Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisor: Alan M. Lambowitz. Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 56-59). Also available for download via the World Wide Web; free to University of Oregon users.

The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer for reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base pairing of the intron RNA. Consequently, Ll.LtrB introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, I show that an Escherichia coli gene disruption library obtained using randomly inserted Ll.LtrB introns contains most viable E. coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. I also demonstrate that Ll.LtrB introns can be used for efficient gene targeting in a variety of Gram-negative and positive bacteria, including E. coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Bacillus subtilis, and Staphylococcus aureus. Ll.LtrB introns expressed from a broad-host-range vector or an E. coli-S. aureus shuttle vector yielded targeted disruptions in a variety of test genes in these organisms at frequencies of 1-100% without selection. By using an Ll.LtrB intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, I disrupted the essential gene hsa in S. aureus. Because the splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32oC, but not at 43oC. Finally, I developed high-throughput screens to identify E. coli genes that affect either the splicing or retrohoming of the Ll.LtrB intron. By using these screens, I identified fourteen mutants in a variety of genes that have decreased intron retrohoming efficiencies and additional mutants that have increased intron retrohoming efficiencies, in some cases apparently resulting from increased stability of the intron RNA. / text

The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein), which binds the intron RNA to promote RNA splicing and intron mobility. Mobility occurs by intron RNA reverse splicing directly into a DNA strand and reverse transcription by LtrA. I used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to the cellular poles in both Escherichia coli and L. lactis. This polar localization occurs with or without co-expression of the intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three non-overlapping LtrA subsegments, reflecting dependence on common redundant signals and/or protein physiochemical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. In E. coli, the Ll.LtrB intron inserts preferentially into the chromosomal ori and ter regions, which are pole localized during much of the cell cycle. Thus, the polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in these regions. I established a high throughput method using cellular array and automated fluorescence microscopy for screening transposon-induced mutants, and identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) in which disruptions result in increased proportion of cells having diffuse LtrA distribution. This altered localization is correlated with a more uniform distribution of Ll.LtrB insertion sites throughout the E. coli genome. Finally, I find that altered LtrA localization in all five disruptants is correlated with accumulation and more diffuse intracellular distribution of polyphosphate, and that a ppx disruptant, which also results in polyphosphate accumulation, shows similar LtrA mislocalization. These findings may reflect interaction between LtrA and intracellular polyphosphate. My findings support the hypothesis that the intracellular localization of LtrA is a major determinant of Ll.LtrB insertion site preference in the E. coli genome. Further, they show that alterations in polyphosphate metabolism can lead to protein mislocalization, and suggest that polyphosphate is an important factor affecting intracellular protein localization.

Mobile group II introns are retroelements that site-specifically insert into DNA target sequences. The group II intron mobility pathway is mediated by a ribonucleoprotein particle (RNP) composed of excised intron RNA and an intron-encoded protein (IEP). The intron lariat inserts at a specific DNA target sequence and is then reverse transcribed by the IEP. Both the intron RNA and IEP are required for DNA target site recognition. I have identified the contact sites within the IEP responsible for recognition of two key positions in the DNA target, T+5 and T-23. IEP recognition of T+5 in the 3'-exon is required for endonuclease cleavage of the bottom-strand of the DNA target site, which generates a primer used for initiation of reverse transcription of the intron. The T+5 base is contacted by G498 in the LtrA DNA-binding domain and nearby residues, particularly K499, potentially bolster this interaction. Recognition of T-23 in the distal 5'-exon is required for initial recognition of the DNA target site by the RNP. The T533 side-chain contacts the T-23 base and the L534 side-chain may also contribute to recognition through hydrophobic interactions with the C5 methyl group. A mutant, L534H, that switches target site specificity to T-23G has been characterized. In order for the RNP to make these and other contacts in the 5'- and 3'-exons simultaneously, the DNA must be bent. I have dissected the role of DNA bending in the intron mobility pathway and found that the DNA is bent at two progressively larger angles as the reaction proceeds. The predominant bend angle at earlier time points places the bottom-strand DNA cleavage site at the protein endonuclease active site. The predominant bend angle of later time points places the cleaved DNA site at the RT domain active site for initiation of reverse transcription of intron cDNA. Finally, in a practical application of group II intron mobility, I have used reprogrammed group II introns ("targetrons") to target two genes in Bacillus subtilis to demonstrate the suitability of targetron technology for gene targeting in the Gram-positive Bacillus genus. / text

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 76-84). Also available online in ProQuest, free to University of Oregon users.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [92]-104).