HIGH-FREQUENCY VECTOR-MEDIATED TRANSFORMATION AND GENE REPLACEMENT INTETRAHYMENA

Recently, we developed a mass DNA-mediated transformation technique fo r the ciliated protozoan Tetrahymena thermophila that introduces trans forming DNA by electroporation into conjugating cells (1). Other studi es demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macron ucleus (2). We describe the use of conjugant electrotransformation (CE T) for gene replacement and for the development of new independently r eplicating vectors and a gene cassette that can be used as a selectabl e marker in gene knockout experiments. Using CET, the neomycin resista nce gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of th e H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanki ng region of the H4-I locus. Gene replacement was obtained with non-di gested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy o f the Tetrahymena rDNA replication origin was included on the transfor ming plasmid. However, the efficiency of transformation using CET incr eased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promotor function to within 333 bp upstream of the initiator ATG. Similarly similar to 300 bp of sequence downstre am of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I /neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasm id lacking an origin of replication, but did transform at high frequen cy on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a sele ctable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co -express a gene encoding a cycloheximide resistant ribosomal protein.