ABSTRACT
Karenia brevis is a toxic dinoflagellate responsible for red tides in the Gulf of Mexico. The molecular mechanisms controlling its cell cycle are important to bloom formation because blooms develop through vegetative cell division. This study identifies a suite of conserved S-phase genes in K. brevis-proliferating cell nuclear antigen (PCNA), ribonucleotide reductase 2, replication factor C, and replication protein A-and characterizes their expression at the mRNA and protein level over the cell cycle. In higher eukaryotes, the expression of these genes is controlled by transcription, activated at S-phase entry by the E2F transcription factor, which ensures their timely availability for DNA synthesis. In the dinoflagellate, these transcripts possess a 5'-transspliced leader sequence, which suggests they may be under post-transcriptional control as demonstrated in trypanosomes. Using quantitative polymerase chain reaction (qPCR), we confirmed that their transcript levels are unchanged over the cell cycle. However, their proteins are maximally expressed during S-phase. This suggests their cell-cycle-dependent expression may be achieved at the level of translation and/or stability. Proliferating cell nuclear antigen further undergoes an increase in size of â¼9 kDa that dominates during S-phase. This coincides with a change in its distribution, with prominent staining of chromatin-bound PCNA occurring during S-phase. We hypothesize that the change in the observed size of PCNA is due to post-translational modification. Together, these studies demonstrate post-transcriptional regulation of S-phase genes in K. brevis. Differential expression of these S-phase proteins may be useful in the development of biomarkers to assess bloom growth status in the field.
