Life Sciences Commons^™

Structural Studies Of Membrane-Assembled Popd And Popb, The Pseudomonas Aeruginosa Type 3 Secretion Translocators, Fabian B Romano Chernac

Doctoral Dissertations 1896 - February 2014

Transport of proteins across membranes is essential during many stages of pathogen infection and colonization of human cells. Many Gram-negative pathogens use a Type 3 Secretion (T3S) system to inject proteins into the target cell during infection. Substantial genetic and biochemical evidence suggest that proteins are translocated across the host plasma membrane through a proteinaceous pore or translocon formed by two bacterial secreted proteins: the T3S translocators. Despite its key role in pathogenesis, virtually nothing is known about the assembly mechanism, structure, and composition of this critical transmembrane complex.

To this end, a cell-free system for the structural and functional …

Transposable Elements In Phytopathogenic Verticillium Spp.: Insights Into Genome Evolution And Inter- And Intra-Specific Diversification, Stefan G. Amyotte, Xiaoping Tan, Kayla Pennerman, Maria Del Mar Jimenez-Gasco, Steven J. Klosterman, Li-Jun Ma, Katherine F. Dobinson, Paola Veronese

Li-Jun Ma

Background Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. Whole genome comparative analysis of the recently sequenced strains VdLs.17 and VaMs.102 revealed that non-random insertions of transposable elements (TEs) have contributed to the generation of four lineage-specific (LS) regions in VdLs.17. Results We present here a detailed analysis of Class I retrotransposons and Class II “cut-and-paste” DNA elements detected in the sequenced …

Identification And Expression Analysis Of Methyl Jasmonate Responsive Ests In Paclitaxel Producing Taxus Cuspidata Suspension Culture Cells, Sangram K. Lenka, Nadia Boutaoui, Bibin Paulose, Kham Vongpaseuth, Jennifer Normanly, Susan C. Roberts, Elsbeth L. Walker

Jennifer Normanly

Background Taxol® (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation …

Foldeco: A Model For Proteostasis In E. Coli, Evan T. Powers, David L. Powers, Lila Gierasch

Lila Gierasch

To gain insight into the interplay of processes and species that maintain a correctly folded, functional proteome, we have developed a computational model called FoldEco. FoldEco models the cellular proteostasis network of the E. coli cytoplasm, including protein synthesis, degradation, aggregation, chaperone systems, and the folding characteristics of protein clients. We focused on E. coli because much of the needed input information—including mechanisms, rate parameters, and equilibrium coefficients—is available, largely from in vitro experiments; however, FoldEco will shed light on proteostasis in other organisms. FoldEco can generate hypotheses to guide the design of new experiments. Hypothesis generation leads to system-wide …

Purification And Characterization Of Novel Nucleases From A Thermophilic Fungus, Kyle S. Landry

Masters Theses 1911 - February 2014

A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 ^oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined …

Proteolytic Regulation Of Ctra, The Master Regulator Of Cell Cycle In Caulobacter Crescentus, Amber M. Cantin

Masters Theses 1911 - February 2014

Cell cycle progression in Caulobacter crescentus depends on the master regulator, CtrA. During the transition from swarmer to stalk cell (G1 to S phase), CtrA is degraded by the AAA+ protease ClpXP and levels rise again in the predivisional stage. The focus of this work is to explore how cyclic, regulated degradation is controlled. CtrA is known to bind to the origin of replication, thereby suppressing replication, so we first asked if DNA binding had an effect on CtrA stability. CtrA is readily degraded by ClpXP on its own, but when bound to DNA containing the proper binding sites, degradation …

Contribution Of Taxane Biosynthetic Pathway Gene Expression To Observed Variability In Paclitaxel Accumulation In Taxus Suspension Cultures, Rohan A. Patil, Martin E. Kolewe, Jennifer Normanly, Elsbeth L. Walker, Susan C. Roberts

Jennifer Normanly

Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In this work, cultures with different aggregation profiles were established to create predictable differences in paclitaxel accumulation upon MeJA elicitation. Expression of known paclitaxel biosynthetic genes in MeJA-elicited cultures exhibiting both substantial (15-fold) and moderate (2-fold) differences …