SEQUENCE OF THE MACRONUCLEAR DNA ENCODING LARGE SUBUNIT RIBOSOMAL PROTEIN-29 (L29) IN EUPLOTES-CRASSUS AND CYCLOHEXIMIDE SENSITIVITY

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibit ory concentration (MIC) for cell division in the presence of cyclohexi mide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines, Because site-dir ected mutagenesis of the RPL29 gene encoding the large subunit ribosom al protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc . Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded pe ptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance i n both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organism s. Thus, E. crassus RPL29 is not a convenient source of a selectable m arker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.