Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 78-98).

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 145-165). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references (p. 122-142).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 121-135).

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 129-136.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 108-121).

Cytoplasmic dynein is a multisubunit complex involved in retrograde transport of cellular components along microtubules. The heavy chains (HC) are very large catalytic subunits which possess microtubule binding ability. The intermediate chains (IC) are responsible for targeting dynein to its appropriate cargo by interacting with the dynactin complex. The light intermediate chains (LIC) are previously unexplored subunits that have been proposed to modulate dynein activity by regulating the motor or the IC-dynactin interaction. The light chains (LC) are a newly identified class of subunit which are also thought to have regulatory functions. In the first part of this work, I analyzed the relationship between the four SDS-PAGE gel bands that comprise the light intermediate chains. 1- and 2-D electrophoresis before and after alkaline phosphatase treatment revealed that the four bands are derived from two different polypeptides, each of which is phosphorylated. Peptide microsequencing of these subunits yielded sequences that indicated similarity between them. cDNA cloning of the rat LICs revealed the presence of a conserved P-loop sequence and a very high degree of homology between the two different rat LICs and among LICs from different species. The second series of experiments was designed to analyze the association of pericentrin with cytoplasmic dynein. First, various dynein and dynactin subunits were co-associate with pericentrin in these experiments. Co-precipitation from 35S labeled cell extracts revealed a direct interaction between LIC and pericentrin. Comparison of pericentrin binding by LICl and LIC2 showed that only LICl was able to bind. Further investigation of the relationship between LICl and LIC2 demonstrated that each LIC will self-associate, but they will not form heterooligomers. Additionally, using co-overexpression and immunoprecipitation of LICl, LIC2, and HC, I have shown that binding of the two LICs to HC is mutually exclusive. Finally, I investigated the relationships between dynein HC, IC, and LIC by examining the interactions among the subunits. IC and LIC were both found to bind to the HC, but not to each other. Despite the lack of interaction between IC and LIC, they are, in fact, present in the same dynein complexes and they have partially overlapping binding sites within the N-terminal sequence of the HC. The HC dimerization site was determined to extend through a large portion of the N-terminus, and it includes both the IC and LIC binding sites, although these subunits are not required for dimerization. Together these studies implicate the light intermediate chains in dynein targeting. Targeting of dynein to its cargo has been thought to be performed by the dynactin complex, and for one particular cargo, the kinetochore, there is considerable evidence to support this model. The results presented here suggest that the light intermediate chains appear to function in a separate, non-dynactin-based targeting mechanism.

The lcrF gene of Yersinia pestis, the etiological agent of plague, encodes a transcription activator responsible for inducing expression of several virulence-related proteins (Yops) in response to temperature. The mechanism of this thermoregulation was investigated. Using a yopE::lacZ reporter fusion, lcrF-mediated thermal regulation was observed in Y. pestis and Escherichia coli. The lcrF gene was sequenced, the 30.8 kDa. LcrF protein identified and purified, and LcrF-dependent yopE-specific DNA binding activity was detected. A sequence similarity search revealed that LcrF exhibits 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcription activators. During localization studies, a significant proportion of LcrF was found associated with the membrane fraction in E. coli. However, pulse-chase experiments indicated that this result is an artifact of fractionation. lcrF-mediated thermal induction of the yopE::lacZ reporter fusion remains intact in a Shigella flexneri virR mutant. The virR mutation is known to affect thermal induction of Shigellavirulence genes, which are also controlled by an activator in the AraC family. As a first step toward identifying the temperature-sensitive step in the regulation of yop expression, lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. To confirm these results, attempts were made to identify both the native lcrF message in Y. pestis, and a lcrF-lacZ hybrid message in Y. pestis and E. coli. These attempts were unsuccessful. Examination of LcrF protein production revealed temperature-dependent expression in Y. pestis. Surprisingly, high-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at both 26 and 37°C, suggesting that translation rate or message degradation is thermally controlled. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37°C in E. coli indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented.

The Dam-dependent mismatch repair system of Escherichia coli is part of a large network of DNA surveillance and error avoidance systems that identify and repair DNA damage. In this thesis, I have investigated the Dam-dependent mismatch repair system of E. coliand its role in the recognition and repair of DNA substrate molecules containing small insertion/deletion heterologies. This investigation was divided into two parts: the first part utilized genetic techniques to evaluate the specificity of repair and the second part utilized biochemical approaches into the recognition of insertion/deletion heterologies. I have developed a sensitive in vivo transformation system to rapidly evaluate the repair of small insertion/deletion heterologies by Dam-dependent mismatch repair. Heteroduplexes were constructed, for each state of methylation of d(GATC) sequences, by annealing single strand DNA to the linearized complementary strand of duplex DNA. The unmethylated single strand DNA was isolated from f1 phage (R408) propagated on a strain of E. coli containing the dam-16 allele (Chapter 2) to eliminate the possibility of residual Dam-methylation of d(GATC) sequences. Tranformation of E. coli indicator strains with heteroduplexes containing 1, 2, 3, 4 and 5 base insertion/deletion heterologies were scored for repair based on colony color. The results of these experiments show that the Dam-dependent mismatch repair system can recognize and repair 1, 2 and 3 base heterologies as well as repairing G/T mispairs (Chapters 3 and 4). The repair of 4 base heterologies was marginal, while no repair was observed with 5 base heterologies (Chapters 3 and 4). Repair of the 1, 2, 3 and 4 base heterologies proceeded in a Dam-dependent process that required the gene products of mutL, mutS, and I have demonstrated that MutS protein from both Salmonella typhimurium and E. coli can recognize and bind in vitro to the same 1, 2, 3 and 4 base heterologies used for the genetic studies above (Chapters 4 and 5). In fact, MutS protein binds to 1, 2 and 3 base heterologies with greater affinity than it binds to a G/T mismatch. The in vitro observation that MutS does not bind to 5 base heterologies is consistent with the in vivoobservation that 5 base heterologies are not subject to repair. I have also shown that MutS protein specifically binds to 1, 2 and 3 base heterologies since MutS protects about 25 base pairs of DNA flanking the site of the heterology from DNaseI digestion. The results of the genetic and biochemical experiments described in this thesis (and summarized above) serve to re-emphasize the importance of the role that methyl-directed mismatch repair plays in mutation avoidance, and hence in the preservation of genetic integrity.