Title

Isolation and characterization of proliferating cell nuclear antigen and the small subunit ribosomal RNA genes in dinoflagellates: Insights into dinoflagellate genome evolution

Date of Completion

January 2008

Keywords

Biology, Oceanography|Biology, Bioinformatics

Degree

Ph.D.

Abstract

Dinoflagellates are important protists with many unusual characteristics compared to other eukaryotes. Documented dinoflagellate genomes contain 3-250 pg of DNA, equivalent to about 1-77 folds that of a human genome. This study aimed at gaining insights into the composition and evolutionary history of these enormous genomes. I first established an empirical equation correlating gene content with genome size in eukaryotes, which deviates from that for other life forms, and from the equation estimated that dinoflagellate genomes contain about 61,000-383,000 genes. This high gene content in dinoflagellates is attributed to high copy numbers. To gain a glimpse into dinoflagellate genome evolution, I analyzed two essential genes, proliferating cell nuclear antigen gene (pcna) and the small subunit ribosomal RNA gene (18S rDNA), from representative species of major dinoflagellate lineages. In eukaryotes, pcna typically occurs in 1-2 copies and 18S rDNA in many. Extensive gene sequencing in this study showed that dinoflagellate pcna is highly polymorphic but 18S rDNA is less so. To further quantify pcna and 18S rDNA copy numbers, I developed a real-time PCR assay and found that linear DNA standard is a standard of choice whereas commonly used supercoiled plasmid standard led to dramatic overestimation. With linear DNA standards, unusually high copy numbers were found for both genes: pcna in 4-280 copies, and 18S rDNA on average about 10,000 copies. These results indicate that genomic duplication has occurred repetitively in dinoflagellate evolution. I also observed that dinoflagellate pcna copy number increased with genome size significantly, but the same trend was not found for 18S rDNA. Nevertheless, the linear relationship between pcna CN and genome size suggests that the high gene copy numbers and the large genome sizes in dinoflagellates may be a result of successive whole genome duplications, and the widely varied contemporary dinoflagellate genome sizes might offer snapshots of different times of duplications. Yet, based on my pcna phylogeny, dinoflagellates pcna copy number (hence genome size) variation did not show a phylogenetic trend. Other factors including environmental conditions may contribute to genome size variation but they remain to be identified.^

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