Identification and preliminary characterization of PCNA gene in the marine phytoplankton Dunaliella tertiolecta and Isochrysis galbana.

Abstract

The gene coding for proliferating cell nuclear antigen (PCNA) was identified in Dunaliella tertiolecta (Chlorophyceae) and Isochrysis galbana (Prymnesiophyceae). Southern blot hybridization using a PstI fragment of rat PCNA gene (pCR-1) as a probe showed that there is apparently a single copy of this gene per haploid genome in both species. On the Northern blot pCR-1 probed a single messenger RNA for each species of a molecular size close to rat PCNA mRNA (1.1 kilobases [kb]). Polymerase chain reaction (PCR) with a set of degenerated primers produced a fragment of about 610 base pairs [bp] from genomic DNA of both species; the PCR products appeared close in size to the amplified from rat PCNA and hybridized to the pCR-1 probe. Further analysis with reverse transcription-PCR (RT-PCR), cloning, and sequencing revealed a complementary DNA of a similar size (616 and 576 bp) that possesses an open reading frame encoding 205 and 192 amino acids, respectively, for Dun (D. tertiolecta) and Iso (I. galbana). Surprisingly, the polypeptides deduced from the two cDNA shared no higher homology to each other (71%) than to animals such as Xenopus (Dun 72%; Iso 73%), rat (Dun 73%; Iso 74%), and human (Dun 73%; Iso 74%), and to higher plants such as soybean (Dun 78%; Iso 72%), Zea mays (Dun 77%; Iso 73%), and rice (Dunn 77%; Iso 72%), although D. tertiolecta has a higher homology (77%) to the Prasinophyceae alga Tetraselmis chiu than does I. galbana (71%). The homology to PCNA in budding and fision yeasts (63% and 53%, respectively) is also lower than to animals and higher plants. It is thus suggested that with regard to PCNA genes, the three algae are as different from each other as they are from higher plants and animals. In a partially synchronized exponential culture of D. tertiolecta grown with a photocycle of 12 h light and 12 h dark, the abundance of the transcript appeared to be low at hours 3 and 9 (hour 0 = the onset of light period), and increased about 2- to 3-fold at hours 15 and 21 (i.e., during the dark period). Western blotting and immunofluorescence analysis on concurrent diel samples showed an over 2-fold increase in PCNA protein abundance (in proportion to total cellular protein) and the percentage of cells labeled by PCNA antibody. A similar trend was found for I. galbana grown under the same conditions. The results suggest that the gene transcription was in pace with PCNA synthesis, which was lower in the light period when G1 phase was dominant and higher in the dark period when S (and probably G2 and early M) phase was dominant, and that the expression of this gene may be regulated at the transcriptional level in these two algae.