MUTATIONAL ANALYSIS OF THE GENE FOR SCHIZOSACCHAROMYCES-POMBE RNASE MRP RNA, MRP1, USING PLASMID SHUFFLE BY COUNTERSELECTION ON CANAVANINE

Reverse genetics in fission yeast is hindered by the lack of a versati le established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene f or the RNA component of RNase MRP, mrp1, we have developed a system fo r plasmid shuffling in fission yeast using counterselection on canavan ine. The system lakes advantage of the ability of the Saccharomyces ce revisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mu tation. Two general use plasmids were constructed that allow direction al cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries a uxotrophic markers for ade6, leu1, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces ces p ombe RNase MRP RNA for their ability to complement a chromosomal delet ion of the mrp1 gene. The degree of background canavanine resistance a s well as plasmid-plasmid recombination encountered in these experimen ts was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmi ds will be widely useful.