Chez les bactéries en contact direct avec leur milieu, la transcription des gènes et la synthèse des protéines sont régulées de manière efficace à chaque changement des paramètres environnementaux afin de permettre la survie cellulaire. Dans le cas des bactéries commensales de l’intestin, ces régulations doivent aussi permettre les interactions symbiotiques et la colonisation dont les mécanismes moléculaires, encore peu connus, sont probablement liés, entre autres, à la surface des bactéries (molécules exposées et sécrétées…). Lactobacillus casei, bactérie commensale, possède environ 330 gènes prédits comme intervenant dans la composition et la fonctionnalité de la surface cellulaire. Afin d’avoir une vue globale de la totalité des gènes qui interviennent dans l’établissement de L. casei dans l’intestin, une approche de génétique inverse a été réalisée. Pour cela, une banque de mutants aléatoires étiquetés de L. casei par « Signature-Tagged Mutagenesis » a été créée puis annotée et réorganisée grâce au séquençage des régions d’insertion du transposon. Les mutants ont été criblés quant à leur capacité à s’établir dans l’anse iléale ligaturée de lapin et quantifiés par qPCR. Parmi les 47 gènes identifiés comme étant impliqués dans l’établissement in vivo, trois gènes en opéron codant pour un système à deux composants et une « penicillin-binding protein » ont été caractérisés. Ces trois gènes sont impliqués dans la modulation de la surface cellulaire et plus particulièrement dans la régulation des hydrolases du peptidoglycane qui sont nécessaires à la protection de la bactérie dans l’environnement intestinal. / In bacteria which are in direct contact with their environment, genes transcription and proteins synthesis are efficiently regulated at each change of environmental parameters to allow cell survival. For intestinal commensal bacteria, these regulations must also allow symbiotic interactions and colonization whose molecular mechanisms, so far little known, are probably related, among others, to the bacteria surface (molecules exposed and secreted…). Lactobacillus casei, a commensal bacterium, has about 330 predicted genes involved in the composition and functionality of the cell surface. To have a global view of the whole genes involved in the establishment of L. casei in the gut, a reverse genetics approach was performed. For that, a library of L. casei random labeled-mutants by Signature-Tagged Mutagenesis was generated then annotated and reassembled thanks to the sequencing of transposon insertion sites. Mutants were screened for their ability to establish themselves in the rabbit ligated ileal loop and quantified by qPCR. Among the 47 genes identified as involved in the in vivo establishment, three genes in an operon encoding a two-component system and a penicillin-binding protein were characterized. These three genes are involved in the cell surface modulation and particularly in the regulation of peptidoglycan hydrolases which are required for the bacteria protection in the intestinal environment.

Indiana University-Purdue University Indianapolis (IUPUI) / The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Eremothecium gossypii has the ability to utilize vegetable oils as a carbon source to produce riboflavin. This organism has been known to produce as much as 40 000 times more riboflavin than it requires after genetic modification on simple sugars. Adaptation of this organism to various oil substrates for riboflavin production has been poorly investigated. The aim of this research was thus to investigate the production of riboflavin by Eremothecium gossypii, on various oils and to improve production by mutating the organism and optimising media components using Design of Experiments (DOE). Nine overproducing mutants were obtained after mutating with various concentrations of ethylmethane sulphonate (EMS), n-methyl-n‟-nitro-n-nitrosoguanidine (MNNG) and Ultraviolet light. Riboflavin overproducing mutants were screened on an itaconate-containing medium; the colonies appeared yellow instead of white in the case of the wild-type. The itaconate screening medium isolated mutants with an isocitrate lyase that was insensitive to feedback inhibition. Mutations performed using EMS increased the ability of E. gossypii to produce riboflavin by 611% (7-fold) compared to the wild-type. This was achieved with soybean oil as a carbon source and was better than the other five oils used. Using DOE, fractional factorial experiments were carried out to optimise media components for riboflavin production on soybean oil. The total riboflavin produced by E. gossypii mutant EMS30/1 increased from 59.30 mg l-1 on a standard O&K medium using soybean oil as a carbon source to 100.03 mg l-1 on a DOE improved O&K medium, a 69% increase. The final optimised growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 5.0 g l-1 peptone, 5.0 g l-1 malt extract, 5.1 g l-1 yeast extract, 0.64 g l-1 K2HPO4, 0.6 g l-1 MgSO4 and 20 g l-1 soybean oil. Fractional factorial and central composite media optimization designs increased riboflavin production by several fold over their iterations. There was an overall increase of 1099% (12-fold) in riboflavin production by the mutant grown in an optimized medium compared to the initial riboflavin produced by the wild-type.

In the context of the somatic mutation theory of chemical carcinogenesis, mutations are thought to arise during the replication of DNA past carcinogen-DNA adducts. The work described in this thesis deals with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with the ultimate carcinogenic form of benzo (a) pyrene, 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo (a) pyrene (BPDE I). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. The data obtained were, therefore, supportive of the 'block-gap' model of DNA bypass replication in carcinogen damaged mammalian cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. It was determined that the integrated human c-Ha-ras gene was hypomethylated, and expressed at the messenger level. The human c-Ha-ras protein, p21, was also detected in these transfected cell lines. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-N-nitroso-N'-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines. Screening of the in vitro and in vivo treated, transformed cell lines by RNA:RNA duplex mismatch analysis led to the detection of no mutations within the first exon of the human c-Ha-ras oncogene.

C. difficile is an obligate anaerobic, Gram-positive, rodshaped and spore-forming bacterium. It is a well-recognised causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. C. difficile has emerged as an important nosocomial pathogen in recent years, associated with considerable morbidity, mortality and economic burden. Despite its importance, functional genomic studies have been lagging behind in comparison to other enteric pathogens. This is attributed to the fact that C. difficile is difficult to manipulate genetically and the lack of robust, reproducible mutagenesis systems for many years. The ideal mutation for robust functional genomic studies is a markerless, in-frame deletion of the gene of interest. All systems developed for C. difficile, up to the start of this study, involve insertional inactivation of the gene of interest. This study describes the development of a novel genetic system for C. difficile, to create precise and markerless chromosomal deletions, using the meganuclease ISceI. For validation of the system, the addBA genes in C. difficile were deleted. The AddAB enzyme complex is important in the survival of many bacteria, since it maintains genome integrity, by the repair of double-strand breaks. Deletion of addBA in C. difficile did not significantly affect growth and viability, but the mutant strains were sensitive to DNA damaging agents. In addition, it was shown that C. difficile is capable of initiating the SOS response after DNA damage and that AddAB is not necessary for the induction of this response. The genetic system was further optimised to delete type IV pili (TFP)- associated genes, particularly pilT (CD3505) and pilA (CD3507), to investigate twitching motility. TFP are important in virulence and pathogenesis of many bacteria and twitching motility is often involved. TFP in C. difficile may be expressed in vivo during infection and may be involved in biofilm formation and colonization. To study potential TFP-mediated motility, a non-flagellated C. difficile strain was first constructed by deleting the fliC gene. The pilT gene, predicted to encode a protein involved in TFP retraction, was then deleted in the ΔfliC strain. A ΔpilT strain was also generated. Preliminary experimental work using these strains did not show any evidence for twitching motility and no difference between the ΔpilT strains and the parental strains. Examination of cells from the ΔfliC strain, under various conditions, did not reveal any pili, which indicates that TFP are regulated in C. difficile and that the TFP locus might be repressed at the transcriptional level. Preliminary work to investigate an intergenic region located upstream of the TFP locus in C. difficile, that might be involved in regulation, suggested that transcription is being initiated within a 500 bp region upstream of the CD3513 gene.

<p>Antibodies are proteins capable of specific interactions to a wide range of molecules. These interactions are facilitated by the complementary determining regions (CDR).</p><p>Carcinomas are the most common of human cancers and they release significant amount of cytokeratins (CK) in the necrotic areas of the tumors. The CKs stay in the tumor, since they have low solubility. The antibody studied in this thesis, the anti-CK 8 antibody TS1, has shown to be effective in tumor targeting and is proposed to be useful in therapy.</p><p>Single-chain antibodies (scFv) are recombinant antibodies which are much smaller than the intact IgG. This is an advantage when used in tumor therapy, since they can penetrate the tumors more easily than the larger IgG. Moreover, they are expressed by one single gene which make them easy to modify, for example by site-directed mutagenesis.</p><p>The anti-idiotypic antibody αTS1 can be used to clear the TS1 form the circulation and thereby clear the body from non-tumor bound TS1 in therapy. To be able to modify the binding of an antibody to its antigen and or anti-idiotype, these interactions must be studied. In this study this is accomplished by chemical modifications of the IgGs TS1 and αTS1 and the antigen CK 8. Guided by these results, amino acid residues were mutated by using site-directed mutagenesis in the TS1-218 scFv and the effects were studied. From mutational study results, the functional epitope could be mapped and it was found that there are mainly tyrosines, but also charged residues, serine and a tryptophan that are important for both interactions. The binding of TS1-218 to both αTS1 and CK 8 could be improved by changing the negatively charged side-chains by mutations to their corresponding amide or alanine.</p><p>Both the IgG and scFv versions of TS1 were administered in vivo. The IgG αTS1 was used to clear the TS1 from the circulation by forming immune complexes. The immune complexes, consisting of four or more antibodies, were mainly metabolized by the liver. The scFv TS1-218 could localize to the tumor in a tumor xenograft mouse model, although a higher uptake would be desired in a therapeutic strategy. The scFv was cleared rapidly by the kidneys, but the clearance could be slowed by pre-formed immune complexes with anti-TS1 scFv in vitro, prior to administration in vivo.</p>

La transcétolase (TK) permet de synthétiser des cétoses D-thréo par formation stéréospécifique d'une liaison C-C. L'objectif de ces travaux vise à modifier la spécificité de substrat de la TK de Saccharomyces cerevisiae par mutagenèse afin d'élargir le potentiel synthétique aux aldoses D-thréo et cétoses L-érythro. Notre stratégie a consisté à créer des banques de TK mutées à partir de courtes séquences du gène TKL1 (identifiées d'après la structure 3D) qui ont été dégénérées grâce à une approche de type "cassette mutagenesis". Pour identifier les TK recherchées, nous avons développé un test de sélection in vivo basé sur l'auxotrophie vis-à-vis d'un acide aminé. Dans ce but, nous avons synthétisé des sondes appropriées comportant un motif reconnu par la TK naturelle (cétose D-thréo) ou par les TK mutées recherchées (cétose L-érythro ou aldose D-thréo) et la chaîne latérale d'un acide aminé (alanine, valine leucine, méthionine, thréonine). La faisabilité de ce test a été étudiée en présence des composés cétose D-thréo et de la TK sauvage. In vitro, nous avons montré que ces différents composés sont des sustrats de la TK. Pour le développement du test de sélection in vivo dans E. coli, les substrats précurseurs de la leucine et la méthionine ont été retenus en raison de la stabilité de l'auxotrophie pour ces acides aminés. Les meilleurs taux de croissance ont été obtenus avec la sonde cétose D-thréo précurseur de la méthionine.

<p>Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated.</p><p>In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype.</p><p>The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome.</p><p>In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.</p>

No description available.

The overriding objective of this study was to induce genetic variation in Vernonia (Vernonia galamensis variety ethiopica) using ethylmethanesulfonate (EMS) and select mutants for subsequent selective breeding. Vernonia is an underutilised, potential novel oilseed crop with multiple applications in industry mostly due to the production of naturally epoxidised vernolic acid oil. Commercial cultivation of vernonia is significantly hampered by non-uniform seed maturity, tall plant height, seed shattering and lack of appropriate technologies for mechanical harvesting, seed threshing and cleaning. Mutations of a single or few genes possessing target traits are invaluable in crop improvement programs. Chemical mutagenesis using EMS is an important, affordable and effective method to induce random useful genetic mutations in crop plants. Ethylmethanesulfonate mutagenesis has previously been reported to affect various agronomic traits, induce a wide variety of phenotypic mutations and alter both seed oil content and fatty acid profile on several crops. The objectives of this study were: (i) to determine an optimum EMS treatment combination i.e. exposure duration, temperature and dose that would enable 50-60% germination at minimum days to emergence in selected V. galamensis var. ethiopica lines (Vge-1, Vge-4, Vge-7 and Vge-10), (ii) to induce genetic variation using predetermined optimal treatment conditions and select mutants in V. galamensis variety ethiopica lines (Vge-1 and Vge-4) and (iii) to evaluate oil content and fatty acid compositions among seeds of chloroplast mutants, EMS treated seeds and untreated controls of Vge-1 and Vge-4. Before any mutation is administered in plants, it is important that the optimal mutation dose is determined. The lethal dose 50 (LD50) was the standard used in this study to find optimal treatment conditions. Significant interactions (P<0.001) existed between EMS, line, time and temperature with respect to days to 50% emergence, germination percentage and seedling height. Optimal days to 50% emergence (10-12 days) and germination (50- 58%) was achieved for Vge-1, Vge-7 and Vge-10 when treated with 0.372% EMS at 350C for 1 hour treatment. The optimal treatment combination for Vge-4 was 0.372% EMS at 32.50C for 2hr. The treatment combinations that yielded optimum results in the tested lines were utilized to induce large scale mutations in V. galamensis to select target mutants in the field. Large scale mutation was conducted using the observed optimal treatment conditions. Ethylmethanesulfonate mutagenesis significantly delayed days to head formation, days to flowering and days to maturity on both lines. Delays in days to emergence were only significant in Vge-4. EMS treatment also significantly reduced germination percentage, number of seeds per head, number of fertile plants, plant height and plot yield for both Vge-1 and Vge-4. Thousand seed weight significantly increased in treated seeds of the two lines. Chlorophyll mutants were observed for tested lines associated with high count of sterility for both lines. Ethylmethanesulfonate successfully induced phenotypic mutation in selected vernonia lines, however at this stage the effect of mutation on vernonia seed oil content and fatty acid was unknown. Liquid gas chromatography method was employed for oil and fatty acids analysis. In Vge-1, significant differences were observed in composition of linoleic and oleic acid due to the mutagenesis. Significant increases in linoleic and oleic acid composition were found in chloroplast mutants due to EMS mutagenesis. No significant differences were detected in fatty acid compositions in Vge-4 after the EMS treatment. Differential responses were observed when lines were compared at various EMS mutation levels showing significant effect on vernolic, linoleic and oleic acids compositions. In both lines no differences were detected on seed oil content, palmitic acid, steraic acid and arachidic acid compositions after the treatment. Oil content significantly and positively correlated with vernolic acid for Vge-1 (P<0.001; r= 0.898) and Vge-4 (P<0.05; r= 0.65). Vernolic acid significantly and negatively correlated with other fatty acids. The study found that EMS mutagenesis significantly changed the oleic acid and linoleic acid compositions in vernonia. However, the oil content and vernolic acid composition were not significantly affected by EMS treatment. This study established that EMS was successful in inducing genetic variation (in agronomic traits, seed oil content and fatty acid composition) in the two tested lines of V. galamensis. Data from a single planting generation is insufficient to conclude fully on the effect of EMS on V. galamensis; therefore it is highly recommended that further multigenerational studies should be conducted with an increased number of testing lines from a wide range of environmental backgrounds. / Thesis (M.Sc.Agric.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.