Sequence and intranuclear location of the extrachromosomal rDNA plasmid of the amoebo-flagellate Naegleria gruberi

Abstract

Several lower eukaryotic genomes have distinctive organization of rDNA on extrachromosomal molecules: the rDNAs of the amoebo-flagellate Naegleria gruberi (Heterolobosea) are encoded on an extrachromosomal circular plasmid. Although the presence of a circular rDNA plasmid in N. gruberi has now been accepted, its sequence and intracellular location are still unclear. We have now sequenced the entire 14,128 bp of the extrachromosomal circular rDNA plasmid. It contains a single rRNA gene unit composed of 18S, 5.8S, and 28S rRNA genes, but no tRNA or 5S RNA genes. We predict that there are two open reading frames. The region that flanks the rRNA gene unit is A/T-rich, except for a highly G/C-rich region that is approximately 900 bp upstream of the rRNA genes. Fluorescence in situ hybridization of N. gruberi cells revealed that the rDNA plasmids cluster within the nucleolus, suggesting that they are highly organized for the efficient transcription of rRNAs. The N. gruberi rDNA plasmid has a unique high-order cluster structure that provides both a molecular basis for understanding chromosomal organization in basal eukaryotes, and a vehicle for constructing stable transgenic vectors.