Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus

Abstract

Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5'-non-translated region of the TBP gene of E. crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macronucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E. crassus.