The rapidly increasing emergence of antibiotic resistance amongst pathogenic bacteria is a major clinical and public health problem. The increase in resistant pathogens, accompanied with the small number of new antibiotics introduced in recent years, has limited the number of effective antimicrobials. The classical paradigm suggests that antibiotic resistance emerges by selection for pre-existing mutants in the bacterial population exposed to antibiotics. In contrast, recent data suggested that mutations evolve after cells encounter antibiotic therapy. This kind of mutation is known as adaptive mutation, which is activated by the SOS DNA repair and mutagenesis pathways. Accumulation of single-stranded DNA (ss-DNA) is the signal that induces the SOS response by promoting the formation of the RecA filament, which in turn activates the auto-cleavage activity of LexA and allows expression of SOS genes, including the SOS error-prone polymerases. In this project, phthalocyanine tetrasulfonic acid (PcTs)-based RecA inhibitors were characterized. PcTs molecules were found to potentiate the activity of bactericidal antibiotics and reduce the ability of bacteria to acquire antibiotic resistance mutations. This study highlights the ability of RecA inhibitors to potentiate the activity of antibiotics and provides a strategy for prolonging the life span of existing and newly developed antibiotics. We predicate that RecA inhibitors will be part of an antibiotic “cocktail” that enhances the activity of antibiotics and blocks resistance, which will ultimately prolong antibiotic lifespan.

<p>Different mutations within the <em>NALP3</em> gene are thought to be associated with development of several types of hereditary auto inflammatory disorders such as neonatal onset multisystem inflammatory disorder (NOMID) and muckle-wells syndrome (MWS). In this work two separate mutations E688K and G569R were supposed to be constructed by site-directed mutagenesis in the cloned wild type <em>NALP3</em> genes and further expressed in bacterial and mammalian host cells for functional studies in protein -protein interaction models.</p>

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 32-36).

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Genetically modified (GM) crops attracted interest globally when use of these crops resulted in significant increases in yield and production. These increases were due to protection of crops from pests, weeds and diseases. However, evolution of resistance by pests threatens the continued efficacy of GM crops. One such example is the resistance to Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). Resistance in this pest was due to a mutation in the aminopeptidase N1 (APN) Cry receptor gene, encoding the receptor for Cry1Ac. Laboratory studies have indicated that species in families Noctuidae, Pyralidae and Plutellidae can develop resistance to Bttoxins. To date, field-evolved resistance has only been reported in Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in South Africa, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in the south-eastern United States, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Puerto Rico, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in northern China and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) in The Philippines and Hawaii. Resistance development in lepidopteran species is thus a common phenomenon. The stem borer B. fusca is a major insect pest to Bt-maize in the Vaalharts irrigation scheme (South Africa). The first official report of B. fusca resistance to Cry1Ab toxin was recorded in 2007, although farmers observed increased damage to Bt-maize from stem borers as early as 2004. A second report of resistance in an area nearby followed in 2009. No study has yet been done to determine the molecular mechanism of B. fusca resistance to Cry1Ab. As mentioned, a mutation in the APN receptor gene is responsible for H. armigera resistance to Cry1Ac. Although B. fusca has developed resistance to the B. thuringiensis Cry1Ab toxin, the binding-patterns and -sites of Cry1Ac and Cry1Ab are similar. Thus a similar mutation may be responsible for B. fusca resistance to Cry1Ab. Aminopeptidase, cadherin and alkaline phosphatase are the major Cry toxin receptors that have been identified in lepidopteran species. The present study was concerned with the investigation of mutations in these receptor genes. However, in order to study mutations, sequence data of receptor genes are essential. Degenerate primers were designed based on conserved regions observed in multiple protein sequence alignments of aminopeptidase N (isogenes 1 to 6), cadherin and alkaline phosphatase of several lepidopteran species. Primers were degenerate to take into consideration the variant regions in receptor gene sequences among lepidopteran species. These primers were used to amplify genomic DNA (gDNA) from susceptible and resistant larvae by using PCR. Sequences of PCR amplicons were determined through Sanger sequencing reactions and subjected to BLAST searches. Results of the BLAST searches showed some similarities to the respective receptor genes. These sequences were also used in phylogenetic analysis. This analysis intended to determine the phylogenetic relationship of the respective receptor genes between B. fusca and other lepidopteran species. Mutations could not be identified in the present study, due to a lack in receptor gene sequence data for B. fusca. Thus a goal of the present study was to generate sequence data for B. fusca. In addition to the proposed objectives, cytochrome b gene sequences of B. fusca were used to determine the phylogenetic relationship between B. fusca and other lepidopteran species. Genome sequencing of B. fusca is recommended, as this will provide a platform for genomic, transcriptomic and proteomic studies on this species. These studies will provide much needed information, which can be used to formulate strategies to prevent resistance development in and spread of resistance to other B. fusca populations in sub-Saharan Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Genetically modified (GM) crops attracted interest globally when use of these crops resulted in significant increases in yield and production. These increases were due to protection of crops from pests, weeds and diseases. However, evolution of resistance by pests threatens the continued efficacy of GM crops. One such example is the resistance to Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). Resistance in this pest was due to a mutation in the aminopeptidase N1 (APN) Cry receptor gene, encoding the receptor for Cry1Ac. Laboratory studies have indicated that species in families Noctuidae, Pyralidae and Plutellidae can develop resistance to Bttoxins. To date, field-evolved resistance has only been reported in Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in South Africa, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in the south-eastern United States, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Puerto Rico, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in northern China and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) in The Philippines and Hawaii. Resistance development in lepidopteran species is thus a common phenomenon. The stem borer B. fusca is a major insect pest to Bt-maize in the Vaalharts irrigation scheme (South Africa). The first official report of B. fusca resistance to Cry1Ab toxin was recorded in 2007, although farmers observed increased damage to Bt-maize from stem borers as early as 2004. A second report of resistance in an area nearby followed in 2009. No study has yet been done to determine the molecular mechanism of B. fusca resistance to Cry1Ab. As mentioned, a mutation in the APN receptor gene is responsible for H. armigera resistance to Cry1Ac. Although B. fusca has developed resistance to the B. thuringiensis Cry1Ab toxin, the binding-patterns and -sites of Cry1Ac and Cry1Ab are similar. Thus a similar mutation may be responsible for B. fusca resistance to Cry1Ab. Aminopeptidase, cadherin and alkaline phosphatase are the major Cry toxin receptors that have been identified in lepidopteran species. The present study was concerned with the investigation of mutations in these receptor genes. However, in order to study mutations, sequence data of receptor genes are essential. Degenerate primers were designed based on conserved regions observed in multiple protein sequence alignments of aminopeptidase N (isogenes 1 to 6), cadherin and alkaline phosphatase of several lepidopteran species. Primers were degenerate to take into consideration the variant regions in receptor gene sequences among lepidopteran species. These primers were used to amplify genomic DNA (gDNA) from susceptible and resistant larvae by using PCR. Sequences of PCR amplicons were determined through Sanger sequencing reactions and subjected to BLAST searches. Results of the BLAST searches showed some similarities to the respective receptor genes. These sequences were also used in phylogenetic analysis. This analysis intended to determine the phylogenetic relationship of the respective receptor genes between B. fusca and other lepidopteran species. Mutations could not be identified in the present study, due to a lack in receptor gene sequence data for B. fusca. Thus a goal of the present study was to generate sequence data for B. fusca. In addition to the proposed objectives, cytochrome b gene sequences of B. fusca were used to determine the phylogenetic relationship between B. fusca and other lepidopteran species. Genome sequencing of B. fusca is recommended, as this will provide a platform for genomic, transcriptomic and proteomic studies on this species. These studies will provide much needed information, which can be used to formulate strategies to prevent resistance development in and spread of resistance to other B. fusca populations in sub-Saharan Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Studies were undertaken to evaluate the potential of random amplified polymorphic DNA (RAPD) to detect DNA effects (including DNA damage and mutations) in aquatic invertebrates, following their exposure to a variety of environmental contaminants under laboratory conditions. After rigorous optimisation of the RAPD method, the protocol, which used a high annealing temperature (50&quot;C for 10-mer primers), was found to generate good-quality DNA profiles from groups of organisms belonging to the bacterial, plant and animal kingdoms. The RAPD method was initially used to detect benzo(a)pyrene [B(a)P] and copper-induced DNA effects in the water flea Daphnia magna and ultraviolet-mediated DNA effects in the marine alga Palmaria palmata. The results clearly showed that changes occurred in RAPD profiles obtained from the exposed populations when compared to controls. In these studies, the effect of the genotoxins at higher levels of biological organisation (e.g. Darwinian parameters and/or fitness parameters) were also investigated and were compared with genomic DNA template stability (GTS), a qualitative index representing clear changes in panems compared to control RAPD profiles. The results from these experiments revealed that GTS could be more sensitive than growth parameters and showed at least equal or even greater sensitivity than other measures of fitness. Changes in RAPD profiles were believed to be the result of DNA effects, namely adduct formation, DNA breakage, oxidative damage and mutations and possibly other effects (e.g. variation in gene expression). Nevertheless, the nature and amount of DNA effects could only be speculated because diverse events may induce the same category of changes (i.e. variation in band intensity, appearance of bands, and disappearance of amplicons) in RAPD patterns. Further studies confirmed that RAPD had the potential to qualitatively detect oestrogen and xeno-oestrogen -induced DNA effects in barnacles. Additional experiments emphasised that oxygen radicals and variation in gene expression may induce significant changes in RAPD profiles. To further understand the effects of DNA lesions and mutations on RAPD patterns, individual types of DNA damage were created in vitro. The results clearly indicated that BaP DNA adducts, DNA photoproducts. and DNA breakages had significant effects on RAPD profiles but that diverse types of DNA damage may induce the same category of changes in RAPD patterns which render the interpretation of the results difficult. It was also concluded that mutations could be detected provided they do not arise in a random fashion. Finally, an attempt was made to determine the kinetics of DNA damage and DNA repair and whether changes in patterns obtained from B(a)P exposed Daphnia magna could be transmitted to successive generations. This strategy was developed to distinguish between mutations and DNA damage. The results showed that some bands obtained from the exposed populations were transmitted to the first and/or second generation but not to the third. It was concluded that the transmission of modified genetic material to the offspring was more likely to be the result of large genomic rearrangements and/or base methylation (epigenetic processes) rather than point mutations. In conclusion, the results presented in this research project show the potential of the RAPD assay as a useful method for the qualitative assessment of DNA effects including genotoxicity and changes in gene expression. The main advantage of this technique is that it can be applied to any species without requiring any information about the nucleotide sequence. In the field of ecogenotoxicology, its main advantage lies in its sensitivity and speed to detect a wide range of DNA damage including DNA breakage, DNA adducts, oxidative damage as well as mutations (including point mutations and large rearrangements). On the other hand, RAPD only allows a qualitative assessment of the DNA effects and the nature of the changes occurring in profiles can only be speculated. Finally, a great deal of further experimentation and validation are required in order to assess the applicability of the technique to a variety of other species and pollutants, particularly under field conditions.

Studies have discovered higher frequencies of single nucleotide polymorphisms (SNPs) in different mitochondrial genes are associated with subnormozoospermia. However, the frequencies of SNPs in ND1 and ND2 are not unknown. The present research was aimed to determine the frequencies of SNPs in ND1 and ND2 genes of the mitochondrial genome in fertile and subfertile men and whether changes in codon usage was associated with fertility phenotypes. Total genomic DNA from 157 semen samples was extracted using the proteinase K/SDS digestion procedure, followed by phenol/chloroform purification and ethanol precipitation. ND1 and ND2 genes were amplified respectively from 80 and 92 DNA samples from different fertility groups. Each PCR product was sequenced to identify mutations. Codon change resulting from a nucleotide substitution was determined by comparison with a reference mtDNA sequence obtained from the NCBI database. The frequency of codon usage in the reference mtDNA was determined by the computer program MEGA version 2.1. Eleven synonymous nucleotide substitutions and two non-synonymous substitutions were found in this study. Four SNPs were previously characterized; all SNPs were homoplasmic. None of the SNPs were likely to affect the function of the proteins on the basis of the hydrophobicity plots or secondary structure predictions. Sixty two percent of synonymous mutations were found to change from a high to a low relative codon usage values; 37% of synonymous mutations changed from a low to a high relative usage value. Chi-square (χ²) test (χ²= 0.067 with 1 d.f.) showed that there was no significant difference at the 5% level between these changes. Thus, change in codon usage was not related to semen quality in men. Further, there were no statistically significant differences in the observed frequencies of SNPs of fertile and subfertile men. However, the sample size was small and this study was only focused on a single NZ Caucasian population. Further study including larger and more diverse population samples may provide further insight into the functional importance of codon usage and its relevance to fertility

- / -

Synonymous mutations have traditionally been thought to have no significant effect on fitness. However, a growing body of recent research has shown that this is not always the case. In an experimentally evolved population of Pseudomonas fluorescens grown in minimal glucose media, synonymous mutations arose in a glucose transport gene that resulted in beneficial fitness effects comparable to those of non-synonymous mutations. We found that the increase in fitness was a direct result of increased gene expression; however, the precise mechanism was unclear. Synonymous mutations have been shown to affect gene expression on transcriptional and translational levels through changes in mRNA secondary structure and codon usage. Our study investigates the underlying mechanisms in which these evolved synonymous mutations lead to increased gene expression. In addition to the evolved mutations, we have a library of 42 strains with single synonymous mutations within the glucose transport gene and found a positive correlation between fitness and gene expression. To determine whether these mutations affect transcript levels, translational efficiency or a combination of both, we systematically incorporated transcriptional and translational fusions of a yellow fluorescent protein within the glucose transport operon. We found that the evolved mutations predominantly act on the level of transcription and have strong polar downstream effects. Additionally, through manipulation of the local genetic sequence, we investigated the specific molecular requirements necessary for the increased expression. We found that for one of our evolved synonymous mutants, mRNA secondary structure does not play an essential role, but we speculate that the mutation may strengthen a weak internal promoter sequence to confer its increased expression. Our study provides evidence of the adaptive mechanisms of beneficial synonymous mutations in an experimentally evolved setting.