Characterization of the Euplotes crassus macronuclear rDNA and its potential as a DNA transformation vehicle

We have cloned the macronuclear linear DNA molecule carrying the ribosomal RNA genes from the ciliated protozoan Euplotes crassus. DNA sequence analys is was carried out to locate coding regions and to determine whether sequen ces 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' noncoding sequences fro m E. crassus were compared to the corresponding sequences from the macronuc lear linear rDN4 molecules from the following euplotid species: Euplotes va nnus, Euplotes minuta, Euplotes raikovii and Euplotes rariseta. A DNA trans formation 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 c ells resulted in exconjugant cell lines with increased resistance to anisom ycin. The injected rDNA with the restriction site polymorphism is detectabl e in the anisomycin resistant cells and appears to represent a minor fracti on of the rDNA.