Gene Expression Profiling of Flagellar Length Control in Chlamydomonas Reinhardtii

• Other Authors: Chamberlain, Kara Lista (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Biology
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-0158

Complex organelles, such as cilia and flagella, play an integral part in how a cell interacts with the environment. The model system Chlamydomonas reinhardtii is a biflagellate alga that uses its flagella for swimming and sensing the environment. C. reinhardtii monitor and respond to environmental cues through changes in cellular morphology and gene expression. I used gene expression profiling to characterize the global response of stimulus-induced changes in flagellar morphology and gene expression in C. reinhardtii. Previous investigations have demonstrated coordination between changes in flagellar morphology with changes in gene expression. When C. reinhardtii encounters various stimuli the cell responds through changes in flagellar length, such as assembly, disassembly, and elongation. I created a custom microarray assay to examine the transcript regulation associated with these changes in flagellar length. Using this technique, approximately 1000 genes were evaluated in a single experiment to create a global gene expression profile. While the transcriptional regulation of flagellar assembly is well characterized, that of flagellar disassembly and elongation is less understood. Using high throughput microarrays, I analyzed the changes in gene expression associated with flagellar assembly, disassembly and elongation. Microarrays were probed with fluorescently labeled cDNAs synthesized from RNA extracted from cells before and during stimulation. Evaluation of the gene expression profiles identified ~100 clones showing at least a 2-fold change in expression during changes in flagellar length. Products of these genes are associated not only with flagellar structure and motility but also with other cellular responses including signal transduction and metabolism. Expression of specific genes from each category was further characterized with high resolution quantitative real-time PCR (qRT-PCR). Comparison of the gene expression profiles demonstrated coordinate expression of cell motility component genes during flagellar assembly and disassembly; whereas comparison of the assembly and elongation expression profiles demonstrated that these morphological changes are very different responses at a transcriptional level. Furthermore, transcript regulation of genes involved in other cellular activities revealed a new and uncharacterized whole cell response to stimulation. This analysis lays the groundwork for a more comprehensive understanding of the cellular and molecular networks regulating flagellar length changes. === A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Degree Awarded: Fall Semester, 2007. === Date of Defense: November 1, 2007. === Microarray, Gene Expression, Cilia, Chlamydomonas Reinhardtii, Flagella, Real-Time PCR === Includes bibliographical references. === Laura R. Keller, Professor Directing Dissertation; Cathy W. Levenson, Outside Committee Member; Hank W. Bass, Committee Member; Lloyd M. Epstein, Committee Member; Thomas A. Houpt, Committee Member.