Transformation of ciliates with a circular plasmid derived from an overamplified macronuclear DNA of Stylonychia lemnae

Abstract

Transformation of ciliates was attempted with a circular plasmid containing a chromosomal DNA of the hypotrichous ciliate Stylonychia lemnae by calcium phosphate-mediated transfection. The plasmid, designated Tubingen-3, was constructed from a bacterial plasmid, a gene for neomycin resistance derived from a vector for the cellular slime mold Dictyostelium, and a 1.2 kb macronuclear chromosomal DNA of Stylonychia. Transformants were obtained at a frequency of one per 104 to 105 of input cells in Stylonychia lemnae and at a lower frequency, one per 10 6, in Tetrahymena thermophila. The vector when simply linearized by digestion with restriction enzymes failed to give transformants, whereas when the vector was linearized so as to have short telomeric sequences at both ends, effective transformation was again observed. The Dictyostelium vector, pCERF DRpl4, with a segment of DNA that contained the putative origin of replication was found, unexpectedly, to be effective in transforming Stylonychia cells. The origin of replication in Stylonychia and Dictyostelium might thus be functionally similar.