A high level of thymine replacement by 5-hydroxymethyluracil in nuclear DNA of the primitive dinoflagellate...

The nuclei of dinoflagellate protists display several distinctive features which make it difficult to assign these organisms as either eukaryotes or prokaryotes. We investigated some physical properties of purified nuclear DNA from the primitive species Prorocentrum micans. Nuclear DNA was separated on a CsCl gradient, into two components, which banded with relative densities of 1.7240 g/cm3 for the main peak and 1.7301 g/cm3 for the heavy shoulder. Thermal denaturation of nuclear DNA displayed a broad profile with a Tm of 71 degrees C. A large discrepancy was thus revealed between the apparent (G + C) content as determined from density (65.4%) and that from Tm (41.7%) while the actual (G + C) content determined by 32P nucleotide chromatography was shown to be 57.1%. The abnormal behaviour of this DNA was due to the presence of an unusual nucleotide which was identified as 5-hydroxymethyluridylate (HOMedUMP) from its chromatographic and U.V. spectral characteristics. It amounted to 13.4% of the total nucleotides and replaced an average of 62.8% of the expected thymidylate (dTMP). Composition analysis of different fractions of the CsCl gradient revealed that the unusual pyrimidine, 5-hydroxymethyluracil, was not uniformly interspersed with thymine in the DNA; the substitution rate increased with the relative density of the DNA. A minor component was also found, tentatively identified as 5-methylcytidylate (MedCMP) from its chromatographic properties, which amounted to less than 0.5 mol percent.