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.

Azithromycin, a macrolide antibiotic, specifically binds to the 50S ribosomal subunit of bacterial ribosomes and inhibits translation. Azithromycin also prevents 50S ribosomal subunit assembly by binding to a 50S ribosomal subunit precursor particle. When exposed to azithromycin, several ribonucleases in wild-type Escherichia coli cells degrade antibiotic-bound 50S precursor particles. Presumably, cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound precursor less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonucleaseûdeficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability, growth rates, and protein synthesis rates were measured. Degradation of 23S rRNA was examined by hybridization with a 23S specific probe. Ribonuclease II and polynucleotide phosphorylase mutants demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation, suggesting that these two ribonucleases are important for 23S rRNA turnover in azithromycin-treated Escherichia coli.

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. We are interested in investigating the true substrate for methylation because it is known from our work and the work of others that fully assembled 50S subunits are not substrates for methylation. We have published a model for 50S ribosomal subunit formation where, the precursor particle that accumulates in erythromycin treated cells is a target for methyl transferase activity. Current studies are aimed at investigating the role of the precursor particle as substrate for ermE methyltransferase activity and the competition between this enzyme and erythromycin for the 50S precursor particle. Slot-blot hybridization experiments have identified the presence of 23S rRNA in the 50S precursor region. Quantitation of the 23S rRNA in these blots also revealed that the percentage of the precursor increased as the concentration of erythromycin was increased in the growth media. Ribosomal proteins of S. aureus were studied by two-dimensional gel electrophoresis. Protein content of the 50S precursor particle was analyzed by MALDI-TOF. These studies have identified 16 50S ribosomal proteins in the precursor region. Methyltransferase assays showed that 50S precursor particle was a substrate for ermE methyltransferase. Importantly, RNA that is already assembled into 50S subunits was not a substrate for the enzyme. Inhibition curves showed that macrolide, lincosamide, and streptogramin B (MLSB) drugs bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. Competition experiments suggested that the enzyme can displace erythromycin from the 50S precursor particle and that erm methyltransferase has a lower association constant for the precursor particle compared to that of the erythromycin. This suggests that higher concentrations of erythromycin are needed to combat erm induced resistance. These studies shed light on the interaction of ermE methyltransferase and erythromycin in the clinically important pathogen S. aureus.

L’objectif de cette recherche est d’étudier comment le champ littéraire latino-américain des années 50 est devenu un lieu de création symbolique alternatif. Le projet de modernisation en Amérique latine a abouti au questionnement de la représentation des sujets. Certains discours ont ainsi essayé d’intégrer des minoritaires en leur donnant la parole, comme par exemple les femmes et les paysans. Le champ littéraire a donc ouvert des espaces pour des individualités désireuses de participer à la construction d’un nouveau projet de nation, où des identités hétérogènes seraient acceptées. Dans cette perspective, nous nous intéresserons à l’œuvre de deux écrivains, Gloria Stolk et Juan Rulfo, qui dans les années 50 au Venezuela et au Mexique, ont réussi à construire un nouvel espace de création plurielle et à transformer la figure d’auteur. Notre analyse porte sur deux niveaux : l’un sur le discours de chacun et l’autre sur leur profil. D’une part, ils élaborent leur fiction sur la base de sujets qui les concernent. D’autre part, ils émergent en tant qu’auteurs avec de multiples facettes. / This research centers its study in how the Latin-American literature in the 50’s became a place of alternative symbolic creation. The modernization project in Latin-America resulted in the questioning of the subject representation. Some speeches intended to combine minorities, like women and peasants. The literary field has thus opened space for individuals willing to participate in the development of new project of nation, where diversity would be accepted. From this perspective, we are interested in the work of the Venezuelan writer Gloria Stolk, and Mexican Juan Rulfo. During the 50’s, they were able to establish a new space that diversify and transform the traditional role of the author. The analysis in this study considers two dimensions: the speeches and the profiles of these two authors. On one side they developed fiction around characters of their interest, on the other side they emerged as authors with multiple roles

<p>Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein.</p><p>Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated <i>Escherichia coli</i> individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ.</p><p>Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA.</p><p>We have here compared how an SD⁺ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD⁺ is confirmed. A downstream SD⁺ gives decreased gene expression. If an SD⁺ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD⁺ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD⁺ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD⁺ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD⁺or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.</p>

Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein. Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated Escherichia coli individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ. Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD+ or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.

The broad subject of this diploma thesis would be the Czechoslovak Secret Police (StB) during the years 1945-1969; and, more narrowly, certain aspects of its organization and activities in relation to the then applicable legislation. The overriding objective, which was reflected in this legislation, was the protection of the Communist political system, the regime. This regime protection goal clearly influenced the evolution and organization of the StB and the related policies on the imprisonment of those individuals that represented a threat to the State and the State System. Similar general principles and policies have basically remained in effect into the current period. The paper begins with an Introduction and Preface, which take a look at the events and nature of the society in this post-World War II period. This material is derived from an examination of historical source materials. The remainder of the paper is divided into four additional Chapters with related subsections. The Introduction and Preface focus on the nature and state of the society in the immediate post-war period, which provided the context and background used for the development of the new legislation and the newly created institutions developed for the protection of this new social order and the punishment and incarceration...