Genome size-dependent pcna gene copy number in dinoflagellates and molecular evidence of retroposition as a major evolutionary mechanism.

Abstract

Proliferating cell nuclear antigen (PCNA) plays critical roles in eukaryotic DNA replication and replication-associated processes. It is typically encoded by one or two gene copies (pcna) in eukaryotic genomes. Recently reported higher copy numbers of pcna in some dinoflagellates raised a question of how this gene has uniquely evolved in this phylum. Through real-time PCR quantification, we found a wide range of pcna copy number (2-287 copies) in 11 dinoflagellate species (n=38), and a strong positive correlation between pcna copy number and genome size (log10 -log10 transformed). Intraspecific pcna diverged up to 21% and are dominated by nonsynonymous substitutions, indicating strong purifying selection pressure on and hence functional necessity of this gene. By surveying pcna copy numbers in eukaryotes, we observed a genome size threshold at 4pg DNA, above which more than two pcna copies are found. To examine whether retrotransposition is a mechanism of pcna duplication, we measured the copy number of retroposed pcna, taking advantage of the 22-nt dinoflagellate-specific spliced leader (DinoSL) capping the 5' end of dinoflagellate nuclear-encoded mRNAs, which would exist in the upstream region of a retroposed gene copy. We found that retroposed pcna copy number increased with total pcna copy number and genome size. These results indicate co-evolution of dinoflagellate pcna copy number with genome size, and retroposition asa major mechanism of pcna duplication in dinoflagellates. Furthermore, we posit that the demand of faithful replication and maintenance of the large dinoflagellate genomes might have favored the preservation of the retroposed pcna as functionalgenes.