Date of Award

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Fran Van Dolah

Second Advisor

Michael Janech

Third Advisor

Lauren Ball

Fourth Advisor

David Kurtz

Fifth Advisor

Paul J. McDermott

Abstract

The dinoflagellate, Karenia brevis, produces harmful algal blooms in the Gulf of Mexico that cause extensive marine animal mortalities and human illness nearly annually. The molecular mechanisms controlling cell cycle entry in this dinoflagellate are important because bloom development occurs through asexual cell division. The cell cycle of K. brevis is phased to the diel cycle such that any cells entering the cell cycle on a given day do so synchronously. However, a microarray study of gene expression over a diel/cell cycle found no change in cell cycle transcript levels in actively dividing populations, including genes that code for replication fork proteins, typically activated in eukaryotes by transcription at the G1/S transition. There are many examples of post­ transcriptional regulation of physiological processes in dinoflagellates and this suggests that cell cycle progression may also be regulated post-transcriptionally. The studies presented in this dissertation address the question of whether the cell cycle in K. brevis is regulated at the level of translation. We first characterized the global pattern of translation activity over the diel cycle using polysome profiling. We found that, unlike Lingulodinium polyedrum, a dinoflagellate widely used to study diurnal rhythms, translation was not limited to the dark phase. We next used a 2-DE gel proteomic approach to identify diel patterns of global and specific changes in protein expression, with particular interest in cell cycle proteins. Although a number of proteins displayed diel patterns of expression, we were unable to characterize the behavior of cell cycle proteins using this method. We therefore selected four DNA replication fork genes (PCNA, Replication factor C, Replication protein A, and Ribonucleotide reductase 2) that are classically regulated via transcription at the G1/S phase transition, and characterized their expression by qPCR, western blotting and immunolocalization. Sequence analysis revealed a 5'spliced leader sequence on each of these transcripts, indicative that they are likely under post-transcriptional control, and qRT-PCR confirmed that their transcript levels were indeed unchanged over the cell cycle. Sequence analysis and protein modeling were next used to develop peptide antibodies for western blotting to investigate their protein abundance over the cell cycle. The K. brevis replication fork proteins, PCNA, RFC, RPA and RnR2 were shown to change over the cell cycle with highest expression at S-phase, suggesting translational control. Furthermore, a post­ translational modification appeared to occur on the K. brevis PCNA protein in S-phase cells, resulting in a ~9 kDa increase in size. We hypothesize that this represents modification by either SUMO or ubiquitin, two post-translational modifications observed on PCNA in other eukaryotes during S-phase. The occurrence of this post-translational modification coincided with a shift in nuclear location of PCNA from peripheral to chromatin bound, as determined by immunolocalization. Since the expression of replication fork proteins appears to change over the cell cycle without changes in transcription, CDKs were investigated to begin to understand how these drivers of the cell cycle may be controlling S-phase entry at the translational level. Both the CDK4- specific inhibitor, fascaplysin, and the pan-CDK inhibitor, olomoucine, inhibited progression through the G1/S transition as well as the production of the PCNA protein. These results lead us to propose the dinoflagellate utilizes CDK-dependent translational control of cell cycle entry as opposed to transcriptional control which is seen in most eukaryotes.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS