tRNA). These modifications are posttranscriptionally introduced by modification enzymes during the complex process of tRNA maturation. The function of these modified nucleosides is not well known, but it seems that when present in the anticodon region, they play a direct role in increasing translational efficiency and fidelity, while modifications outside the anticodon region would be involved in the maintenance of the structural integrity of tRNA. Among the naturally occurring nucleoside modifications, base and ribose methylations are by far the most frequently encountered. They are catalyzed by tRNA methyltransferases (MTases), using generally the S-adenosyl-L-methionine (AdoMet) as methyl donor. Most of the knowledge about tRNA MTases comes from studies on bacterial and eukaryal model organisms, and very few informations are available about tRNA methylation in Archaea, particularly for thermophilic and hyperthermophilic Archaea whose GC-rich tRNAs are difficult to sequence. Nevertheless, some works on tRNA hydrolyzates from thermophiles or hyperthermophiles highlighted the presence of numerous methylated nucleosides. Furthermore, it has been shown that the only sequenced tRNA from an hyperthermophilic Archaea, the initiator methionine tRNA (tRNAiMet) from the Sulfolobus acidocaldarius, contains ten modified nucleosides, nine of them bearing a methylation on the base, on the ribose or on both base and ribose.<p>Of special interest is the modified nucleoside found at position 9 of this tRNA. It is an adenosine derivative, but the exact nature of the modification is unknown. In the yeast S. cerevisiae, some tRNAs with a guanosine at this position are methylated by the MTase Trm10p to form m1G9 (126). Since Trm10p-related proteins are found in hyperthermophilic archaea, such a homolog could be responsible for modification at position 9 of S. acidocaldarius tRNAiMet. In this work, we showed indeed that the Trm10p-related protein Saci_1677p from S. acidocaldarius methylates position 9 of tRNAs, but is specific for position N1 of adenosine, forming m1A rather than m1G. Interestingly, we demonstrated that Tk0422p from T. kodakaraensis, the euryarchaeal homolog to Saci_1677p, is the first tRNA MTase presenting a broadened nucleoside recognition capability, methylating both position N1 of A and of G to form m1A and m1G at position 9 of tRNAs. <p>This unique tRNA (m1A-m1G) MTase activity was further studied on one hand by site-directed mutagenesis of residues potentially important for the catalytic activity of Tk0422p enzyme, and on the other hand by determining the importance of the pH on the efficacy of the methylation reaction. Indeed, protonation state of atom N1 of A and G differs at physiological pH (N1 of G being protonated contrary to N1 of A), and we showed that m1G formation was increased with increasing pH. This could reflect the need of the enzyme to deprotonate G to be able to catalyze de methyltransfer. We showed also that the activity of the two archaeal enzymes (Saci_1677p and Tk0422p) present different dependence toward the structure of tRNA, the euryarchaeal Tk0422p requiring the intact tRNA structure while its crenarchaeal counterpart Saci_1677p being able to modify some truncated tRNAs.<p>Finally, some attempts to unveil the in vivo function of these enzymes, as well as their enzymatic mechanisms were undertaken, but these experiments are very preliminary and underline the needs for the development of genetic tools applicable to Archaea./ Tous les ARN cellulaires contiennent des nucléosides modifiés chimiquement, mais ce sont les ARNt qui en contiennent la plus grande variété et la plus grande proportion. Ces modifications sont introduites post-transcriptionnellement par des enzymes de modification durant le processus complexe de maturation des ARNt. Parmi les nucléosides modifiés, les méthylations de bases ou de riboses sont les plus fréquemment rencontrées. Elles sont catalysées par des ARNt méthyltransférases (MTases) utilisant pour la plupart de la S-adenosyl-L-methionine (AdoMet) comme donneur de méthyle. <p>La plupart des connaissances relatives aux ARNt MTases provient d’études sur des organismes modèles eucaryotes et bactériens, et peu de choses sont connues en ce qui concerne les archées, plus particulièrement les archées thermophiles et hyperthermophiles dont les ARNt GC riches sont difficiles à séquencer. Néanmoins, des travaux sur des hydrolysats d’ARNt de thermophiles et hyperthermophiles ont mis en évidence la présence d’un grand nombre de nucléosides modifiés. De plus, le seul ARNt d’archée hyperthermophile séquencé à ce jour, l’ARNtiMet de S. acidocaldarius contient 10 nucléosides modifiés, essentiellement par méthylation de la base, du ribose, ou des deux à la fois. Le nucléoside présent en position 9 de cet ARNt porte une modification chimique de nature encore inconnue. Or, chez la levure S. cerevisiae, certains ARNt possédant une guanosine à cette position sont méthylés par la MTase Trm10p pour former la 1-méthylguanosine. Etant donné qu’il existe une protéine apparentée à Trm10p chez les archées hyperthermophiles, celle-ci pourrait être responsable de la modification trouvée en position 9 de l’ARNtiMet de S. acidocaldarius. Dans ce travail, nous avons montré qu’effectivement la protéine Saci_1677p de la crénarchée S. acidocaldarius, orthologue à Trm10p, modifie la position 9 des ARNt, mais catalyse la formation de 1-methyladénosine (m1A) plutôt que de m1G dans les ARNt. De façon intéressante, nous avons montré que chez l’euryarchée T. kodakaraensis, l’enzyme Tk0422p homologue à Saci_1677p est capable de méthyler à la fois une adénosine et une guanosine en position 9 des ARNt. A notre connaissance, cette enzyme est la première ARNt MTase présentant une capacité élargie de reconnaissance de substrat.<p>Le présent travail a contribué à la caractérisation fonctionnelle et structurale de ces deux enzymes archéennes, et a permis d’améliorer la connaissance générale de la machinerie de modification des ARNt d’archées.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished