Extra-chromosomal inheritance of rhodamine 6G resistance in Saccharomyces cerevisiae

Molecular Genetics and Genomics - Rhodamine 6G was found to be a specific inhibitor of aerobic growth of yeast, having no effect on fermentative growth. A single step spontaneous mutant of S....     

Nuclear inheritance of resistance to antimycin A in Saccharomyces cerevisiae.

Summary A group of 30 independent mutants of Saccharomyces cerevisiae, resistant to the respiratory inhibitor antimycin A, was investigated from a genetical and biochemical point of view. All the mutants can be grouped into two nuclear loci: AMY1 maps on the VII chromosome, between leu 1 and trp 5; AMY2 is close to its centromere on either chromosome XVIII or XIX. Both genes do not affect mitochondrial structures or functions.     

Orchestrating organelle inheritance in Saccharomyces cerevisiae

The biochemical functions of eukaryotic cells are often compartmentalized into membrane-bound organelles to increase their overall efficiency. Although some organelles can be formed anew, cells have evolved elaborate mechanisms to ensure the faithful inheritance of their organelles. In contrast to cells that divide by fission, the budding yeast Saccharomyces cerevisiae must actively and vectorially deliver half of its organelles to the growing bud. To achieve this, proteins called formins are strategically localized to the bud, where they assemble an array of actin cables that radiate deep into the mother cell. Class V myosin motors use these cables as tracks to transport various organelles, including peroxisomes, a portion of the vacuole and elements of the endoplasmic reticulum and Golgi complex. By contrast, mitochondria do not engage a myosin motor for their movement but instead use Arp2/3-nucleated actin polymerization for their bud-directed motility. The translocation machineries work cooperatively with molecular devices that retain organelles within both mother cell and bud to ensure an equitable division of organelles between them. While organelle inheritance requires specific proteins tailored for the inheritance of each type of organelle, it is becoming apparent that a set of fundamental rules underlies the inheritance of all organelles.     

Mitochondrial DNA inheritance in Saccharomyces cerevisiae

Respiratory metabolism depends on mitochondrial DNA, yet the mechanisms that ensure the inheritance of the mitochondrial genome are largely obscure. Recent studies with Saccharomyces cerevisiae suggest that distinct factors mediate the active segregation of mitochondrial DNA during mitotic growth. The identification of the proteins required for the maintenance of the mitochondrial genome provides clues to the mechanisms of, and molecular machinery involved in, mitochondrial DNA inheritance.     

Extrachromosomal inheritance of paromomycin resistance in Saccharomyces cerevisiae. Genetic and biochemical characterization of mutants

Summary In Saccharomyces cerevisiae, mutants were isolated which show high resistance to the aminoglycoside paromomycin. Amino acid incorporation of mitochondria isolated from such mutant strains proved also to be paromomycin resistant. All of them are cross-resistant to the structurally related antibiotic neomycin. Three independent methods revealed the resistance to be extrachromosomally, presumably mitochondrially inherited.     

A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae.

The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.     

Expression of cryptopleurine resistance in Saccharomyces cerevisiae.

An examination of gene expression in diploids may not always be sufficient for determination of the dominant or recessive character of an allele. In Saccharomyces cerevisiae resistance to cryptopleurine has been attributed to a single recessive nuclear gene, cryl, located on chromosome III. We found, contrary to expectations, that resistance to cryptopleurine is not expressed in diploids that are monosomic for chromosome III. Examination of strains of different ploidy on gradient plates shows that the presence of the sensitive allele in a cell does not affect the level of resistance, but rather the level of resistance is directly related to the ratio of resistant alleles to the number of chromosome sets.     

In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae

Vacuole inheritance is temporally coordinated with the cell cycle and is restricted spatially to an axis between the maternal vacuole and the bud. The new bud vacuole is founded by a stream of vacuole-derived membranous vesicles and tubules which are transported from the mother cell into the bud to form the daughter organelle. We now report in vitro formation of vacuole-derived tubules and vesicles. In semi-intact cells, formation of tubulovesicular structures requires ATP and the proteins encoded by VAC1 and VAC2, two genes which are required for vacuole inheritance in vivo. Isolation of vacuoles from cell lysates before in vitro incubation reveals that formation of tubulovesicular structures requires cytosol as well as ATP. After forming tubulovesicular structures, isolated vacuoles subsequently increase in size. Biochemical assays reveal that this increase results from vacuole to vacuole fusion, leading to mixing of organellar contents. Intervacuolar fusion is sensitive to the phosphatase inhibitors microcystin-LR and okadaic acid, suggesting that protein phosphorylation/dephosphorylation reactions play a role in this event.     

Rapid Evolution of Recombinant Saccharomyces cerevisiae for Xylose Fermentation through Formation of Extra-chromosomal Circular DNA

Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure.     

Transport of 6-deoxyglucose in Saccharomyces cerevisiae.

The uptake of 6-deoxyglucose was measured in wild-type Saccharomyces cerevisiae, in a double mutant strain lacking activity for hexokinases A and B (hxkl hxk2), in a triple mutant strain lacking activity for both hexokinases and glucokinase (hxkl hxk2 glk), and in the triple mutant with high levels of activity of single kinases restored by introduction of the cloned genes. In the wild-type strain, uptake of the glucose analog showed two components, with Km values of ca. 20 mM ("high affinity") and 250 mM ("low affinity"), respectively. The double mutant also had high- and low-affinity components, but the triple mutant showed only low-affinity uptake. Reintroduction of the single kinases to the triple mutant restored high-affinity uptake. (Other experiments on 6-deoxyglucose uptake are also presented, including the apparent use of the galactose transport system when induced.) These results show that the recent implication of the kinases in transport of glucose (L.F. Bisson and D.G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983) applies equally to the nonmetabolized analog 6-deoxyglucose and suggests that the role of the kinases in transport is not merely a consequence of metabolism of the transported compound.     

Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a segregation structure) into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one low molecular weight activity (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.     

Caffeine resistance of Saccharomyces cerevisiae

Four caffeine-resistant haploid isolates, two resistant to 50 mM caffeine and two resistant to 100 mM caffeine, were genetically analyzed. Complementation and tetrad analysis indicated that all four mutations are alleles of the same locus. All four isolates demonstrated incomplete dominance when hybridized to the wild-type strain and dominance of high to low resistance when hybridized to one another. Differences in caffeine resistance were found between wild-type grande cells and its petite derivative.     

Fredericamycin A affects mitochondrial inheritance and morphology in Saccharomyces cerevisiae

Fredericamycin A (FMA) is an antibiotic product of Streptomyces griseus that exhibits modest antitumor activity in vivo and in vitro, but, its functions in vivo are poorly understood. We identified this compound as an inducer of G1 arrest in the yeast, Saccharomyces cerevisiae. FMA exhibits an IC50 of 24 nM towards the growth of a disruptant of multi-drug resistance genes, W303-MLC30, and its cytotoxicity is a function of the time of exposure as well as drug dose. Addition of 0.8 microM of FMA caused aggregation of mitochondria within 10 min of incubation and the drug induced petites at high frequency after 4 h of incubation. Rho(-) cells were about 20 times more resistant to FMA than isogenic rho(+) cells. Overexpression of topoisomerase I, a previously suggested target of the drug, did not alleviate the sensitivity of the cells to FMA or the aggregation of mitochondria. Our results suggest that mitochondria are the primary target site of FMA.