Dans un premier temps, je me suis intéressé à la sous-unité ε de l’ATP synthase mitochondriale chez la levure, un organisme qui se prête bien à l’étude des fonctions mitochondriales. Cette protéine fait partie d’un élément de l’ATP synthase appelé la tige centrale. Celui-ci permet de coupler le domaine translocateur de protons de cette enzyme (FO) à son secteur catalytique (F1) où l’ATP est synthétisé. En utilisant un système d’expression régulable (répressible par la doxycycline), j’ai montré qu’en l’absence de la sous-unité ε les secteurs F1 et FO ne sont plus couplés, avec pour résultat des fuites massives de protons à travers la membrane interne des mitochondries. J’ai ensuite montré que l’absence de la sous-unité ε peut être compensée par des mutations ralentissant l’activité du FO. Ces données permettent de conclure que la sous-unité ε est nécessaire au maintien de l’intégrité physique de l’ATP synthase lors de son fonctionnement. Dans un second temps, j’ai cherché à identifier de nouveaux facteurs intervenant dans la biogenèse du FO. Pour cela, j’ai utilisé un crible génétique où la survie des cellules de levure est conditionnée à des mutations inactivation le FO. Un millier d’isolats a été analysé. Les mutations ont été localisées dans les génomes mitochondrial et nucléaire. Dix-huit clones, issus de mutations n’affectant pas des facteurs connus pour être nécessaires à l’expression de l’ATP synthase, ont été entièrement séquencés. Plusieurs nouveaux systèmes cellulaires potentiellement impliqués dans la biogenèse du FO ont été identifiés. / At first, I am interested in the ε subunit of mitochondrial ATP synthase in yeast, an organism that is well suited for the study of mitochondrial functions. This protein is a part of the ATP synthase called central stalk. This allows the coupling of proton translocator domain of this enzyme (FO) to its catalytic domain (F1) where ATP is synthesized. Using a tetO expression system, I showed that in the absence of the ε subunit, F1 and FO domains are no longer coupled. It results in a massive proton leakage across the inner membrane of mitochondria. I then showed that the absence of the ε subunit can be compensated by mutations slowing the activity of FO. These data allow to conclude that the ε subunit is necessary to maintain the physical integrity of the ATP synthase for oxydative phosphorylation. Later, I tried to identify new factors involved in the biogenesis of the FO. For this, I used a genetic screen where the survival of yeast cells is conditioned by mutations inactivating the FO. About a thousand clones were analyzed. The mutations were localized in mitochondrial and nuclear genomes. Eighteen clones with mutations in genes encoding not yet known ATP synthase expression factors were completely sequenced. Several new cellular systems that are potentially involved in the biogenesis of FO were identified.

Abstract Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels. By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases. At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors. To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15675号 / 工博第3333号 / 新制||工||1503(附属図書館) / 28212 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 跡見 晴幸, 教授 濵地 格 / 学位規則第4条第1項該当

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The growth of bacteria by binary division is a key characteristic of these organisms. This growth depends on two types of factors: endogenous and exogenous. Endogenous factors make up the molecular apparatus of cells. Among important endogenous factors belong also those involved in gene expression and its regulation. Exogenous factors are external conditions such as nutrient availability, temperature, pH, various stresses or the presence of antibacterial agents. The main aim of my Thesis was to study the effects of selected endogenous and exogenous factors on bacterial growth. As endogenous factors I studied RNase J1 in Bacillus subtilis and a small RNA called Ms1 in Mycobacterium smegmatis, which are involved in regulation of gene expression at the transcriptional level. I showed that RNase J1 can, besides its role in RNA degradation, play a role in genome integrity by removing stalled RNA polymerase (RNAP) complexes from DNA. I further showed that Ms1 binds to the RNAP core and affects the level of RNAP in the cell. The results revealed new mechanistic aspects of the transcription apparatus and show how individual components or their combinations affect bacterial growth. As exogenous factors I studied the recently discovered antibacterial compounds, called lipophosphonoxins, their interaction...

One of the etiological theories of schizophrenia is dysregulation of the immune system. Autoantibodies specific for the α7 subunit of the nicotinic receptor could potentially contribute to the pathophysiology of the disease. In this study, positive antibodies specific for the receptor were found to exist in 23% of the patients diagnosed with schizophrenia (n = 21). On the average, levels for the antibody were elevated in the schizophrenia patient population than in controls. The data also suggests that there is a significant correlation between antibody titer and age, lending support to the neurodegenerative nature of the disease.

Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by 3H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.

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Soybeans with large seed size, uniformity, clear hilum, and high 11S/7S ratio are favored for soymilk and tofu making. In order to find ideal soybean lines for food making, sixty-eight soybean lines, which were selected from thousands of accessions in the USDA-Soybean Germplasm Collection, were planted in three successive seasons. Eight lines were identified from twenty-two lines harvested in 2014 (Stoneville, MS) to be suitable for tofu making as judged by chemical composition and sensory quality of tofu. The results provided important food quality information to the growers, breeders and tofu industries for their selection of soybean to improve food quality. In the filled tofu making and texture analysis study, the correlation between A3 subunit percentage and tofu firmness was significant (N=22. r = 0.77, P < 0.001). The result proved that the percentage of A3 subunits could be an indicator for predicting the firmness of tofu.

Several antibiotics that prevent translation by binding to ribosomal subunits have been shown to also inhibit ribosomal subunit assembly (Champney and Tober 2003). The aminoglycoside hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal subunit assembly. The streptogramin antibiotics quinupristin-dalfopristin and XRP 2868 (NXL 103) were examined for similar effects on these 2 cellular functions in antibiotic-resistant strains of Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. Pulse chase experiments were performed which verified slower rates of ribosomal subunit formation in drug treated cells. Hygromycin B exhibited a concentration dependent inhibitory effect on viable cell number, growth rate, protein synthesis and 30S and 50S subunit formation. 16S rRNA specific probes hybridized to rRNA fragments in cells treated with hygromycin B. RNase II and RNase III deficient strains of E. coli exhibited the most accumulation of 16S rRNA fragments upon treatment with hygromycin B. Examination of total RNA from treated cells showed an increase in RNA corresponding to precursor to the 16S rRNA while 16S rRNA decreased. There was also an increase in small fragment RNA. Hygromycin B was a more effective inhibitor of translation than ribosomal subunit formation in E. coli. Two streptogramin antibiotics were compared for inhibitory effects in antibiotic-resistant Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. IC50 values for XRP 2868 were several fold lower than those of quinupristin-dalfopristin for inhibition of cell viability, protein synthesis, and ribosomal subunit formation. Both antibiotics revealed a concentration dependent inhibitory effect on cellular functions including 50S ribosomal subunit formation in the three organisms examined. XRP 2868 inhibited both 50S ribosomal subunit assembly and translation. XRP 2868 was effective against MRSA and was a better inhibitor in each of the antibiotic resistant strains examined compared with quinupristin-dalfopristin.