Life Sciences Commons^™

Novel Mechanistic Insight Into Ciliary Regulation: Old Pathways Yield New Mechanisms, Larissa L. Dougherty

Dartmouth College Ph.D Dissertations

Cilia are structures present on most eukaryotic cells which provide important signaling and motile components to cells from early development to fully differentiated and matured cells. Regulation of these structures is critical to proper functioning of the cell and is known to be tied to the cell cycle. Preparation for ciliary assembly following cell cycle exit and ciliary disassembly following cell cycle reentry requires components throughout the cell body and within the cilium to facilitate this process. Here I identify how the cell adapts to ensure modifications to cilia occur for assembly or disassembly using the model organism Chlamydomonas reinhardtii. …

Identification And Characterization Of Novel Transporters Involved In The Co2 Concentrating Mechanism Of Chlamydomonas Reinhardtii, Ananya Mukherjee

LSU Doctoral Dissertations

The green alga Chlamydomonas reinhardtii possesses a CO₂ concentrating mechanism (CCM) which helps in successful acclimation to low CO₂ conditions. One of the main aspects of the CCM is bringing in inorganic carbon (Ci) into the cell as bicarbonate using Ci transporters. Current models of the CCM postulate that a series of ion transporters bring HCO₃^- from outside the cell to the thylakoid lumen where the carbonic anhydrase, CAH3, dehydrates accumulated HCO₃^- to CO₂, raising the CO₂ concentration for Rubisco. Previously, HCO₃^- transporters have been identified at both the …

Making Chlamydomonas Reinhardtii A Better Model Organism: Tackling The Inefficiency Of Nuclear Transgene Expression And Improving Methods For The Generation And Characterization Of Insertional Mutant Libraries, Thomas M. Plucinak

Department of Biochemistry: Dissertations, Theses, and Student Research

The green algal species Chlamydomonas reinhardtii possesses many beneficial features that have made it a useful model organism for many decades. Many types of experimentation however are difficult to conduct with this organism due to the relative under-development of genetic tools available for use. Tasks such as transgene expression, overexpression of proteins of interest (POIs) or site specific genomic modification that are routine in other more facile microbial model organisms such as Escherichia coli and yeast are difficult to accomplish in C. reinhardtii. The second chapter of this thesis describes the development of a novel nuclear transgene expression system …

Transcriptomic Analyses Of The Co2-Concentrating Mechanisms And Development Of Molecular Tools For Chlamydomonas Reinhardtii, Andrew J. Brueggeman

Department of Biochemistry: Dissertations, Theses, and Student Research

Microalgae, such as Chlamydomonas reinhardtii, account for a large percentage of photosynthesis that occurs on the planet. Many algae possess a Carbon-Concentrating Mechanism, or CCM, that actively transports inorganic carbon (C_i) into the cell to create artificially high internal levels of CO₂, enhancing their rate of carbon fixation. The production of biofuels from algal sources can serve as both a renewable and carbon-neutral energy source. This thesis details research in Chlamydomonas, in the effort to both better understand the CCM in algae and improve laboratory and industrial manipulations with algae.

In the first chapter of this …

Phylogenetic Engineering Of The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large Subunit In Chlamydomonas Reinhardtii, Boon Hoe Lim

Department of Biochemistry: Dissertations, Theses, and Student Research

Thirty-four residues in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) may account for the kinetic differences between Rubisco enzyme from green algae and land plants. By substituting these "phylogenetic residues" as groups and combinations of groups in the large subunit of the green alga Chlamydomonas reinhardtii with those of land-plant Rubisco, the functions and relationships of these "phylogenetic groups" were determined.

A phylogenetic-group substitution at the base of catalytic loop 6 of the large subunit decreases the CO₂/O₂ specificity of the enzyme, but function is restored by a further phylogenetic-group substitution at the carboxy-terminal tail. Therefore, these …

Studies On The Small Ubiquitin-Like Modifier (Sumo) E2 Conjugases Of The Sumoylation System In Chlamydomonas Reinhardtii And Their Role In Stress Physiology, Amy R. Knobbe

Department of Biochemistry: Dissertations, Theses, and Student Research

The eukaryotic protein post-translational modification by SUMOylation is involved in a diverse array of cellular processes, including various stress responses. A fully functional SUMOylation system is present in the unicellular green alga Chlamydomonas reinhardtii, and SUMOylation of multiple high molecular weight proteins is induced in response to abiotic stress in this organism. We report here the characterization of a SUMO E2 conjugase deletion mutant in C. reinhardtii, mut5. SUMO E2 conjugase enzymes are responsible for the conjugation of the protein SUMO to a lysine residue within a target protein. C. reinhardtii mutants in which the SUMO E2 …

The Csc Is Required For Complete Radial Spoke Assembly And Wild-Type Ciliary Motility, Erin E. Dymek, Thomas Heuser, Daniela Nicastro, Elizabeth F. Smith

Dartmouth Scholarship

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and …

Photosynthetic Electron Transport In Genetically Altered Photosystem Ii Reaction Centers Of Chloroplasts, Robin A. Roffey, John H. Golebeck, C. Russ Hille, Richard T. Sayre

Department of Biochemistry: Faculty Publications

Using a cotransformation system to identify chloroplast transformants in Chlamydomonas reinhardtii, we converted histidine-195 of the photosystem H reaction center D1 protein to a tyrosine residue. The mutants were characterized by a reduced quantum efficiency for photosynthetic oxygen evolution, which varied in a pH-dependent manner, a reduced capacity to oxidize artificial donors to photosystem II, and P680⁺) reduction kinetics (microsecond) that were essentially similar to wild type. In addition, a dark-stable radical was detected by ESR in mutant photosystem II particles but not in wild-type particles. This radical was similar in g value and lineshape to chlorophyll …