MOLECULAR-GENETICS OF CRYPTOPLEURINE RESISTANCE IN SACCHAROMYCES-CEREVISIAE - EXPRESSION OF A RIBOSOMAL-PROTEIN GENE FAMILY

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subun it protein rp59 and confers sensitivity to the protein synthesis inhib itor cryptopleurine. A yeast strain containing the cry1-DELTA1=URA3 nu ll allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bac teriophage lambda using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 tha t differs at five residues from rp59 encoded by the CRY] gene. The CRY 2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A an d PPL46. Ribosomal protein 59 is an essential protein; upon sporulatio n of a diploid doubly heterozygous for cryl-DELTA2=TRP1 cry2-DELTA1=LE U2 null alleles, no spore clones containing both null alleles were rec overed. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transfo rmation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cryl-DELTA 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2 -DELTA1=LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher l evels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in r esistance to cryptopleurine. Because CRY2 (and cry2) is expressed at l ower levels than CRY1 (and cryl), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cryl-DELTA null allele.