snoRNA Gene Disruptions

snoRNA disruptions were generated by homologous gene replacement in S. cerevisiae haploid strain yM4585 (Mat a his3$\Delta$200 lys2-801 leu2-3,2-112 trp1-901 tyr1-501 URA3+ ADE2+ CAN^S) and diploid strain yM4587 (Mat a/Mat $\alpha$ his3$\Delta$200/his3$\Delta$200 lys2-801/ lys2-801 leu2-3,2-112/ leu2-3,2-112 trp1-901/ trp1-901 tyr1-501/ tyr1-501 URA3+/ URA3+ ADE2+/ ADE2+ CAN^S/ can^r) kindly provided by M. Johnston. The disruption scheme is as described by Baudin et al. (1993), using a protocol provided by L. Riles. Disruption constructs were generated by the polymerase chain reaction (PCR) using forward and reverse primers with 5' ends containing 41 bp of genomic sequence flanking the predicted snoRNA, plus 19 bp matching a bacterial vector pBM2815 (from M. Johnston) containing the HIS3 marker gene. The resulting constructs contained the HIS3 gene bordered by 41 bp of genomic sequence found just upstream and downstream of the target snoRNA gene. Both haploid and diploid strains were transformed with disruption constructs using a standard LiOAc transformation protocol [Schiestl et al., 1993]. Transformants growing on YPD His- plates were picked and assayed by PCR for correct integration of the HIS3 marker gene replacing the target snoRNA. In several cases, diploid transformants were sporulated to obtain haploid disruption mutants.