Stable transfection of the hypotrichous ciliate Stylonychia lemnae with tagged alpha 1 tubulin minichromosomes

Macronuclei of Stylonychia lemnae contain millions of gene sized minichromo somes. These minichromosomes in general harbour only one gene. Their nontra nscribed regions are usually less than 400 bp long and most likely carry th e signals for DNA replication and transcription. Therefore, S. lemnae seems to be an ideal object to study these processes in a protist organism. By m icroinjecting defined linear minichromosomal DNA directly into macronuclei of vegetative cells, we have developed a suitable in vivo assay system for S. lemnae. Transfections were carried out with al tubulin minichromosomes w hose gene was internally tagged by a 19 bp-long insertion. Although the end ogenous al tubulin minichromosome is present in approximately 100,000 copie s per cell, clonal cell lines were generated which stably contained tagged minichromosomes in up to a fivefold excess of endogenous copy numbers. As d emonstrated by Southern blot analysis, the copy number of endogenous al tub ulin minichromosomes was not reduced in these cell lines, and transfected m inichromosomes were not noticeably rearranged by the cells. Moreover, tagge d al tubulin genes were unequivocally expressed. Transcription of these gen es was initiated correctly, and the amount of transcript present in steady- state RNA correlated with gene copy number.