High-throughput genome processing, sophisticated protein sequence analysis, programming, and information management were used to achieve two major advances in the comparative genomics of microbial signal transduction. First, an integrated and flexible bioinformatics platform and the Microbial Signal Transduction database (MiST) were developed, which facilitated the genome-wide analysis of bacterial signal transduction. This platform was used successfully for the high-throughput identification and classification of signal transduction proteins in more than 300 archaeal and bacterial organisms. Second, analysis of information encoded in prokaryotic genomes revealed that the majority of signal transduction systems consist of one-component systems a single protein containing both input and output domains but lacking phosphotransfer domains typical of two-component systems. The prevalence of one-component systems is a paradigm-shifting discovery because two-component systems are currently viewed as the primary mode of signal transduction in prokaryotes. One-component systems are more widely distributed among bacteria and archaea and display a greater diversity of domains than two-component systems. Additionally, in-depth bioinformatic analyses were performed that further characterized the function of two, input, signaling domains. In summary, this systematic, high-throughput delineation of microbial signal transduction is another step forward in our understanding of the genomic basis of life.

The general goal of this project was to use computational biology to understand signal transduction mechanisms in prokaryotes. Its specific focus was to characterize the cytoplasmic domain of methyl-accepting chemotaxis proteins (MCP_CD), a protein domain central to the function of chemotaxis, the most complex signaling network in prokaryotes. Chemotaxis enables cells to sense and respond to multiple external and internal stimuli by actively navigating to an optimal environment. MCP_CD is a central part of this circuit, but its coiled coil structure is difficult to analyze using traditional tools of computational biology. In this project, a new method for analysis of the domain was developed and used to gain insight into its function and evolution. Research advance 1: Characterization of the MCP_CD protein domain. Before this work, MCP_CD was known to have two distinct functional regions: the signaling region that activates the histidine kinase CheA and the methylation region where adaptation enzymes CheB and CheR store information about recent stimuli. The result of this project is classification of ~2000 MCP_CDs into twelve subfamilies. The unique mechanism of evolution of the domain has been clarified and precise boundaries of the adaptation and signaling regions determined. A new functional region, the flexible bundle subdomain, was identified and its contribution to the signaling mechanism elucidated by analysis of conserved sequence features. Conserved and variable sequence features in the adaptation and signaling subdomains led to a better understanding of the evolutionary history of the adaptation mechanism and of alternative higher-order arrangements of receptors within the membrane. Research advance 2: Development of a sensor / kinase correlation algorithm to couple diverse MCP_CD and kinase subfamilies. The receptor diversity discovered in this work is complemented by diversity in the kinases with which they interact. In this work, an algorithm was developed to associate receptor / kinase pairs which facilitated understanding of the function and evolution of chemotaxis. Research advance 3: Development of Cheops, a database of chemotaxis pathways. The Cheops (Chemotaxis operons) database presents the results of the sensor / kinase correlation algorithm and the information about receptor and kinase diversity in an integrated and intuitive way.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 195 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 182-190).

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 326-347). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 88-98.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 103-126. Also available online.

<p>  </p> <p>The phospholipase C (PLC) family of enzymes canonically hydrolyzes the inner plasma membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control cellular function and behavior. The PLCe subfamily is essential for normal cardiovascular function, where it is activated through direct interactions with the RhoA and Rap1A small GTPases, linking lipase activity to the stimulation of G protein-coupled receptors (GPCRs. RhoA activates PLCe at the plasma membrane, whereas Rap1A translocates and activates PLCe at the perinuclear membrane, where phosphatidylintol-4-phosphate (PI4P) is hydrolyzed. The domains of PLCe involved in G protein binding, activation, and translocation to different subcellular membranes are largely unknown. In this work, we use cell-based activity assays, epifluorescence, and confocal microscopy to identify the domains of PLCe involved in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Our preliminary studies demonstrate that the unique N- and C-terminal regulatory domains of PLCe dictate its location within the cell and contribute differently to basal and G protein-dependent activity. These studies will provide needed insights into the regulation and localization of PLCe in cells, which is critical for its roles in cardiovascular function. </p> <p>Lipids on membranes are known to regulate the function of lipases in cells, however their contribution towards this phenomenon is not well understood. It is difficult to explore this mechanism due to the dynamic behavior of lipid bilayers in cells and most importantly the unknown variable of the amount of lipids present locally when lipases are activated. This study utilizes an approach, to use in vitro reconstitution methods to understand the contribution of lipids in activation of PLCb3 which is known to hydrolyze PIP­2 ­in cell to generate IP3 and DAG. We also use single enzyme kinetics using total internal reflection microscopy to understand recruitment of single enzyme to the membrane, its association and disassociation rates at the membrane. </p>

BACKGROUND:The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid.RESULTS:Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background.CONCLUSIONS:These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid.

published_or_final_version / abstract / Anatomy / Master / Master of Philosophy