Isolation and Characterization of Proliferating Cell Nuclear Antigen from the Dinoflagellate Pfiesteria piscicida

Abstract

Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases delta and epsilon, is essential for DNA replication and repair. Understanding the structure and expression characteristics of this gene in dinoflagellates would enable us to gain insights into how the cell cycle in these enigmatic eukaryotes is regulated and whether this gene can be a growth marker of these ecologically important organisms. We analyzed pcna and its encoded protein from Pfiesteria piscicida (Ppi_PCNA). Using reverse transcription-polymerase chain reaction (RT-PCR) and RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) methods, Ppi_pcna cDNA was isolated; it contained a coding region for 258 amino acid residues (aa) preceded by various 5'- and 3'-untranslated ends. The deduced protein length was similar to that of typical vertebrate and plant PCNA. PCR using genomic DNA as the template yielded multiple products whose sequences revealed multiple copies of pcna in tandem repeats separated by an unknown sequence. Using real-time PCR, we estimated 41+/-7 copies of this gene in each P. piscicida cell. Reverse transcription real-time PCR indicated a similar pcna mRNA level between the exponential and the stationary growth phases. Western blot analysis revealed a slightly higher PCNA level (<2-fold) in the exponential than in the stationary growth phases. We conclude that (1) P. piscicida possesses a typical eukaryote PCNA; (2) unlike in other eukaryotes, pcna in P. piscicida occurs in multiple copies arranged in tandem; and (3) regulation of P. piscicida PCNA probably lies in post-translational modification.