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 versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leul, 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 pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid-plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.
Paluh
J L
JL
Department of Cell and Molecular Biology, University of California, Berkeley 94720-3200, USA.
Clayton
D A
DA
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002172:Canavanine / Q000494:pharmacology; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis / Q000379:methods*; D004352:Drug Resistance, Microbial; D004722:Endoribonucleases / Q000235:genetics*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics*; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics*
D012331:RNA, Fungal; D002172:Canavanine; D004722:Endoribonucleases; C058330:mitochondrial RNA-processing endoribonuclease