The MSS51 gene product is required for the translation of the COX1 mRNA in yeast mitochondria

Summary The MSS51 gene product has been previously shown to be involved in the splicing of the mitochondrial pre-mRNA of cytochrome oxidase subunit I (COX1). We show here that it is specifically required for the translation of the COX1 mRNA. Furthermore, the paromomycin-resistance mutation (P _inf454 ^supR ) which affects the 15 S mitoribosomal RNA, interferes, directly or indirectly, with the action of the MSS51 gene product. Possible roles of the MSS51 protein on the excision of COX1 introns are discussed.     

Cloning of the two essential yeast genes, PRP6 and PRP9, and their rapid mapping, disruption and partial sequencing using a linker insertion strategy.

Summary In the yeast Saccharomyces cerevisiae, some thermosensitive (ts) mutants have been shown to be impaired in pre-mRNA splicing (prp mutants). From a yeast genomic library, we have isolated plasmids that complement prp6 or prp9 is mutations. These plasmids also complement the is growth defect of additional independent mutants identified as new prp6 and prp9 is alleles, indicating that the cloned DNAs encode PRP6 and PRP9 genes, respectively. Here, we describe the restriction maps of these loci which are localized on chromosome II and IV, respectively. The limits of open reading frames (ORFs) within the cloned inserts have been determined using a linker insertion strategy combined with the is complementation assay. Double-strand DNA sequencing was also performed directly on the yeast expression vector from the inserted linkers. Gene disruption experiments demonstrate that both genes are essential for viability.     

A novel yeast mutant that is defective in regulation of the Anaphase-Promoting Complex by the spindle damage checkpoint

The accurate segregation of sister chromatids at the metaphase to anaphase transition in Saccharomyces cerevisiae is regulated by the activity of the anaphase-promoting complex or cyclosome (APC/C). In the event of spindle damage or monopolar spindle attachment, the spindle checkpoint is activated and inhibits APC/C activity towards the anaphase inhibitor Pds1p, resulting in a cell cycle arrest at metaphase. We have identified a novel allele of a gene for an APC/C subunit, cdc16-183 , in S. cerevisiae. cdc16-183 mutants arrest at metaphase at 37°C, and are supersensitive to the spindle-damaging agent nocodazole, which activates the spindle checkpoint, at lower temperatures. This supersensitivity to nocodazole cannot be explained by impairment of the spindle checkpoint pathway, as cells respond normally to spindle damage with a stable metaphase arrest and high levels of Pds1p. Despite showing metaphase arrest at G2/M at 37°C, cdc16-183 mutants are able to perform tested G1 functions normally at this temperature. This is the first demonstration that a mutation in a core APC/C subunit can result in a MAD2-dependent arrest at the restrictive temperature. Our results suggest that the cdc16-183 mutant may have a novel APC/C defect(s) that mimics or activates the spindle checkpoint pathway.Communicated by C. P. Hollenberg     

The yeast RNA1 protein, necessary for RNA processing, is homologous to the human ribonuclease/angiogenin inhibitor (RAI)

Summary Mutations in theRNA1 gene ofSaccharomyces cerevisiae, which encodes an essential cytosolic protein, affect the production and processing of all major classes of RNA. The mechanisms underlying these effects are not at all understood. Detailed comparative sequence analyses revealed that the RNA1 protein belongs to a superfamily, the members of which contain repetitive leucine-rich motifs (LRM). Within this superfamily RNA1 is most closely related to the ribonuclease/angiogenin inhibitor (RAI), which is a tightly binding inhibitor of ribonucleolytic activities in mammals. These results not only provide important clues to the structure, function and evolution of the RNAI protein, but also have intriguing implications for possible novel functions of RAI.     

Mak5p,which is required for the maintenance of the M1 dsRNA virus,is encoded by the yeast ORF<Emphasis Type="Italic"> YBR142w</Emphasis> and is involved in the biogenesis of the 60S subunit of the ribosome

In this study, we show that the Saccharomyces cerevisiae ORF YBR142w, which encodes a putative DEAD-box RNA helicase, corresponds to MAK5. The mak5-1 allele is deficient in the maintenance of the M1 dsRNA virus, resulting in a killer minus phenotype. This allele carries two mutations, G218D in the conserved ATPase A-motif and P618S in a non-conserved region. We have separated these mutations and shown that it is the G218D mutation that is responsible for the killer minus phenotype. Mak5p is an essential nucleolar protein; depletion of the protein leads to a reduction in the level of 60S ribosomal subunits, the appearance of half-mer polysomes, and a delay in production of the mature 25S and 5.8S rRNAs. Thus, Mak5p is involved in the biogenesis of 60S ribosomal subunits.Communicated by F. Messenguy     

Lap4, a vacuolar aminopeptidase I, is involved in cadmium-glutathione metabolism

In Saccharomyces cerevisiae, accumulation of cadmium-glutathione complex in cytoplasm inhibits cadmium absorption, glutathione transferase 2 is required for the formation of the complex and the vacuolar gamma-glutamyl transferase participates of the first step of glutathione degradation. Here, we proposed that Lap4, a vacuolar amino peptidase, is involved in glutathione catabolism under cadmium stress. Saccharomyces cerevisiae cells deficient in Lap4 absorbed almost 3-fold as much cadmium as the wild-type strain (wt), probably due to the lower rate of cadmium-glutathione complex synthesis in the cytoplasm. In wt, but not in lap4 strain, the oxidized/reduced GSH ratio and the Gtt activity increased in response to cadmium, confirming that the mutant is deficient in the synthesis of the complex probably because the degradation of vacuolar glutathione is impaired. Thus, under cadmium stress, Lap4 and gamma-glutamyl transferase seem to work together to assure an efficient glutathione turnover stored in the vacuole.     

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation

Summary A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli SecA gene as a probe. The deduced amino acid sequence showed 58% identity to the aminoterminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secA ^ts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.     

Studying nuclear protein import in yeast

The yeast Saccharomyces cerevisiae is a common model organism for biological discovery. It has become popularized primarily because it is biochemically and genetically amenable for many fundamental studies on eukaryotic cells. These features, as well as the development of a number of procedures and reagents for isolating protein complexes, and for following macromolecules in vivo, have also fueled studies on nucleo-cytoplasmic transport in yeast. One limitation of using yeast to study transport has been the absence of a reconstituted in vitro system that yields quantitative data. However, advances in microscopy and data analysis have recently enabled quantitative nuclear import studies, which, when coupled with the significant advantages of yeast, promise to yield new fundamental insights into the mechanisms of nucleo-cytoplasmic transport.     

Donation: a new, facile method of gene replacement in yeast

Summary We describe here a new method for the introduction of non-selectable alleles into Saccharomyces cerevisiae, gene replacement by donation. This method only requires the availability of an autonomously replicating, selectable plasmid containing the allele to be introduced into yeast. The plasmid is digested at a restriction site (or sites) within this allele, and introduced into yeast by transformation. In the course of double-strand break repair, the entering plasmid donates genetic information to the chromosome, replacing the chromosomal allele in a gene conversion-like event. Gene replacement events are identified by a phenotypic screen of the transformants. When necessary, the transforming plasmid may be subsequently lost by segregation during permissive growth. We have studied several parameters affecting the utility of this protocol as a method of gene replacement. Together with our previous results, the results show gene replacement by donation to be a useful, facile method, yielding gene replacement in up to 1.5% of transformants.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

Amplification and loss of repeat units of the human minisatellite MS1 integrated in chromosome III of a haploid yeast strain

Minisatellites comprise arrays of tandemly repeated short DNA sequences which show extensive variation in repeat unit number. The mechanisms that underlie this length variation are not understood. In order to study processes influencing length changes of minisatellites, we integrated the human minisatellite MS1 into a haploid strain of the yeast Saccharomyces cerevisiae. Frequent spontaneous generation of MS1 alleles with new lengths were observed in this yeast strain. Hence it is concluded that recombination between members of a pair of homologous chromosomes is not a prerequisite for the generation of length changes in MS1 in yeast.     

Molecular cloning of Saccharomyces cerevisiae MGC1/YDR473c gene which is essential for cell growth

Saccharomyces cerevisiae haploid cells undergo morphological changes in response to mating pheromones, a- and -factors, during sexual conjugation. As a first step to elucidate the mechanism, I had previously identified the mgc1 mutation which affected the morphogenesis induced by mating pheromones. The mutation had been designated mgc1 for morphogenesis control. In the present study I cloned the MGC1 gene. Sequencing analysis indicates that the MGC1 gene corresponds to the YDR473c gene. The MGC1 gene was shown to be essential for cell growth and required for the transition from the G1 to S phase of cell cycle. Protein-protein interaction of Mgc1 protein was shown by using yeast two-hybrid system. Mgc1 protein was also proposed to be localized in the nucleus in yeast cells.