Characterization of the Euplotes crassus Macronuclear rDNA and its Potential as a DNA Transformation Vehicle

Abstract

We have cloned the macronuclear linear DNA molecule carrying the ribosomal RNA genes from the ciliated protozoan Euplotes crassus. DNA sequence analysis was carried out to locate coding regions and to determine whether sequences that have been mutated to confer antibiotic resistance are conserved in the E. crassus genes. The beginning and end of the primary transcript were mapped. In order to determine whether conserved sequences that might serve as replication origins were present, the 5' and 3' non-coding sequences from E. crassus were compared to the corresponding sequences from the macronuclear linear rDNA molecules from the following euplotid species: Euplotes vannus, Euplotes minuta, Euplotes raikovii and Euplotes rariseta. A DNA transformation construct was made by generating a putative anisomycin resistant mutation along with a mutation generating a restriction site polymorphism. Microinjection of the construct into the developing macronucleus of mated cells resulted in exconjugant cell lines with increased resistance to anisomycin. The injected rDNA with the restriction site polymorphism is detectable in the anisomycin resistant cells and appears to represent a minor fraction of the rDNA.

Origin Usage during Euplotes Ribosomal DNA Amplification

“…Following transfer to nylon membrane, the rDNA molecules were identified by Southern hybridization. The Euplotes rDNA is an ϳ7.6-kb molecule that encodes the 17S, 5.8S, and 26S rRNAs (8). Figure 2A shows its organization and the positions of the probes used in this study, while Fig.…”

“…This is ϳ100-fold higher than the average copy number of other macronuclear DNA molecules (3), so the rDNA replication intermediates should be easier to detect if this differential amplification occurs during macronuclear development. Previous sequence analysis of the nontranscribed regions of the rDNA molecules from different euplotid species did not identify conserved sequences beyond the 50 bp immediately upstream or 30 bp downstream of the rDNA transcription unit (8). Thus, potential replication control elements were not identifiable (8).…”

“…Previous sequence analysis of the nontranscribed regions of the rDNA molecules from different euplotid species did not identify conserved sequences beyond the 50 bp immediately upstream or 30 bp downstream of the rDNA transcription unit (8). Thus, potential replication control elements were not identifiable (8). Since two-dimensional (2D) gels provide a convenient method for identifying sites of replication initiation (6, 11), we used this approach to determine whether the replication origins on Euplotes rDNA molecules are telomeric or internally located and whether the location differs during development and vegetative growth.…”

“…The final period occurs late in macronuclear development at the time when the anlagen start to elongate (34,36). Differential amplification of the individual DNA molecules starts during the third period of replication (7; this paper) and results in copy numbers of 200 to 100,000 molecules per macronucleus (3,7,8).…”

“…We chose to examine the rDNA origins because in Euplotes this molecule is amplified to a copy number of ϳ100,000 molecules per macronucleus (8,26). This is ϳ100-fold higher than the average copy number of other macronuclear DNA molecules (3), so the rDNA replication intermediates should be easier to detect if this differential amplification occurs during macronuclear development.…”