Molecular cloning and analysis of the nuclear gene MRP-L6 coding for a putative mitochondrial ribosomal protein from Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear gene MRP-L6 was cloned by complementation of the respiratory-deficient mutant pet-ts 2523 with a library of wildtype yeast genomic DNA. The isolated gene was part of a 3.8-kb sequenced DNA fragment containing, in addition to MRP-L6, two unassigned reading frames, ORF1 and ORF2. MRP-L6 codes for a basic protein of 205 amino acids and a molecular mass of 22.8 kDa. The protein exhibits significant sequence similarity to the ribosomal protein L6 of bacteria and chloroplasts. Unlike the corresponding bacterial proteins, however, the MRP-L6 protein (MRP-L6p) contains at its N-terminus a 16 amino-acid leader sequence exhibiting the known characteristics of mitochondrial import signals. Disruption of MRP-L6 leads to the phenotype of a mitochondrial translation-defective, rho-negative yeast mutant. The results are consistent with MRP-L6p representing an essential component of yeast mitochondrial ribosomes. Expression of MRP-L6 was examined, under conditions of glucose repression and derepression, in wild-type cells and in a series of catabolite repression-defective yeast mutants. In most cases, a distinct though small influence of the carbon source on the expression of an MRP-L6/lacZ reporter construct was observed.     

A novel mutation occurring in the PHO80 gene suppresses the PHO4 c mutations of Saccharomyces cerevisiae

Summary We have isolated suppressors of a PHO4 ^c (a positive regulator) mutant which normally confers weak constitutivity for acid phosphatase production on the Saccharomyces cell. One dominant suppressor (PHO80-2) was found to be an allele of PHO80 (a negative regulator) that changes G to A, resulting in substitution of isoleucine for methionine 42 of the Pho80 protein. Substitution of valine (PHO80-3) or leucine (PHO80-4) for the same methionine by site-directed mutagenesis also suppressed PHO ^c. Suppression by PHO80-2) did show some allele specificity. From these results we were able to delimit the region of PHo80 which may interact with the Pho4 protein.     

Effects of the kar gene on cytoplasmic mixing and mitochondrial genome suppressiveness,and consequences for cytoduction of petite DNA in Saccharomyces cerevisiae

Summary During a series of cytoduction experiments to transfer Saccharomyces cerevisiae mitochondrial genomes from one nuclear background to another, using the karl-1 nuclear fusion mutation, one of the five petite genomes used proved difficult to transfer. This genome, ^- F13, was highly suppressive (90%) in its original nuclear background. Molecular and genetic studies on the putative karl-1 ^–F13 cytoductant were done to discover the nature of this difficulty. They showed that while the ^–F13 was maintained in a karl-l background, zygotes from a mating with a ⁰ strain showed poor cytoplasmic mixing and therefore inefficient ^–F 13 DNA transfer into first zygotic buds. This also caused a reduction of ^–F13 suppressiveness to 20–30% in crosses with different ⁺ strains. The effect was genome specific since another highly suppressive petite in the karl-l background did not show suppressiveness reduction when crossed to ⁺. The nature of suppressiveness modulation is discussed. Since the ^–F13 genome was eventually transferred using a modification of the original scheme, the problems were not caused by the inability of the acceptor nuclear background to maintain the ^–F13 genome.     

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae

Summary A pattern of active accumulation of trehalose during growth on glucose medium, TAC(+) phenotype, is controlled by a polymeric series of maltose fermentation (MAL) genes. An essential requirement for expression of the TAC(+) phenotype is that the MAL gene be in the constitutive state, MAL ^c. Mutation of a constitutive MAL allele to a maltose- inducible or nonfermenting (mal) state, alters the pattern of trehalose metabolism so that little or no trehalose accumulation occurs during growth on glucose medium. The TAC(+) phenotype is obtained in MAL ^c strains whether or not -glucosidase formation is sensitive or resistant to carbon catabolite repression. However, trehalose accumulation is sensitive to glucose levels even in MAL ^c strains in which -glucosidase formation is insensitive to catabolite repression. The effects of constitutive MAL genes on trehalose accumulation cannot be accounted for by an increase in trehalose-6 phosphate synthase or a decrease in trehalase as determined in vitro. A mechanism is proposed in which the gene-product of a MAL gene serves as a common positive regulator for expression of four genes coding respectively for maltose permease, maltase, -methylglucosidase and a component of the trehalose accumulation system.Paper I appeared in Cell. and Molec. Biology 25: 345–354, 1979     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

The regulation of mitochondrial DNA levels in Saccharomyces cerevisiae

Summary Mitochondrial DNA (mtDNA) synthesis can continue under conditions which block cell division and nuclear DNA (nDNA) synthesis, producing cells with several times the normal level of mtDNA. We have examined mtDNA synthesis in cultures recovering from such cell cycle blocks. Our results show that the rate of mtDNA synthesis is not affected either during a block of the cell cycle with -factor or during recovery from a perturbation in the amount of mtDNA/cell induced by blocking the cell cycle with -factor or cdc4. The normal mtDNA content was restored a period of several generations when permissive conditions were restored. These results suggest that mtDNA synthesis is coupled to cell growth.     

An α-specific gene,SAG1 is required for sexual agglutination in Saccharomyces cerevisiae

Summary Seven -specific mutants specifically defective in sexual agglutinability were isolated. The other mating functions exhibited by these mutants, designated sag mutants, such as the production of pheromone and response to a mating pheromone, were normal. While the MAT sag1 cells did not agglutinate with wild-type a cells, the MAT sag1 cells did, indicating that the SAG1 gene is expressed only in cells. The mutations were semi-dominant and fell into a single complementation group, SAG1, which was mapped near met3 on chromosome X. Complementation analysis showed that sag1 and aga1, the latter being a previously reported -specific mutation, were mutations in the same gene.     

The MAL63 gene of Saccharomyces encodes a cysteine-zinc finger protein

Summary Inducible maltose fermentation by tSaccharomyces carlesbergensis requires the product of the MAL63 gene of the MAL6 locus. It has been suggested that this gene product is an activator protein controlling the expression of the structural genes encoding the maltose fermentative enzymes perhaps by binding to DNA sequences upstream of these genes. We report the sequence of the MAL63 gene. A single open reading frame is seen capable of encoding a protein of 470 amino acid residues. The deduced sequence of this protein indicates that it is a cysteine-zinc forger protein supporting the hypothesis that the MAL63 gene product is a DNA binding protein.     

Identification and characterization of CEN12 in the budding yeast Saccharomyces cerevisiae

In this paper we report the cloning, sequencing and functional characterization of CEN12 and an associated autonomously replicating sequence (ARS) from the budding yeast Saccharomyces cerevisiae. In the course of studying a dynamin-related gene, DNM1, we previously physically mapped the gene to chromosome 12. Genetic mapping showed that the gene was tightly linked (0.35 cM) to the centromere. Subcloning experiments revealed that a centromerelike activity was included in a small segment of DNA immediately downstream from the DNM1 gene. Mitotic centromere activity was discerned by the ability of the region to de-stabilize a centromere-containing plasmid, and to stabilize an ARS-containing plasmid. Meiotic centromere activity was determined by the first-division segregation in crosses of ARS plasmids containing this region. The DNA sequence of this region revealed a sequence with strong homology to the consensus for yeast centromeres.