Thesis (Ph. D.)--University of Queensland, 2001. / Includes bibliographical references.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas at Arlington, 2008.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2016. Adviser: Dean Sheppard.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2084. Adviser: David P. Toczyski.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 72-80).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

T7 RNA polymerase is a single protein capable of driving transcription from a simple promoter in virtually any context. This has made it a powerful tool in a range of biotechnology applications. In this work, previous efforts to evolve or engineer T7 RNA polymerase are reviewed. This work is then expanded upon, first with the development of a method for the cell-free evolution of T7 RNA polymerase based on the functioning of an autogene. The autogene is a transcriptional feedback circuit in which active T7 RNA polymerase proteins transcribe their own gene, resulting in exponential amplification of their genetic information. While this system is doomed by an error catastrophe, this can be delayed by the use of in vitro compartmentalization. In response to the limits of the autogene, a novel directed evolution approach termed compartmentalized partnered replication (CPR) is presented. CPR couples the in vivo functionality of a gene to its subsequent in vitro amplification by emulsion PCR. The use of CPR to generate a panel of six versions of T7 RNA polymerase, each specific to one of six promoters, is described. Separately, a rational engineering approach, taken to facilitate the high-yield transcription of fully 2′-modified RNA, is detailed. Two sets of mutations to T7 RNA polymerase, previously known to confer thermal stability and enhance promoter clearance respectively, can be used to enhance the activity of existing T7 RNA polymerase mutants that utilize non-standard nucleotides as their substrates. Next, CPR and random mutagenesis is used to populate the functional fitness landscape of T7 RNA polymerase. This neutral drift library is then challenged to increase the processivity of T7 RNA polymerase, enabling long-range transcription. Finally, the lessons that can be learned about T7 RNA polymerase specifically and molecular evolution and protein engineering generally are discussed. / text

<p> <i>Myo-inositol</i> is a six-carbon sugar alcohol. It is essential as a precursor of the phospholipid membrane component phosphatidylinositol (PI) and the phosphoinositide signaling pathway in all eukaryotes. It aids in cellular metabolism, osmoregulation, and plays an important role in fertilization and diseases such as diabetes, bipolar disorder, and Alzheimer&rsquo;s disease. <i> Myo</i>-inositol metabolism is comprised of synthesis, transport, catabolism, and recycling. <i>Myo</i>-inositol synthesis is catalyzed by myo-inositol-3-phosphate synthase (MIPS). Surprisingly, synthesis of <i>myo</i>-inositol and its role in fertilization has not yet been studied in the model organism <span style="text-decoration:overline"> Drosophila melanogaster</span> (fruit fly). We hypothesize that MIPS expression is essential for growth and development of <i>D. melanogaster.</i> In this study, a precise deletion of the entire MIPS gene was generated and confirmed through PCR amplification and sequencing of the resultant DNA fragments. The lack of the MIPS transcript in homozygous MIPS deletion flies was confirmed by RT-PCR. During that experiment, two additional isoforms of MIPS were identified in wild-type flies (CS). Supplementation of chemically defined food with 0.5mM inositol was required to sustain all homozygous MIPS deletion fly strains. Fully-grown homozygous deletion flies could live without additional inositol in the food, but newly emerged larvae only survived to the first instar larval stage. However, even while on rich media supplemented with 170mM inositol, a homozygous MIPS deletion stock was unable to produce viable offspring. Homozygous MIPS deletion strains were identified as male-sterile, incapable of producing offspring when mated to any strain of females (including wild-type). Homozygous female MIPS deletion flies were fertile and maintained a high fecundity rate when mated to any strain (with an exception of homozygous male MIPS deletion flies). The male-sterility was complemented with the addition of a wild-type MIPS gene to chromosome 3. Testes dissections of homozygous male MIPS deletion flies revealed improper progression of spermatogenesis, specifically during sperm individualization. These studies contribute to the understanding of the role of inositol synthesis in growth, development, and fertilization.</p>