At present, species are faced with a host of human-induced impacts that have already led to a loss of biodiversity and shifts in species’ ranges. Knowledge of the ecological and evolutionary processes of dynamic species’ ranges, that are either shifting or expanding, will be key to understanding the ability of species to persist and adapt during periods of environmental change. The aim of this thesis is to investigate the effects of range expansion on the genetics and evolution of species, and understand the processes that facilitate the spatial spread of populations. Using individual-based simulation models, this work demonstrates that the unique selective environment of range expansion can have a significant effect on spatial genetics and the evolution of life-history traits. It is found that the survival and spread of mutations, of various fitness effects, is influenced substantially by landscape features encountered by the expanding range. The incorporation of more specific genetic architecture shows that a substantially increased frequency of fitness peak shifts may occur on the edge of an expanding range than would arise within a stationary population. Range expansion is shown to select for increased dispersal and reproduction at the expense of competitive ability on the front of the expanding wave which results in an accelerating spread rate. The survival and spread of a population during range expansion is also affected substantially by the specific movement behaviour of individuals. The exact nature of suitable habitat, in terms of the number of suitable habitat patches and level of fragmentation, will also greatly affect the ability of a population to survive and spread during a period of environmental change. These findings are synthesised within a conceptual framework that is proposed for the evolution of populations during range expansion, where a flattening of the fitness landscape leads to adaptive revolutions.

Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.

<p>Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.</p> / Dissertation

Mutation, whether spontaneous or induced, is the premier tool for understanding gene function. One approach is to create mutations in a specific gene, and then use the resulting cell or organism to search for a phenotype. An alternative is to create mutations at random, and focus first on the identification of phenotypes. The mutation that underlies a phenotype can then be tracked down, forming the foundation of testable hypotheses. Using random chemical mutagenesis in mice, I have identified 20 heritable phenotypes affecting either the innate or adaptive branches of immunity. The genetic basis of 18 of these phenotypes was solved, caused by mutations in at least 16 unique genes. Five of these genes were not previously known to be involved in immunity, and a detailed analysis of four of them is provided in this thesis. These include genes encoding the following proteins: the inactive rhomboid protease iRhom2, which is specifically required for the secretion of tumour necrosis factor alpha; the hypothetical phospholipid flippase ATP11C, required for B cell development in the adult bone marrow; the folliculin-interacting protein FNIP1, also required for B cell development; and the zinc finger transcription factor ZBTB1, essential for the development of all lymphocyte lineages. These findings uncover new entry points for the understanding of mammalian immunity, and highlight the value of mouse forward genetics in the understanding of mammalian phenomena in general.

The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.

A dissertation submitted to the Faculty of Science, University of' the Witwatersrand, in partial fulfilment of the requirements of the Degree of Master of Science in the field ·of Biotechnology. February 1991. / Nocardioform bacteria were studied for their ability to interconvert bile acids. From the studies of utilisation and resistance curves, the most suitable donor and recipient strains for complementary gene cloning, were Arthrobacter oxydans strain C1 and Rhodococcus erythropolis strain ATCC 4217-1 respectively. [Abbreviated Abstract. Open document to view full version] / MT2016

Thesis advisor: Evan R. Kantrowitz / The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of cellular regulation can be related in some manner to protein conformational changes such as the quaternary conformational changes of allosteric enzymes that alter enzyme activity to regulate metabolism. The control of metabolic pathways by allosteric enzymes is analogous to a molecular valve with "on" and "off" positions. In the "off" position, flow through the pathway is severely hindered, while in the "on" position the flow is normal. For a comprehensive understanding of allosteric regulation we must elucidate in molecular detail how the allosteric signal is transmitted to the active site to alter enzyme activity. In this work we use unnatural amino acid mutagenesis to introduce a fluorescent amino acid into the allosteric binding site of aspartate transcarbamoylase (ATCase), the enzyme responsible for regulation of pyrimidine nucleotide biosynthesis. The fluorescence from the amino acid is exquisitely sensitive to the binding of the allosteric effectors ATP, CTP, UTP, and GTP. In particular we show how the asymmetric nature of the allosteric sites of the enzyme are used to achieve regulatory sensitivity over a broad range of mixed heterotropic effector concentrations as is observed in the cell. Furthermore, employing the method of random sampling - high dimensional model representation (RS-HDMR) we derived a model for how ATCase is regulated when all four nucleotides are present at fluctuating concentrations, consistent with physiological conditions. We've discovered the fundamental requirements to induce the allosteric transition to the R state by showing that although ATCase can accept L-asparagine as an unnatural substrate, the transition to the R allosteric state requires the correct positioning of the alpha-carboxylate of its natural substrate L-aspartate. However, linking the functionalities of L-asparagine and carbamoyl phosphate into a single molecule is sufficient to correctly position the bi-substrate analog in the active site to induce the allosteric transition to the R-state. The cooperative nature of ATCase was further investigated through the isolation of a unique quaternary structure of ATCase consisting of two catalytic trimers linked covalently by disulfide bonds. By relieving the quaternary constraints imposed by the bridging regulatory subunits of the native holoenzyme, the flexibility of the c6 subunit significantly enhanced enzyme activity over the native holoenzyme. Unlike the native c3 catalytic subunit, the c6 species displays homotropic cooperativity for L-aspartate demonstrating that, when two catalytic trimers are linked, a binding event at one or more active sites can be transmitted through the molecule to the other active sites in the absence of regulatory subunits. The catalytic reaction of ATCase follows an ordered sequential mechanism that is complicated by the transition from the T state to the R state upon the binding of the second substrate L-aspartate. Acquiring X-ray crystal structures at each step along the pathway has advanced our understanding of the catalytic mechanism, yet R-state structures are difficult to obtain. Using a mutant version of ATCase locked in the R-allosteric state by disulfide bonds we captured crystallographic images of ATCase in the R state bound to the true substrates (CP and Asp), products (CA and Pi), and in the process of releasing the final product (Pi) prior to reversion of the molecule to the T state. These structures depict the steps in the catalytic cycle immediately before the catalytic reaction occurs, immediately after the reaction, and after the first product has been released from the active site. This work also focuses on developing allosteric inhibitors of the enzyme fructose-1,6-bisphosphatase (FBPase), one of the enzymes responsible for regulation of the gluconeogenesis pathway. Inhibitors of FBPase could serve as potential therapeutic agents against type-2 diabetes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

Chan Kam Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 175-182). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Drosophila as a model for studying development --- p.1 / Chapter 1.2 --- The formation of the body axis in Drosophila --- p.2 / Chapter 1.3 --- The maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.11 / Chapter 1.5 --- The formation of the biocid protein gradient from anterior pole to posterior pole of the embryo --- p.13 / Chapter 1.6 --- The bed protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.15 / Chapter 1.7 --- The formation of the bed protein gradient in embryo --- p.17 / Chapter 1.8 --- Components required for bcd mRNA localization at anterior pole of oocyte --- p.21 / Chapter 1.8.1 --- Cis-acting elements --- p.21 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.9 --- The properties of exuperantia protein --- p.25 / Chapter 1.9.1 --- The function of exu protein --- p.25 / Chapter 1.9.2 --- Exuperantia is a phosphoprotein --- p.26 / Chapter 1.9.3 --- Phosphorylation pattern of exuperantia protein is stage-specific --- p.28 / Chapter 1.9.4 --- Reversible phosphorylation is one of the major mechanisms to control protein activity in all eukaryotic cells --- p.29 / Chapter 1.9.5 --- The relationship between the exu protein phosphorylation and the bcd mRNA localization --- p.30 / Chapter 1.10 --- Aim of project --- p.31 / Chapter CHAPTER 2 --- Preparation of the exuperantia genomic DNA and complement DNA (cDNA) mutant Constructs / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- DNA preparation methods --- p.35 / Chapter 2.2.1.1 --- Preparation of double-stranded DNA by polyethylene glycol6000 --- p.35 / Chapter 2.2.1.2 --- Preparation of M13mp8 single-stranded DNA --- p.37 / Chapter 2.2.1.3 --- "Preparation of double-stranded DNA by Biol prep (Modified from Maniatis et al.,1989)" --- p.38 / Chapter 2.2.2 --- "Preparation of DH5α，JM109, TG1 competent cells" --- p.39 / Chapter 2.2.3 --- Bacteria transformation --- p.40 / Chapter 2.2.4 --- Restriction enzyme digestion --- p.40 / Chapter 2.2.5 --- Phenol/chloroform extraction --- p.41 / Chapter 2.2.6 --- Purification of DNA fragment by electro-elution --- p.42 / Chapter 2.2.7 --- DNA ligation --- p.43 / Chapter 2.2.8 --- DNA dephosphorylation --- p.43 / Chapter 2.2.9 --- In vitro site-directed mutagenesis --- p.44 / Chapter 2.2.9.1 --- The Sculptor´ёØ in vitro mutagenesis --- p.44 / Chapter 2.2.9.2 --- The GeneEditor´ёØ in vitro site-directed mutagenesis --- p.47 / Chapter 2.2.10 --- The double-stranded or single-stranded DNA sequencing by T7 DNA polymerase sequencing system --- p.50 / Chapter 2.2.11 --- Denatured polyacrylamide gel electorphoresis --- p.51 / Chapter 2.2.11 --- Nucleotide sequence of the sequencing primers and the mutageneic oligonucleotides --- p.54 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Design exuperantia mutant constructs --- p.55 / Chapter 2.3.1.1 --- Comparison of exu protein amino acids sequence with different Drosophila species --- p.56 / Chapter 2.3.2 --- The exu genomic mutant constructs --- p.63 / Chapter 2.3.3 --- The exu cDNA mutant constructs --- p.63 / Chapter 2.4 --- Discussion --- p.76 / Chapter CHAPTER 3 --- Epitope tagging of exuperantia protein with c-myc eptiope / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Materials and methods --- p.84 / Chapter 3.2.1 --- Preparation of the c-myc eptiope DNA fragment --- p.84 / Chapter 3.2.2 --- End-filling of 5'overhang DNA fragment by Klenow fragment --- p.86 / Chapter 3.2.3 --- In vitro translation of protein by TNT® Quick coupled transcription and translation system --- p.86 / Chapter 3.2.4 --- Immunoprecipitation of recombinant exu protein --- p.87 / Chapter 3.2.5 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 3.2.5.1 --- SDS-PAGE preparation --- p.88 / Chapter 3.2.5.2 --- SDS-PAGE electrophoresis --- p.90 / Chapter 3.2.6 --- Western blot analysis --- p.90 / Chapter 3.2.6.1 --- Transfer the protein to a nitro-cellulose membrane by semi-dried blotting --- p.90 / Chapter 3.2.6.2 --- Western blot blocking and antibody recognition --- p.91 / Chapter 3.3 --- Results --- p.92 / Chapter 3.3.1 --- Construction of the plasmid containing exu cDNA tagging with a c-myc epitope --- p.92 / Chapter 3.3.2 --- In vitro translation of c-myc epitope tagged exu protein --- p.102 / Chapter 3.3.3 --- Immunoprecipitation of c-myc labeled exu protein by a polyclonal rabbit anti-exu antibody and monoclonal mouse anti-myc antibody --- p.104 / Chapter 3.4 --- Discussion --- p.109 / Chapter CHAPTER 4 --- In vitro phosphorylation of exuperantia Protein / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Materials and methods --- p.113 / Chapter 4.2.1 --- Exogenous kinase phsophorylation reactions --- p.113 / Chapter 4.2.2 --- Separation of the phosphorylated exu protein variants by SDS- PAGE --- p.114 / Chapter 4.3 --- Results --- p.115 / Chapter 4.3.1 --- Western blot analysis of in vitro translated exu protein variants --- p.115 / Chapter 4.3.2 --- Phosphorylation of in vitro translated exu protein variants by exogenous cAMP-dependent protein kinase --- p.118 / Chapter 4.3.3 --- Phosphorylation of in vitro translated exu protein variants by exogenous cGMP-dependent protein kinase --- p.123 / Chapter 4.3.4 --- Phosphorylation of in vitro translated exu protein variants by exogenous protein kinase C --- p.128 / Chapter 4.4 --- Discussion --- p.133 / Chapter CHAPTER 5 --- Introduction of the exuperantia genomic constrcuts into the germline of Drosophila by P element-mediated transformation / Chapter 5.1 --- Introduction --- p.136 / Chapter 5.2 --- Materials and methods --- p.138 / Chapter 5.2.1 --- Construction of a genomic construct for production of transgenic flies --- p.138 / Chapter 5.2.2 --- Preparation of double-stranded DNA by ultra-centrifugation --- p.142 / Chapter 5.2.3 --- P-element mediated transformation --- p.143 / Chapter 5.2.3.1 --- Eggs collection --- p.143 / Chapter 5.2.3.2 --- Dechorionating the eggs --- p.143 / Chapter 5.2.3.3 --- Orientating the eggs --- p.144 / Chapter 5.2.3.4 --- Microinjection --- p.145 / Chapter 5.2.4 --- Collecting virgin female Drosophila --- p.146 / Chapter 5.2.5 --- Setup a crossing experiment --- p.146 / Chapter 5.2.6 --- Preparation of total ovaries and testes extracts exu protein from Female and male Drosophila --- p.147 / Chapter 5.2.7 --- Immunohistochemical distribution of exuperantia protein --- p.147 / Chapter 5.3 --- Results --- p.150 / Chapter 5.3.1 --- Insertion of the mutated exu fragments into the Drosophila Transformation vector (pCaSpeR) --- p.150 / Chapter 5.3.2 --- Introduction of the mutated exu gene into the genome of Drosophila by P-element mediated transformation --- p.153 / Chapter 5.3.3 --- Western blot analysis of the exu protein in the exu (ES2.1) transgenic fly --- p.160 / Chapter 5.3.4 --- Immunohistochemical distribution of exu protein in exuES21 mutants --- p.162 / Chapter 5.3.5 --- Rescue test of exuES2.1 trangenic flies --- p.165 / Chapter 5.4 --- Discussion --- p.168 / Chapter CHAPTER 6 --- General Discussion --- p.171 / References --- p.173 / Chapter Appendix I: --- List of reagents --- p.183 / Chapter Appendix II: --- Publication --- p.187

by Zhong Sheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 186-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.