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Thesis (Ph. D.)--University of Texas at Austin, 2005. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 191-208). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 136-144). Available also in a digital version from Dissertation Abstracts.

Diss. (sammanfattning) Stockholm : Univ., 2004. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 95-109).

No description available.

The role of the T7-induced exonuclease (gene 6) in recombination was studied using both molecular and genetic techniques. In the molecular method the fate of parental DNA during parent-to-progeny recombination was examined. A comparison of infections with T7⁺, T7am6 (amber gene 6), or T7ts6 (temperature sensitive gene 6) under permissive and nonpermissive conditions was made. CsCl density gradient analysis of replicative DNA indicated that the T7 exonuclease is necessary for recombination to occur, i.e., in the absence of the exonuclease the parental DNA replicated continuously as a hybrid molecule and did not recombine. Analysis of denatured replicative DNA by CsCl density gradient centrifugation indicated that the exonuclease also may be needed for a limited amount of covalent repair of recombinants. Further confirmation of the essential role which the exonuclease plays in recombination came from genetic analysis. The T7 exonuclease was shown to be necessary for intragenic and intergenic recombination in several areas of the T7 genetic map; genetic recombination frequencies were found to be decreased from 3 to 18-fold under conditions nonpermissive for the exonuclease. The role of the T7-induced endonuclease (gene 3) in molecular recombination was studied by examining the fate of parental DNA during parent-to-progeny recombination using a shear technique. The T7 endonuclease was found to be necessary for the dispersion of parental DNA in the newly replicated DNA. Concatemers synthesized by either T7⁺ or T7am3 (amber gene 3) phage containing the newly replicated DNA were sheared to the size of mature phage DNA and also to quarter size molecules. In the presence of gene 3 protein, parental DNA and newly replicated DNA were interspersed, i.e., the 32P-label from the sheared DNA was found to sediment at the density of recombined DNA. In the absence of gene 3 protein, the parental strand of each sheared DNA molecule was usually found intact, i.e., the ³²P-label from the sheared DNA was found to sediment at the density of hybrid DNA. These results support the previous genetic data (52, 83) that the gene 3 protein is essential for T7 recombination. The role of T7 recombination enzymes in the formation of concatemers was studied by examining selected gene 3 and gene 6 mutants. Results of sucrose gradient analysis showed that DNA concatemers were formed when both the T7 exonuclease (gene 6) and the T7 endonuclease (gene 3) were absent. Further results showed that concatemers cannot be maintained in the absence of the exonuclease unless the endonuclease was eliminated. In a T7am6 infection DNA concatemers formed early were prematurely broken down and accumulated as fragments smaller than mature size phage DNA. In a T7am3am6 (amber in both genes 3 and 6) infection concatemers accumulated and were not matured. These results indicate that concatemers are formed by a process other than normal phage recombination. However, selective defects in the recombination system do interfere with the stability of concatemers. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Toxoplasma infection is a serious threat to immunocompromised individuals such as AIDS patients and organ transplant recipients. Side effects associated with current drug treatment calls for identification of new drug targets. DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen species produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host immune response. Most of the oxidative DNA damage is repaired through the base excision repair (BER) pathway, of which, the apurinic /apyrimidinic (AP) endonucleases are the rate limiting enzymes. Toxoplasma possesses two different AP endonucleases. The first, TgAPE, is a magnesium-dependent homologue of the human APE1 (hAPE1), but considerably divergent from hAPE1. The second, TgAPN, is a magnesium-independent homologue of yeast (Saccharomyces cerevisiae) APN1 and is not present in mammals. We have expressed and purified recombinant versions of TgAPE and TgAPN in E. coli and shown AP endonuclease activity. Our data shows that TgAPN is the more abundant AP endonuclease and confers protection against a DNA damaging agent when over-expressed in Toxoplasma tachyzoites. We also generated TgAPN knockdown Toxoplasma tachyzoites to establish that TgAPN is important for parasite protection against DNA damage. We have also identified pharmacological inhibitors of TgAPN in a high-throughput screen. The lead compound inhibits Toxoplasma replication at concentrations that do not have overt toxicity to the host cells. The importance of TgAPN in parasite physiology and the fact that humans lack APN1 makes TgAPN a promising candidate for drug development to treat toxoplasmosis.

Thesis (M.S.)--University of Texas at El Paso, 2007. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.