MRX protects telomeric DNA at uncapped telomeres of budding yeast cdc13-1 mutants

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.     

Isolation of pleiotropic yeast mutants requiring ergosterol for growth

Mutant strains of Saccharomyces cerevisiae which require ergosterol for growth have been isolated. These mutants are all petite and require a fatty acid. Several mutants require methionine in addition. These mutants have been classified into 6 complementation groups. For one of the mutants the enzymatic block has been localized after lanosterol. These mutants do not show a stringent requirement for ergosterol, as sitosterol, stigmasterol or cholesterol also support growth. Mutants of this type will be of value not only in studies of sterol biosynthesis, but also in assessing the biological role of sterols in the cytoplasmic yeast membrane. Similar mutants but without a stringent requirement for a sterol have been previously isolated by Resnick and Mortimer (8).     

Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase

Yeast mutants lacking mitochondrial NAD⁺-specific isocitrate dehydrogenase (idhΔ) or aconitase (aco1Δ) were found to share several growth phenotypes as well as patterns of specific protein expression that differed from the parental strain. These shared properties of idhΔ and aco1Δ strains were eliminated or moderated by co-disruption of the CIT1 gene encoding mitochondrial citrate synthase. Gas chromatography/mass spectrometry analyses indicated a particularly dramatic increase in cellular citrate levels in idhΔ and aco1Δ strains, whereas citrate levels were substantially lower in idhΔcit1Δ and aco1Δcit1Δ strains. Exogenous addition of citrate to parental strain cultures partially recapitulated effects of high endogenous levels of citrate in idhΔ and aco1Δ strains. Finally, effects of elevated cellular citrate in idhΔ and aco1Δ mutant strains were partially alleviated by addition of iron or by an increase in pH of the growth medium, suggesting that detrimental effects of citrate are due to elevated levels of the ionized form of this metabolite.     

Expulsion of live pathogenic yeast by macrophages

Phagocytic cells, such as neutrophils and macrophages, perform a critical role in protecting organisms from infection by engulfing and destroying invading microbes . Although some bacteria and fungi have evolved strategies to survive within a phagocyte after uptake, most of these pathogens must eventually kill the host cell if they are to escape and infect other tissues . However, we now demonstrate that the human fungal pathogen Cryptococcus neoformans is able to escape from within macrophages without killing the host cell by a novel expulsive mechanism. This process occurs in both murine J774 cells and primary human macrophages. It is extremely rapid and yet can occur many hours after phagocytosis of the pathogen. Expulsion occurs independently of the initial route of phagocytic uptake and does not require phagosome maturation . After the expulsive event, both the host macrophage and the expelled C. neoformans appear morphologically normal and continue to proliferate, suggesting that this process may represent an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation .     

Cold-sensitive mutants of p34cdc2 that suppress a mitotic catastrophe phenotype in fission yeast.

Summary The p34^cdc2 protein kinase plays a central role in the regulation of the eukaryotic cell cycle, being required both in late G1 for the commitment to S-phase and in late G2 for the initiation of mitosis. p34^cdc2 also determines the precise timing of entry into mitosis in fission yeast, where a number of gene produts that regulate p34^cdc2 activity have been identified and characterised. To investigate further the mitotic role of p34^cdc2 in this organism we have isolated new cold-sensitive p34^cdc2 mutants. These are defective only in their G2 function and are extragenic suppressors of the lethal premature entry into mitosis brought about by mutating the mitotic inhibitor p107^wee1 and overproducing the mitotic activator p80^cdc25. One of the mutant proteins p34^cdc2-E8 is only functional in the absence of p107^wee1, and all the mutant strains have reduced histone H1 kinase activity in vitro. Each mutant allele has been cloned and sequenced, and the lesions responsible for the cold-sensitive phenotypes identified. All the mutations were found to map to regions that are conserved between the fission yeast p34^cdc2 and functional homologues from higher eukaryotes.     

Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector. The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells. The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization. These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector. We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.     

Human SNM1A suppresses the DNA repair defects of yeast pso2 mutants

Pso2/Snm1 plays a key role in the repair of DNA interstrand cross-links in yeast. Human cells possess three orthologues of Pso2; SNM1A, SNM1B/Apollo and SNM1C/Artemis. Studies using mammalian cells disrupted or depleted for these genes have yielded equivocal evidence that any of these is a true functional homologues of the yeast gene. Here we show that ectopic expression of only one of the three human orthologues, hSNM1A, effectively suppresses the sensitivity of yeast pso2 (snm1) disruptants to cross-linking agents. Two other phenotypes of the pso2 mutants are also partially rescued by ectopic expression of hSNM1A, namely the double-strand repair break defect observed during cross-link processing in pso2 cells, as well as the spontaneous intrachromatid recombination defect of pso2 msh2 double mutants. Finally, we show that recombinant hSNM1A is a 5'-exonuclease, as also recently reported for the yeast Pso2 protein. Together our data suggest that hSnm1A is a functional homologue of yeast Pso2/Snm1.     

Induction of petite yeast mutants by membrane-active agents

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Isolation of ethanol-tolerant mutants of yeast by continuous selection

Summary Mutants of Saccharomyces uvarum, 5D-cyc with increased tolerance to ethanol have been isolated by a continuous selection technique which allows the culture itself to determine the intensity of selection via a feedback control circuit. The output of CO₂ from a continuous culture of the yeast was monitored using an infrared analyser and the signal from that analyser fed to a potentiometric controller which regulated the introduction of a concentrated ethanol solution into the culture vessel. The frequency of ethanol addition to the culture thus increased as the tolerance of the organisms improved.The use of this system permitted the selection of mutants of yeast which were viable in the presence of 12% w/v ethanol and which showed higher fermentation rates (as measured by CO₂ production) than the wild-type in the presence of 10% w/v ethanol and above. The technique of continuous selection with feedback should be generally applicable to the isolation of mutants of any microorganism to improved tolerance to any inhibitory condition of either its physical or chemical environment.     

Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.     

Induction of petite yeast mutants by membrane-active agents.

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Mitotic hyperphosphorylation of the fission yeast SIN scaffold protein cdc11p is regulated by the protein kinase cdc7p

The fission yeast septation initiation network (SIN) triggers the onset of septum formation and cytokinesis. SIN proteins signal from the spindle pole body (SPB), to which they bind in a cell cycle-dependent manner, via the scaffold proteins sid4p and cdc11p. cdc11p becomes hyperphosphorylated during anaphase, when the SIN is active. We have investigated the phosphorylation state of cdc11p during mitosis in various mutant backgrounds. We show that association of cdc11p with the spindle pole body is required for its phosphorylation and that ectopic activation of the SIN results in hyperphosphorylation of cdc11p. We demonstrate that mitotic hyperphosphorylation of cdc11p requires the activity of cdc7p and that its dephosphorylation at the end of mitosis requires PP2A-par1p. Furthermore, spindle checkpoint arrest prevents cdc11p hyperphosphorylation. Finally, we show that the septation inhibitor byr4p interacts preferentially with hypophosphorylated cdc11p. We conclude that cdc11p hyperphosphorylation correlates with activation of the SIN and that this may be mediated primarily by cdc7p in vivo.     

Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis.

Elimination of plasmids from regenerating S. aureus protoplasts occurred when the regeneration medium contained sucrose but not when it contained sodium succinate. This difference was caused by the occurrence of cell division prior to regeneration of the cell wall on sucrose but not on succinate. Coexisting compatible plasmids were cured independently; coexisting incompatible plasmids were cured jointly. These results support the hypothesis that plasmid pools exist as physically sequestered units in protoplasts and that curing is a consequence of the segregation of such units during abnormal division of wall-less organisms.     

Degradation of wild-type vasopressin precursor and pathogenic mutants by the proteasome

Mutations in the gene encoding the antidiuretic hormone arginine vasopressin cause autosomal dominant neurogenic diabetes insipidus. Autoptic data in affected individuals suggest that the neurons expressing mutant vasopressin undergo selective degeneration. Expression studies have shown that the mutants are retained in the endoplasmic reticulum, but how this trafficking defect is linked to neurotoxicity is unknown. One possibility is that unsecreted mutant precursors, or degradation products thereof, are cytotoxic. We therefore investigated the fate of endoplasmic reticulum-retained pathogenic mutants. Our data show that the mutants are retrotranslocated to the cytosol and degraded by the proteasome. In the presence of proteasomal inhibitors, three distinct un- or deglycosylated cytosolic species of vasopressin precursors were stabilized: pre-pro-vasopressin, pro-vasopressin, and an N-terminally truncated form. In addition to the retrotranslocated forms, a fraction of the newly synthesized precursor was not translocated, but was synthesized into the cytosol due to inefficient function of the vasopressin signal peptide. As a result, cytosolic pre-pro-vasopressin and its degradation product were also recovered when wild-type vasopressin was expressed. Cytosolic forms of vasopressin might trigger cytotoxicity in vivo, as has been proposed in the case of prion protein, which also contains an inefficient N-terminal signal peptide.