by Yiu-kuen Kam. / Bibliography: leaves 108-118 / Thesis (M.Phil.) -- Chinese University of Hong Kong, 1983

Sui-lam Wong. / Thesis (M.Phil.) -- Chinese University of Hong Kong. / Includes bibliographies.

Leung Kwan-chi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 215-223). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.iii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Brief description of Momordica charantia --- p.2 / Chapter 1.2 --- Toxicity of RIPs and their potential uses in the treatment of AIDS --- p.3 / Chapter 1.3 --- General mechanism of action of RIPs --- p.6 / Chapter 1.4 --- Structure of αMMC --- p.7 / Chapter 1.5 --- "Antigenicities of αMMC, BMMC and TCS" --- p.13 / Chapter 1.6 --- "Immunosuppressive properties of the abortifacient proteins αMMC, BMMC and TCS" --- p.14 / Chapter 1.7 --- Objectives of our study --- p.15 / Chapter CHAPTER 2 --- EXPRESSION OF FULL-LENGTH αMMC cDNA --- p.20 / Chapter 2.1 --- Expression of αMMC cDNA as a fusion protein --- p.22 / Chapter 2.1.1 --- Materials and methods --- p.22 / Chapter 2.1.1.1 --- Construction of fusion vector pRIT2T/MMC --- p.22 / Chapter 2.1.1.2 --- Preparation of αMMC insert by PCR --- p.26 / Chapter 2.1.1.3 --- Cloning of αMMC cDNA into fusion vector pRIT2T --- p.27 / Chapter 2.1.1.4 --- Transformation --- p.28 / Chapter 2.1.1.5 --- DNA sequencing --- p.29 / Chapter 2.1.1.6 --- Expression of protein A-αMMC fusion cDNA --- p.30 / Chapter 2.1.1.7 --- Preparation of fusion αMMC for affinity chromatography --- p.31 / Chapter 2.1.1.8 --- Affinity chromatography of Protein A-αMMC fusion protein --- p.31 / Chapter 2.1.1.9 --- Cleavage of protein A-αMMC fusion protein by factor Xa --- p.32 / Chapter 2.1.1.10 --- SDS-PAGE analysis --- p.33 / Chapter 2.1.1.11 --- Western blot analysis --- p.33 / Chapter 2.1.1.12 --- Assay of biological activity --- p.35 / Chapter 2.1.2 --- Results --- p.37 / Chapter 2.1.2.1 --- Construction of pRIT2T/MMC --- p.37 / Chapter 2.1.2.2 --- DNA sequencing --- p.40 / Chapter 2.1.2.3 --- Expression of protein A-αMMC fusion cDNA --- p.42 / Chapter 2.1.2.4 --- Purification of protein A-αMMC fusion protein --- p.45 / Chapter 2.1.2.5 --- Cleavage of protein A-aMMC fusion protein --- p.49 / Chapter 2.1.2.6 --- Assay of biological activity --- p.49 / Chapter 2.1.3 --- Discussion --- p.51 / Chapter 2.2 --- Expression of αMMC cDNA as an unfused protein --- p.52 / Chapter 2.2.1 --- Materials and methods --- p.52 / Chapter 2.2.1.1 --- Construction of the plasmid pET/MMC --- p.52 / Chapter 2.2.1.2 --- Preparation of αMMC insert by PCR --- p.56 / Chapter 2.2.1.3 --- Enzyme digestions --- p.57 / Chapter 2.2.1.4 --- Ligation --- p.58 / Chapter 2.2.1.5 --- Transformation --- p.59 / Chapter 2.2.1.6 --- Screening for αMMC inserts --- p.59 / Chapter 2.2.1.7 --- DNA sequencing --- p.60 / Chapter 2.2.1.8 --- Expression of unfused aMMC cDNA --- p.60 / Chapter 2.2.1.9 --- SDS-PAGE analysis --- p.61 / Chapter 2.2.1.10 --- Western blot analysis --- p.62 / Chapter 2.2.1.11 --- Purification of recombinant αMMC --- p.62 / Chapter 2.2.1.12 --- Biological activity of recombinant αMMC --- p.63 / Chapter 2.2.1.13 --- Radioimmunoassay --- p.63 / Chapter 2.2.2 --- Results --- p.67 / Chapter 2.2.2.1 --- Screening of pET/MMC --- p.67 / Chapter 2.2.2.2 --- DNA sequencing --- p.69 / Chapter 2.2.2.3 --- Expression of unfused αMMC cDNA --- p.69 / Chapter 2.2.2.4 --- Radioimmunoassay --- p.72 / Chapter 2.2.2.5 --- Purification of recombinant αMMC --- p.74 / Chapter 2.2.2.6 --- Biological activity of recombinant αMMC --- p.74 / Chapter 2.2.3 --- Discussion --- p.80 / Chapter CHAPTER 3 --- EXPRESSION OF MODIFIED FORMS OF αMMC cDNA --- p.82 / Chapter 3.1 --- Expression of deletion fragments of αMMC cDNA --- p.83 / Chapter 3.1.1 --- Materials and methods --- p.83 / Chapter 3.1.1.1. --- Construction of deletion mutants --- p.83 / Chapter 3.1.1.1.1 --- Modification of pRIT2T/MMC --- p.86 / Chapter 3.1.1.1.2 --- Preparation of closed circular DNA --- p.86 / Chapter 3.1.1.1.3 --- Alpha-phosphorothioate nucleotide --- p.87 / Chapter 3.1.1.1.4 --- Exo III digestion --- p.89 / Chapter 3.1.1.1.5 --- Ligation --- p.89 / Chapter 3.1.1.1.6 --- Transformation --- p.90 / Chapter 3.1.1.1.7 --- Screening of deletion subclones --- p.91 / Chapter 3.1.1.2 --- Confirmation of sequences --- p.91 / Chapter 3.1.1.3 --- Expression of deletion mutants --- p.92 / Chapter 3.1.1.4 --- Purification of deletion mutants --- p.92 / Chapter 3.1.1.5 --- Cleavage of deletion mutants --- p.93 / Chapter 3.1.1.6 --- Subcloning of the αMMC cDNA fragments --- p.94 / Chapter 3.1.1.7 --- Expression of the unfused deletion --- p.96 / Chapter 3.1.2 --- Results --- p.97 / Chapter 3.1.2.1 --- Designation of the deletion mutants --- p.97 / Chapter 3.1.2.2 --- Screening of deletion mutants --- p.98 / Chapter 3.1.2.3 --- DNA sequencing --- p.100 / Chapter 3.1.2.4 --- Expression of deletion mutants --- p.109 / Chapter 3.1.2.5 --- Purification of the fusion fragments --- p.111 / Chapter 3.1.2.6 --- Digestion of deletion mutants by factor Xa --- p.113 / Chapter 3.1.2.7 --- Subcloning of αMMC deletion fragments --- p.115 / Chapter 3.1.2.8 --- Expression of the unfused aMMC deletion --- p.117 / Chapter 3.1.3 --- Discussion --- p.119 / Chapter 3.2 --- Expression of a chimeric αMMC/TCS cDNA --- p.121 / Chapter 3.2.1 --- Materials and methods --- p.122 / Chapter 3.2.1.1 --- Construction of the MMC/TCS chimeric plasmid --- p.122 / Chapter 3.2.1.1.1 --- Digestion of pfG104 - Preparation of GH1100 --- p.125 / Chapter 3.2.1.1.2 --- Preparation of the GH405 fragment --- p.125 / Chapter 3.2.1.1.3 --- Digestion of pACYC177 --- p.126 / Chapter 3.2.1.1.4 --- "Dephosphorylation, ligation and transformation" --- p.126 / Chapter 3.2.1.1.5 --- Confirmation of insert orientation --- p.127 / Chapter 3.2.1.1.6 --- "Preparation of a fragment without PstI, ScaI" --- p.128 / Chapter 3.2.1.1.7 --- Preparation of the 750-bp TCS fragment --- p.128 / Chapter 3.2.1.1.8 --- Ligation of the TCS fragment --- p.129 / Chapter 3.2.1.1.9 --- Cleavage of pACYC177/TCS with ScaI and PstI --- p.129 / Chapter 3.2.1.1.10 --- Preparation of the PstI/HhaI-digested αMMC --- p.130 / Chapter 3.2.1.1.11 --- Ligation of the 252-bp fragment --- p.131 / Chapter 3.2.1.1.12 --- Cloning of MMC/TCS chimeric fragment --- p.131 / Chapter 3.2.1.2 --- Expression of pET/MMC-TCS --- p.132 / Chapter 3.2.1.3 --- SDS-PAGE analysis --- p.133 / Chapter 3.2.1.4 --- Western blot analysis --- p.134 / Chapter 3.2.1.5 --- Purification of MMC-TCS chimeric protein --- p.134 / Chapter 3.2.2 --- Results --- p.135 / Chapter 3.2.2.1 --- Construction of pET/MMC-TCS --- p.135 / Chapter 3.2.2.2 --- Expression of TCS/MMC chimeric cDNA --- p.140 / Chapter 3.2.2.3 --- Purification of MMC-TCS chimeric protein --- p.142 / Chapter 3.2.2.4 --- Reactivity of MMC-TCS chimeric protein with various antisera --- p.145 / Chapter 3.2.3 --- Discussion --- p.146 / Chapter CHAPTER 4 --- SCREENING OF αMMC IMMUNO-REACTIVE FRAGMENTS FROM A RANDOM FRAGMENT LIBRARY --- p.148 / Chapter 4.1 --- Materials and methods --- p.150 / Chapter 4.1.1 --- Description of the pTOPE vector --- p.150 / Chapter 4.1.2 --- Construction of an αMMC random fragment library --- p.152 / Chapter 4.1.2.1 --- Preparation of the cDNA insert of αMMC --- p.155 / Chapter 4.1.2.1.1 --- Large scale prearation of theE plasmid MMC18p8 --- p.155 / Chapter 4.1.2.1.2 --- Digestion of the plasmid MMC18p8 with EcoRI --- p.156 / Chapter 4.1.2.1.3 --- Electro-elution --- p.157 / Chapter 4.1.2.2 --- DNase I digestion --- p.158 / Chapter 4.1.2.3 --- Fractionation of DNA fragments --- p.159 / Chapter 4.1.2.3.1 --- Electrophoresis --- p.159 / Chapter 4.1.2.3.2 --- Electro-elution --- p.160 / Chapter 4.1.2.4 --- Single dA Tailing --- p.161 / Chapter 4.1.2.5 --- Ligation --- p.162 / Chapter 4.1.2.6 --- Transformation --- p.162 / Chapter 4.1.2.7 --- Controls --- p.163 / Chapter 4.1.2.7.1 --- Full-length αMMC cDNA control --- p.163 / Chapter 4.1.2.7.2 --- T-Vector ligation control --- p.164 / Chapter 4.1.2.8 --- Storage of the fragment library --- p.164 / Chapter 4.1.3 --- Immunoscreening of the random fragment library OF αMMC --- p.165 / Chapter 4.1.3.1 --- Anti-αMMC sera --- p.165 / Chapter 4.1.3.2 --- Purification of anti-αMMC sera --- p.165 / Chapter 4.1.3.3 --- Colony lift --- p.167 / Chapter 4.1.3.4 --- Induction of expression --- p.169 / Chapter 4.1.3.5 --- Colony lysis --- p.169 / Chapter 4.1.3.6 --- Immunoscreening --- p.170 / Chapter 4.1.4 --- PCR screening of inserts --- p.170 / Chapter 4.1.5 --- Amplification of positive signals --- p.172 / Chapter 4.1.6 --- Dot blot --- p.173 / Chapter 4.1.7 --- Confirmation of positive signals by Western blotting --- p.174 / Chapter 4.1.8 --- Analysis of positive clones by DNA sequencing --- p.175 / Chapter 4.1.9 --- Analysis of 3-dimensional structure of αMMC --- p.176 / Chapter 4.1.10 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of aMMC --- p.176 / Chapter 4.2 --- Results --- p.178 / Chapter 4.2.1 --- Theoretical considerations --- p.178 / Chapter 4.2.2 --- Construction of a random fragment library of αMMC cDNA --- p.180 / Chapter 4.2.3 --- Screening for immuno-reactive fragments of αMMC --- p.183 / Chapter 4.2.4 --- Confirmation of positive signals by Western blotting --- p.186 / Chapter 4.2.5 --- Estimation of fragment sizes by PCR --- p.188 / Chapter 4.2.6 --- Analysis of the fragment sequences --- p.190 / Chapter 4.2.7 --- Cross-reactivity of the immuno-reactive fragments --- p.194 / Chapter 4.2.8 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of αMMC --- p.196 / Chapter 4.3 --- Discussion --- p.198 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.200 / Concluding remarks --- p.214 / REFERENCES --- p.215 / APPENDIXES GENERAL PROCEDURES --- p.224 / Chapter A.l --- DNA sequencing --- p.224 / Chapter A.2 --- Purification of DNA with Gene Clean --- p.229 / Chapter A.3 --- Purification of primers after synthesis --- p.230 / Chapter A.4 --- Purification of plasmid DNA by Magic Prep (Promega) --- p.232 / Chapter A.5 --- Large-scale preparation of plasmid DNA by QIAGEN --- p.234 / Chapter A.6 --- Lowry protein determination --- p.236 / Chapter A.7 --- Preparation of acid phenol --- p.237 / Chapter A.8 --- SDS-polyacrylamide gel electrophoresis --- p.238

Strains of enteroaggregative E. scherichia colt (EAggEC), characterised by their pattern of adhesion to HEp-2 cells known as the `stacked brick' formation, are a significant cause of chronic diarrhoea in certain under-developed countries. Strains of EAggEC are detected either by a HEp-2 adhesion cell test or by an `aggregative adhesion' gene probe. The pathogenic mechanisms expressed by EAggEC are only poorly understood and the aim of the research described was to obtain a better understanding of how these bacteria cause disease. The adhesion of EAggEC to HEp-2 cells was shown in the majority of strains not to involve fimbriae and was thought to result from physical properties of strains such as charge, since EAggEC adhered to `fixed' HEp-2 cells and readily agglutinated a range of different erythrocytes. Certain strains of EAggEC, which also hybridised with a probe for diffuse adhesion, expressed membrane-associated proteins (MAPs) of 18 or 20 kDa responsible for HEp-2 adhesion. Divalent cations were essential for the expression of the MAPs, which did not contain disulphide bonds or have a quaternary structure. Strains of EAggEC did not express recognised subunit toxins such as Verocytotoxin or E. coil heat-labile toxin, and strains which hybridised with probes for enteroaggregativeh eat-stable toxin-1 did not produce E. coil heat-stable toxin detected by the infant mouse test. Some EAggEC strains (15%) had haemolytic properties. Certain strains expressed type II capsular polysaccharides and approximately 50% of strains expressed an aerobactin-mediated iron uptake system. It was concluded that strains of EAggEC belonged to a very diverse range of serotypes, and it was thought that this heterogeneity resulted from strains of E. coil readily acquiring the genes encoding the EAggEC phenotype. Strains of EAggEC were not associated with a single pathogenic phenotype and the ability of these bacteria to adhere to HEp-2 cells in a `stacked brick'-pattern remains the only common characteristic.

As plantas possuem um arsenal de substâncias utilizadas como defesa contra patógenos e predadores. A possibilidade de utilizar tais substâncias como biopesticidas revolucionou o estudo das proteínas tóxicas. Proteínas inativadoras de ribossomos (RIPs) e as ureases estão entre as proteínas que são abundantes em plantas. RIPs tipo 2 como a ricina, são muito tóxicas, e podem despurinar ribossomos de várias espécies e induzir lesão em DNA, levando à interrupção da síntese protéica e morte de células. Menos tóxicas que a ricina, a maior parte das RIPs conhecidas são do tipo 1 com apenas uma cadeia polipeptídica de 25 - 32 kDa. As ureases (EC 3.5.1.5) são metaloenzimas dependentes de níquel, que catalisam a hidrólise da uréia para formar amônia e dióxido de carbono. A semente do feijão-de-porco, Canavalia ensiformis, é fonte rica de isoformas de urease, entre elas, a canatoxina (CNTX). A proteína CNTX apresenta atividade inseticida contra diferentes espécies de insetos, e sua toxicidade depende da liberação de um peptídeo interno de 10kDa (pepcanatox), que ocorre por ação das catepsinas do sistema digestivo dos insetos suscetíveis. Um peptídeo equivalente ao pepcanatox foi obtido por expressão heteróloga em Escherichia coli - Jaburetox-2Ec, o qual apresentou atividade tóxica contra Dysdercus peruvianus, Rhodnius prolixus e Blatella germanica. Nesse trabalho demonstramos a atividade inseticida de cinco RIPs tipo 1 (PAP-S, gelonina, momordina, saporina-S6 e licnina) em Spodoptera frugiperda e Anticarsia gemmatalis. As RIPs mostraram um efeito entomotóxico espécie-específico para as lagartas, sendo que momordina foi a menos tóxica nos bioensaios. Perda de peso mais pronunciada foi observada em S. frugiperda no 4° dia após o início dos ensaios e para a A. gemmatalis, no 10° dia. A indução da mortalidade (larval e/ou pupal) foi de 57,13% para os tratamentos em A. gemmatalis e 29,45% para S. frugiperda. Para investigar o efeito deterrente de RIPs tipo 1 em insetos, verificou-se, através do teste cometa, o nível de danos ao DNA em tecidos de S. frugiperda e A. gemmatalis que ingeriram um total de 40 μg de RIPs. Os insetos tratados com RIPs mostraram um valor 2 a 3 vezes maior de células com sinais de dano de DNA do que o controle. O dano de DNA poderia ser conseqüência do estresse oxidativo, assim analisou-se atividade de enzimas antioxidantes CAT e SOD e níveis de peroxidação lipídica (TBARS) nos extratos celulares dos insetos, mas não houve uma correlação entre dano de DNA e marcadores de estresse oxidativo. O peptídeo recombinante derivado de urease, jaburetox-2Ec, induziu uma mortalidade de 100% de S. frugiperda após ingestão de 47μg do peptídeo. Em contraste com os dados obtidos com as RIPs, o jaburetox-2Ec não causou lesões no DNA ou alterações em marcadores do balanço redox em S. frugiperda evidenciando um mecanismo de ação distinto. Em linhagens de células de insetos em cultura (Tn5B e UFL-AG-286), a análise citomorfológica sugeriu a ocorrência de citotoxicidade e lise celular com exposição a 80 e 10 μg do jaburetox-2Ec, após 4 e 7 dias de incubação, respectivamente. Para compreender a ação do peptídeo entomotóxico em células de insetos, realizou-se testes de citotoxicidade utilizando o kit CyTotox-GloP TM P incubando-se células UFL-AG-286 e Sf21 com jaburetox-2Ec. Os resultados com esse kit não foram conclusivos, sugerindo que o peptídeo recombinante seria capaz de inibir as proteases intracelulares liberadas na lise celular. Nossos resultados mostraram que RIPs tipo 1 e o jaburetox-2Ec têm efeito inseticida em lepidópteros por mecanismos distintos, sendo que as RIPs tipo 1 induzem lesão de DNA em A. gemmatalis e S. frugiperda, enquanto que jaburetox-2Ec induz alterações ainda não identificadas, que resultam em morte do inseto. / The plants have an arsenal of substances used as a defense against pathogens and predators. The possibility of using these substances as biopesticides revolutionized the study of toxic proteins. Ribosomes-inactivating proteins (RIPs) and ureases are among the proteins that are abundant in plants. Type 2 RIPS, such as ricin, are highly toxic and depurinate ribosomes of different species and induce DNA damage, arresting protein synthesis and leading to cell death. Less toxic, most of the known RIPs are type 1, composed of a single polypeptide chain of 25-32 kDa. Ureases (EC 3.5.1.5) are nickel dependent metalloenzymes that catalyze the hydrolysis of urea into ammonia and carbon dioxide. The seed of jackbean (Canavalia ensiformis) is a rich source of ureases isoforms, one of which is canatoxin (CNTX). The protein CNTX presents insecticidal activity and its toxicity relies on an internal ~10 kDa peptide (pepcanatox) released upon hydrolysis of CNTX by digestive cathepsins of susceptible insects. A peptide equivalent to pepcanatox obtained by heterologous expression in Escherichia coli - Jaburetox-2EC, showed insecticidal activity against Dysdercus peruvianus, Rhodnius prolixus and Blatella germanica. In this study we demonstrated the insecticidal activity of five type 1 RIPs (PAP-S, gelonin, momordin, saporin-S6 and lychnin) in Spodoptera frugiperda and Anticarsia gemmatalis. The entomotoxic effect of RIPs was species-specific and momordin was shown to be the less toxic in the bioassays. S. frugiperda had a more pronounced weight loss on the 4th day of treatment and A. gemmatalis on the 10th day. Mortality (larval and/or pupal) rate reached 57.13% for A. gemmatalis and 29.45% for S. frugiperda. To investigate the deterrent effect of type 1 RIPs in insects, the levels of DNA damage were evaluated using the comet test in tissues homogenates of S. frugiperda and A. gemmatalis fed a total of 40μg of RIPs. The RIPs-treated insects showed 2 to 3 times more cells with DNA damage, as compared to controls. To test whether DNA damage could be consequent to oxidative stress, the activity of the antioxidant enzymes SOD and CAT and levels of lipid peroxidation (TBARS) were assayed in cellular extracts of S. frugiperda and A. gemmatalis fed 40 μg RIPs, but no correlations were found between DNA damage and stress markers. The urease-derived recombinant peptide, jaburetox-2Ec, induced 100% mortality of S. frugiperda fed 47 μg peptide. In contrast to the results observed for type 1 RIPs, treatment of S. frugiperda with jaburetox-2Ec did not cause damage in DNA nor modifications in redox balance markers, indicating a distinct mechanism of action. Microscopic analysis of lepidopteran insect cells in culture (lines Tn5B and UFL-AG-286) suggested cytotoxicity and cell lysis induced by incubation with 80 and 10 μg jaburetox- 2Ec, after 4 and 7 days, respectively. Aiming to understand the mode of action of the entomotoxic peptide in insects cells, the CyTotox-GloP TM Pkit was used to detect cytotoxicity in UFL-AG-286 and Sf21 cells incubated with jaburetox-2Ec. Unexpectedly, the results were not conclusive since it appears that the recombinant peptide is able to inhibit the intracellular proteases released upon cell lysis, preventing the hydrolysis of fluorogenic substrate used in the kit. In conclusion, our results demonstrated that type 1 RIPs and jaburetox-2Ec are insecticidal to lepidopterans acting through distinct mechanisms. Thus type 1 RIPs induce DNA damage in A. gemmatalis and S. frugiperda, while jaburetox-2Ec induce yet to be identified physiological changes that lead to insect death.

Termination of protein synthesis involves the recognition of one of three stop codons (UAG, UAA or UGA) and hydrolysis of the nascent polypeptide chain from the peptidyl-tRNA on the ribosome. Unlike sense codons, which are decoded by aminoacyl-tRNAs, stop codons are decoded by proteins known as release factors. The decoding release factors occupy the same site as aminoacyl-tRNA, interacting directly with the stop codon at the decoding centre and inducing peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Eubacteria have two codon-specific decoding release factors - RF1, which recognizes UAG and UAA, and RF2, which recognizes UGA and UAA. Biochemical studies identified two tripeptide 'anticodon' motifs, PXT in RF1 and SPF in RF2, which structural studies have shown occur in exposed loops (anticodon loops) on the surface of the proteins. Structures of isolated release factors show a compact 'closed' conformation whereas structures of release factors bound to the ribosome show them to be in a highly extended 'open' conformation. This suggests that a large conformational change in the release factor must take place upon or before binding to the ribosome. This transition has been invoked as a mechanism for how translational fidelity is maintained (Rawat et al, 2003), however, small angle X-ray scattering data from E. coli RF1 suggest the decoding release factors are also in the open conformation in solution challenging this mechanism. Mora et al. (2003a) presented evidence that swapping the anticodon loop of RF2 with that of RF1 switched the stop codon specificity of the release factor. Recent structures of the decoding release factors bound to the ribosome showed that there was a second structural element of the release factor, the tip of helix α5, involved in recognition of the first base of the stop codon. The objectives of this thesis were to investigate both the anticodon loop and the helix α5 region for their roles in stop codon recognition, and to investigate whether there is a conformational change in the release factors on binding to the ribosome. The anticodon loop was investigated using chimeras of E. coli RF1/RF2 and E. coli RF1/C. elegans mitochondrial RF1 (MRF1) within the anticodon loop. An RF1 variant containing the RF2-specific SPF tripeptide motif did not switch stop codon specificity showing that the tripeptide motifs are not sufficient determinants for the codon specificity of RF1 and RF2 as was originally proposed. Surprisingly repeating the complete swap of the RF1 anticodon loop to that of RF2 did not switch the stop codon specificity as claimed in Mora et al. (2003a). The studies in this thesis identified additional regions of the anticodon loop of the release factor that are important for stop codon recognition. Two of the RF1/RF2 anticodon loop variants produced showed altered codon specificity recognizing all three standard stop codons and the sense codon UGG. These variants provided unexpected insights into the mechanism of stop codon recognition and can explain why there are two release factors in eubacteria. The C. elegans MRF1 contains a novel anticodon loop that is shorter and lacks the classical PXT motif. E. coli RF1/C. elegans MRF1 chimeras showed that this anticodon loop could function in E. coli RF1 and maintain the same codon specificity. While size and sequence within the loop together are important for recognition clearly there is more than one way RF1-type release factors can recognize the UAG and UAA stop codons. Vertebrate mitochondria use four stop codons, two of the standard stop codons, UAA and UAG, and the reassigned arginine codons AGA and AGG. Two vertebrate mitochondrial release factors have been identified, mtRF1a and mtRF1 (renamed here mRF1[Canonical] and mRF1[Noncanonical]). Bioinformatic studies showed mRF1[C] had similar helix α5 and anticodon loop regions to classical RF1s. mRF1[NC] had different helix α5 and anticodon loop regions and was hypothesized to recognize the non-standard stop codons AGA and AGG. E. coli RF1/Human mRF1[NC] chimeras were constructed that showed that the helix α5 and anticodon loop regions are important for stop codon recognition. Nevertheless the chimeras showed poor activity at the AGA and AGG stop codons on E. coli 70S ribosomes suggesting that mRF1[NC] has evolved to function exclusively on 55S mitoribosomes. A release factor variant of RF2 was designed that had the potential to trap this E. coli factor in the closed conformation in solution by disulphide bond formation. The RF2 double cysteine variant was successfully expressed and purified. The disulphide bond between the two cysteines was detected directly by mass spectrometry in a high proportion of molecules, showing the closed form of RF2 exists in solution. The RF2 closed form variant was shown to have release activity concomitant with the proportion of the open form in the RF preparation showing that the conformational change is required for normal release factor function. Preliminary binding studies have suggested that the RF2 closed form variant can bind to the ribosome. The ability of the closed form of RF2 to bind to the ribosome allowed a mechanism of translational fidelity to be proposed from the studies in this thesis; the release factor would recognize the stop codon in the decoding centre and, if cognate, the conformational change would occur allowing peptidyl-tRNA hydrolysis.

"March, 2005" / Bibliography: leaves 144-155. / ix, 155 p. : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / "The aims of this thesis were to investigate potentially novel aspects of the regulation of B and morphogenetic gene expression in coliphage 186, in order to understand more fully how late gene expression is controlled in this phage, and how gene expression may be regulated in general. Three specific aims were pursued in this project: 1. to characterize E. coli mutants that appear to abolish 186 B protein activity; 2. to determine the role of replication for the provision of late functions during the phage lytic cycle; and 3. to determine the role of CI repression of the 186 B promoter." --p. 41. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, Discipline of Biochemistry, 2005

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Diane S. Herson, Dept. of Biological Sciences. Includes bibliographical references.

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L).  This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association.  Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Many studies have attempted to link antimicrobial use in food animal agriculture with an increased risk of antimicrobial-resistant (AR) bacterial levels in humans. Our data arise from longitudinal aggregated fecal samples in a 3-year cohort study of vertically integrated populations of human workers and consumers, and swine. Human and swine E. coli isolates (N = 2130 and 3485, respectively) were tested for antimicrobial susceptibility using the SensititreTM broth microdilution system. The associations between AR prevalence for each antimicrobial agent, multi-drug resistant E. coli, or multivariate AR E. coli, and the risk factors (host species, production type (swine), vocation (human swine worker versus non-worker), and season) in the study were assessed using generalized estimating equations (GEE), GLM with multinomial distribution, or GEE in a multivariate model using a SAS® macro to adjust for the correlated AR phenotypes. There were significant (p < 0.05) differences in AR isolates: 1) between host-species with swine at higher risk for ceftiofur, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. The prevalence of ciprofloxacin, nalidixic acid, and trimethoprim/sulfamethoxazole resistance were higher among human isolates, 2) swine production group was significantly associated with AR with purchased boars, nursery piglets, and breeding boars at a higher risk of resistance to streptomycin and tetracycline, and 3) human swine worker cohorts exhibited an elevated tetracycline prevalence, but lowered sulfisoxazole prevalence when compared to nonworkers. High variability among seasonal samples over the 3-year period was observed. There were significant differences in multiple resistance isolates between host species, with swine at higher risk than humans of carrying multi-resistant strains; however, no significant differences in multiple resistance isolates within humans by vocation or within swine by production group. The odds-ratios, adjusted for multivariate dependence of individual AR phenotypes, were increased relative to unadjusted oddsratios among 1) swine as compared to human for tetracycline (OR = 21.8 vs. 19.6), and 2) increased significantly among swine-workers as compared to non-workers only for tetracycline (OR = 1.4 vs. 1.3). Occupational exposure to swine-rearing facilities appears to be associated with an increased relative odds for the prevalence of tetracycline resistance compared to non-workers.