Localization and Copy Number of the Protein-Coding Genes Actin, α-Tubulin, and HSP90 in the Nucleus of a Primitive Dinoflagellate, Oxyrrhis marina

Chromosomes of the dinoflagellate Oxyrrhis marina are composed of thin parallel filaments running longitudinally and lack the arched structure common to the dinochromosome. The physicochemical and molecular organization of these chromosomes, including the localization and arrangement of genes, is still unknown. We investigated the locations of three protein-coding genes, actin (AF482402), alpha-tubulin (AF482403), and HSP90 (AY391258), on these chromosomes using fluorescence in-situ hybridization (FISH). Primers for these three genes were designated according to known partial sequences. PCR products amplified from total DNA were labeled with digoxigenin (DIG) by random priming and used as probes. After in-situ hybridization, DIG signals were amplified and visualized with anti-DIG-FITC. The number of signals was 3 +/- 1.3 (n=90) for actin, 4.1 +/- 1.4 (n=70) for alpha-tubulin, and 5.5 +/- 1.7 (n=80) for HSP90. This study is the first to locate protein-coding genes in the nucleus of a dinoflagellate, although the chromosomes were greatly damaged during the FISH process. The copy number of each gene per cell was estimated using real time PCR. Resulting copy numbers of actin, alpha-tubulin and HSP90 were, 33.7, 10.4 and 5.4, respectively.