No description available.

Summary in Chinese. / Thesis (M.S.)--Chinese University of Hong Kong. / Bibliography: l. 84-95.

In the past few years, in vivo recombination technologies have emerged to improve the efficiency and simplicity of genetic engineering in Escherichia coli, Salmonella enterica serovar, and other gram-negative bacteria. Phage λ Red homologous recombination system is used to mediate the accurate replacement of target DNA with PCR-generated ?targeting cassettes? that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Salmonella is far lower than that in Escherichia coli. In this study, I firstly improved the recombineering-based strategy by using linear DNA targeting cassettes that contain long flanking ?arms? of sequence (ca. 1,000 base pairs) homologous to the chromosomal target. This reliable and efficient method enables multiple gene targeting procedures to be performed on a single Salmonella enterica serovar typhi Ty21a (Ty21a) chromosome in a straightforward, sequential manner with high efficiency. Secondly, I applied this improved strategy in construction of Salmonella to be live attenuated oral vaccine and tumor targeting vector. In the first part of this thesis, I describe an improved method in Ty21a. Using this strategy, I inserted three different influenza antigen expression cassettes as well as a green fluorescent protein reporter gene into four different loci on the Ty21a chromosome with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. The immune response of this vaccine was also evaluated by ELISA and ELIspots. In the second part of this thesisi, I use this improved recombineering strategy to engineer bacteria of Salmonella typhimurium (S. typhimurium) as therapeutic agents against solid tumor. In current study, a major challenge for bacterial therapy of cancer is avoiding damage to normal tissues. Consequently the virulence of bacteria must be adequately attenuated for therapeutic use. An alternative approach was developed here. By placing an essential gene under a hypoxia conditioned promoter, S. typhimurium strain SL7207 was engineered to generate strain YB1 that survives only in anaerobic conditions without otherwise affecting its functions. In breast and liver tumor bearing mice models, YB1 grew within tumor, retarding its growth, while being rapidly eliminated from normal tissues. Mice treated with SL7207 were killed by infection within short period time. Inhibition of tumor growth by YB1 was significant and was enhanced by the addition of 5-FU in breast cancer model. The development of an “obligate” anaerobic Salmonella provides a much safer bacterial vector for further development of anti-tumor therapies without compromising the other functions or tumor fitness of the bacterium as attenuation methods normally do. In summary, I have developed an efficient, robust and versatile method in genome-wide Salmonella genetic manipulation. Furthermore, I used this method to construct a recombinant Ty21a antigen-expressing vaccine strain and a tumor targeting YB1 strain. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

Recently, hydrogen peroxide (H₂O₂), and its free-radical product the hydroxyl radical (OH·), have been identified as major sources of DNA damage in living organisms. We examined DNA repair of hydrogen peroxide damage, using a standard bacteriophage T4 test system in which several different types of repair could be determined. Post-replication recombinational repair and denV-dependent excision repair had little or no effect on H₂O₂ damage. Also, an enzyme important in repair of H₂O₂-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H₂O₂ damage to the phage. However, multiplicity reactivation, a form of recombinational repair between multiply infecting phage genomes, was found to repair H₂O₂ damages efficiently. The RAD52 gene of Saccharomyces cerevisiae and genes 46 and 47 of bacteriophage T4 are essential for most recombination and recombinational repair in their respective organisms. The RAD52 gene was introduced into expression vectors which were used to transform E. coli. RAD52 expression was induced, and its ability to complement either gene 46 or gene 47 phage mutants was determined with respect to phage growth, recombination, and recombinational repair. RAD52 gene expression allowed growth of gene 46 and gene 47 mutants under otherwise restrictive conditions, as measured by plaque formation and burst size. The RAD52 gene also restored the ability of gene 46 and gene 47 mutants to undergo recombination of rII markers. Furthermore, the RAD52 gene restored recombinational repair after UV irradiation of gene 46 and gene 47 mutants. The published DNA sequence of RAD52 was compared with the published sequences of genes 46 and 47. Although overall homologies were only marginally significant, RAD52 and gene 46 had substantial sequence similarity over a limited region. These results indicate that the recombinational repair pathway found in phage T4 may be ubiquitous for DNA damage caused by endogenous exidative reactions. Furthermore, they indicated that an essential element of the recombination mechanism in both procaryotic viruses and eucaryotes arose from a common ancestor. Procaryotes and eucaryotes are thought to have diverged at least one billion years ago. Thus, recombination apparently arose early in evolution.

No description available.

φC31 integrase (Int) and other site-specific recombinases enable controlled and precise genetic manipulations of complex genomes. Int mediates integration of the φC31 genome into the genome of its Streptomyces host. Recombination occurs between specific attachment sites; attB and attP. Int binds attP and attB with similar affinities, despite significant sequence differences. The mechanism through which Int recognises its substrates is not fully understood. To study DNA binding in vivo in the absence of recombination, we employed the challenge phage assay. In this assay, binding by Int to attP or attB results in a high frequency of P22-1000 lysogen formation in Salmonella. When Int has lost binding activity, fewer lysogens are generated. A randomly mutated integrase library has been screened using this assay. A number of the mutants showed a reduction in binding to both attB and attP or just to attB. Point mutations in these integrases largely clustered either a putative zinc finger or to the pfam07508 ‘recombinase' domain. To validate the phage challenge assay data, the binding defective Int mutants were purified and tested in in vitro DNA binding experiments. Int mutants displayed reduced binding to attB and/or attP compared to attL or attR. The purified proteins were used in in vitro recombination assays. Mutants in the recombinase domain generally showed reduced integration whilst demonstrating almost wild type gp3 dependant excision. These data combined with data from others suggested two DNA binding domains in Int; the recombinases domain and the zinc finger. A truncated mutant Int, IntV371SUGA had previously been shown to bind DNA with low affinity. The mutations in the recombinase domain were transferred to IntV371SUGA to test their effect on DNA binding. I suggest that the recombinase motif is intimately involved in DNA recognition and discrimination between the att sites required for phage integratation and excision.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Arthur Landy. Includes bibliographical references.

Thesis (Ph. D.)--York University, 1999. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 116-143). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ39313.