Cloning and analysis of the nuclear gene MRP-S9 encoding mitochondrial ribosomal protein S9 of Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear gene MRP-S9 was identified as part of the European effort in sequencing chromosome II. MRP-S9 encodes for a hydrophilic and basic protein of 278 amino acids with a molecular mass of 32 kDa. The C-terminal part (aa 153–278) of the MRP-S9 protein exhibits significant sequence similarity to members of the eubacterial and chloroplast S9 ribosomal-protein family. Cells disrupted in the chromosomal copy of MRP-S9 were unable to respire and displayed a characteristic phenotype of mutants with defects in mitochondrial protein synthesis as indicated by a loss of cytochrome c oxidase activity. Additionally, no activities of the gluconeogenetic enzymes, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, could be observed under conditions of glucose de-repression. The respiration-deficient phenotype could not be restored by transformation of the disruption strain with a wild-type copy of MRP-S9, indicating that MRP-S9 disruption led to rho^- or rho^o cells. Sequence similarities of MRP-S9 to other members of the ribosomal S9-protein family and the phenotype of disrupted cells are consistent with an essential role of MRP-S9 is assembly and/or function of the 30s subunit of yeast mitochondrial ribosomes.     

Yeast ribosomal proteins: XI. Molecular analysis of two genes encoding YL41, an extremely small and basic ribosomal protein,from Saccharomyces cerevisiae

Summary Two genes encoding ribosomal protein YL41 were cloned from Saccharomyces cerevisiae chromosomal DNA. Both genes contain an uniterrupted region of only 75 nucleotides coding for a protein of 3.3 kD. Within the coding regions the nucleotide sequences are virtually identical, whereas in both the 5-and 3-flanking regions the two genes differ significantly from each other. The deduced protein shows an arginine and lysine content of 68 percent, i.e., 17 out of 25 residues, and the basic residues are evenly distributed over the molecule. When compared to the ribosomal protein sequences currently available no counterpart to YL41 could be found in prokaryotes and it seems likely that YL41 is a eukaryotespecific ribosomal protein.     

Genetic mapping of two pairs of linked ribosomal protein genes in Saccharomyces cerevisiae

Summary We have used the 2 mapping method described by Falco and Botstein (1983) and tetrad analysis to map four ribosomal protein genes (two linked pairs) in S. cerevisiae. One pair (rp28–rp55 copy 1) is on chromosome XV, 14 cM proximal to ARG8. The other pair (rp55–rp28 copy 2) is 19 cM from the centromere on the left arm of chromosome XIV. To map copy 1 we used the E. coli -galactosidase gene rather than a yeast gene to mark the ribosomal protein chromosomal locus. This provided a more sensitive color screening assay for chromosome loss in the 2 method. It also removed the restriction that the mapping tester strains must be mutant for the plasmid marker.     

Characterization of the nuclear gene encoding chloroplast ribosomal protein S13 from Arabidopsis thaliana

We have characterised a cDNA clone and a nuclear gene encoding the chloroplast 30 s ribosomal protein S13 from Arabidopsis thaliana. The identification is based on the high similarity of the predicted amino-acid sequence with eubacterial S13 protein sequences, and immunodetection of a 14.5-kDa chloroplast ribosomal polypeptide using antibodies raised against the polypeptide produced from part of the cDNA expressed in bacteria. The predicted amino-acid sequence contains an N-terminal extension which has several features characteristic of chloroplast transit peptides. Experiments suggest there is a single copy of this gene in A. thaliana and multiple copies in Brassica species. The origin of the mitochondrial S13 polypeptide in crucifers is also discussed.     

Isolation,sequencing, and characterization of crp-5, a gene encoding a Neurospora ribosomal protein

A Neurospora crassa cytoplasmic ribosomal protein gene, crp-5, has been isolated and characterized. The cDNA was isolated by a differential screening of a cDNA library for glucose-inducible genes. The cDNA was subsequently used to identify and isolate crp-5 genomic sequences. Computer analysis of the DNA sequences showed that they contain an open reading frame which encodes a protein homologous to the rat ribosomal protein S26. The crp-5 mRNA levels are regulated in a carbon-source-dependent manner. The organization of the gene and the region upstream of the coding sequences are discussed.     

Two group I mitochondrial introns in the cob-box and coxI genes require the same MRS1/PET157 nuclear gene product for splicing

Summary We have studied the role of the product of the nuclear gene PET157 in mitochondrial pre-mRNA splicing. Cytoduction experiments show that a mitochondrial genome deleted for the three introns bI3, aI5 and aI6 is able to suppress the pet157-1 mutation: the strain recovers respiratory competency indicating that the product of the PET157 gene is only required for mitochondrial premRNA splicing. Characterization of the high molecular weight pre-mRNAs which accumulate in the pet157 mutant demonstrate that the product of the PET157 gene is required for the excision of two group I introns bI3 and aI6 (corresponding to aI5) located in the cob-box and coxI genes respectively. Furthermore, the pet157 mutant strain accumulates the bI3 maturase in the form of a polypeptide of 50K (p50) previously observed in mitochondrial mutants defective in the excision of bI3. We have shown by restriction analysis and allelism tests that the pet157-1 mutation is allelic to the nuclear mrs1 mutation, previously described as specifically blocking the excision of bI3. Finally, revertants obtained by the deletion of bI3 or aI6 from the mitochondrial DNA were isolated from the MRS1 disrupted allele, confirming the involvment of the product of the MRS1/PET157 gene in the excision of the two introns bI3 and aI6.     

The yeast ORF YDL202w codes for the mitochondrial ribosomal protein YmL11

The Saccharomyces cerevisiae open reading frame YDL202w has been characterised in the course of the EUROFAN yeast genome analysis program. Disruption of YDL202w causes a respiratory deficient phenotype accompanied by a loss of mitochondrial DNA. This phenotype is usually found in mutants defective in mitochondrial replication or gene expression. YDL202w has the potential to encode a soluble protein of 249 amino acids. It shows significant similarities to the ribosomal protein L10 from various bacteria and to a previously determined amino-terminal peptide sequence of the yeast mitochondrial ribosomal protein L11. The predicted amino-acid sequence of YDL202w starts with a stretch which has neither any correspondence in the bacterial sequences nor in the protein isolated from mitochondrial ribosomes. Furthermore, this stretch matches the requirements for a signal sequence for mitochondrial protein import. A mitochondrial location of the YDL202w gene product was proven by use of a carboxy terminally HA-tagged version. These findings clearly indicate that YDL202w encodes this mitochondrial ribosomal protein (YmL11). Received: 13 January 1997     

Altered ribosomal protein L29 in a cycloheximide-resistant strain of Saccharomyces cerevisiae

Summary A spontaneous high-level cycloheximide-resistant mutant of the yeast Saccharomyces cerevisiae (strain cy32) is found to have an altered protein of the large subunit (60S) of cytoplasmic ribosomes, namely protein L29. The resistance character segregates together with this biochemical defect and is semidominant in heterozygous diploids. Judged from in vitro susceptibility to inhibition by cycloheximide there are at least 50% resistant ribosomes present in such diploid strains. From these results it is concluded that cycloheximide resistance of mutant cy32 is caused by mutation of a single gene and that it is the structural gene for L29 which is affected.Preliminary genetic mapping data are also reported. They indicate a location of cyhx-32 marker on chromosome 7 near met13.Some of the data delt with in this paper were reported on the IX. Steenbock Symposium on Ribosomes, July 5th to 8th, 1979, Madison (Wisc.), U.S.A.     

Cloning and characterization of MRP10, a yeast gene coding for a mitochondrial ribosomal protein

The nuclear gene MRP10 of Saccharomyces cerevisiae was cloned by complementation of a respiratory deficient mutant N518/L1. This mutant is defective in mitochondrial translation and shows a tendency to accumulate deletions in mitochondrial DNA (ρ ^–). Analysis revealed Mrp10p to be a component of the 37 S subunit of the mitochondrial ribosomes. Disruption of MRP10 in a haploid strain of yeast elicits a phenotype identical to that of the original mutant. The respiratory defect of the null mutant is rescued by re-introducing the MRP10 gene in a wild-type mitochondrial DNA background. These results indicate that Mrp10p belongs to the class of yeast mitochondrial ribosomal proteins that are essential for translation. Searches of current databases failed to reveal any homologs among known bacterial or eucaryotic cytoplasmic ribosomal proteins. Some sequence similarity, however, was detected between Mrp10p and Yml37p, previously identified as a component of the yeast mitochondrial 50 S ribosomal subunit. Received: 21 November 1996     

Disruption of the MRP-L23 gene encoding the mitochondrial ribosomal protein L23 is lethal for Kluyveromyces lactis but not for Saccharomyces cerevisiae

The Kluyveromyces lactis nuclear gene, MRP-L23, encodes a polypeptide of 155 amino acids that shares 70% and 43% identity to the ribosomal proteins L23 and L13 of Saccharomyces cerevisiae and Escherichia coli. The deduced protein, designated KlL23, is a likely component of the large subunit of mitochondrial ribosomes as it can complement the respiratory deficient phenotype of a S. cerevisiae mrp-L23 mutant. As in S. cerevisiae, KlMRP-L23 is essential for respiratory growth of K. lactis because disruption of the gene in a “petite-positive” strain carrying a ρ^o-lethality suppressor atp mutation rendered cells unable to grow on a non-fermentable carbon source. However, in contrast to S. cerevisiae, disruption of MRP-L23 in wild type K. lactis is lethal. Meiotic segregants of K. lactis with a disrupted MRP-L23 allele form microcolonies with cell numbers varying from 32 to 300. These data clearly indicate an essential role of mitochondrial protein synthesis for viability of the petite-negative yeast K. lactis. Received: 2 September / 20 October 1999     

A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division

Summary The coding region of a yeast calmodulin gene was fused to a galactose-inducible GAL1 promoter, and a conditional-lethal mutant of Saccharomyces cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, in which the promoter was repressed, it ceased growing after 12–15 h. The growth arrest was associated with a decrease in intracellular calmodulin levels: after 12h, no intracellular calmodulin protein was detectable. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. Thus, the defect was mainly in nuclear division. Bud growth was partially inhibited in these cells: 27% of the cells stopped growing with a small bud. Furthermore, calmodulin-deficient cells showed elevated rates of chromosome loss, possibly as the result of a defect in the precise segregation of chromosomes.     

Cloning and identification of a DNA fragment coding for the sup1 gene of Saccharomyces cerevisiae

Summary A plasmid, pYsup1-1, containing a DNA fragment able to suppress the recessive mutant phenotype of the suppressor locus sup1 (allele sup1-ts36) of Saccharomyces cerevisiae was isolated from a bank of yeast chromosomal DNA cloned in cosmid p3030. The complementing gene was localized on a 2.6 kb DNA fragment by further subcloning. Evidence is presented that the cloned DNA segment codes for the sup1 structural gene (chromosome IIR).