Disorder of the repair of DNA double-stranded breaks in radiosensitive mutants of Saccharomyces cerevisiae yeasts

DNA double-strand break repair and restoration of viability in X-irradiated diploid yeast cells homozygous for rad50, rad51, rad52, rad55 mutations were studies under conditions of keeping the cells in non-nutrient medium, after irradiation. All the cells were synchronized at the G1 stage of the cell cycle. In contrast to the wild-type yeast, this group of mutants are unable to repair DNA double-strand breaks and do not enhance viability, when kept in non-nutrient medium after irradiation.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

Inhibition of the mixed lymphocyte reaction by fractionated niridazole urine dialysate.

Urine dialysate from rats treated orally with 25 mg/Kg 3H-labeled niridazole was fractionated by DEAE-Sepharose column chromatography and was found to contain three radioactive metabolites and no parent compound. When human niridazole urine dialysate (NUD) was fractionated under identical conditions, fractions corresponding to the three rat NUD metabolites were found to inhibit the human one-way MLR. No inhibition was obtained with fractionated control urine dialysate. It was concluded that nonimmunosuppressive niridazole is metabolized by rats and man to produce three active compounds with the ability to suppress the in vitro response to alloantigens.     

Macrophages and resistance to tumors

Summary Delayed-type hypersensitivity (DTH) reactions in mouse feet were depressed by irradiation and by treatment with carrageenan, niridazole, or reserpine. Specific resistance of immunized mice to footpad challenge with a syngeneic methylcholanthrene-induced fibrosarcoma was also depressed by irradiation, carrageenan, niridazole, and reserpine. Growth of the tumor in the feet of normal mice was unaffected by irradiation or niridazole. It could be inhibited or enhanced by carrageenan treatment, depending on the dose of tumor cells injected. Paradoxically, treatment with reserpine inhibited tumor growth in the feet of nonimmune mice. It is suggested that: (a) specific, acquired resistance to this tumor is strongly akin to DTH; (b) mice offer some natural resistance to this tumor; (c) the establishment of an isograft of this tumor may depend on the occurrence of some degree of inflammation.This work was carried out pursuant to Research Contract NO1-CB-63973 with the U.S. National Cancer Institute. It was supported in part by a grant from the Australian National Health and Medical Research Council     

The fate of yeast mitochondrial DNA after ultraviolet irradiation. I. Degradation during post-UV dark liquid holding in non-nutrient medium.

A partial recovery of ultraviolet (U.V., 254 nm) induced petite mutation (?^?) is observed in exponential phase yeast. This process requires a period of dark holding (LH) in non-nutrient medium followed by growth in nutrient medium. At intervals during LH prelabelled DNA was examined by equilibrium cesium chloride gradients. The gradual decrease in ?^? was accompanied by an ongoing degradation of mitochondrial DNA (mitDNA) during the first 24 hours followed by a stabilization. The dose response for mitDNA degradation was biphasic. No new synthesis of mitDNA occured during LH. MitDNA remaiting after degradation showed a) slight shift to a heavier buoyant density indicating a possible selective degradation of A-T regions b) no difference in size when compared to non-irradiated samples. The first step in the recovery of the ?^? mutation is mitDNA degradation followed by other events taking place when growth resumes.     

Immunosuppressive properties of sera and urine dialysates from kidney-graft recipients treated with azathioprine,prednisolone, and niridazole

Niridazole, an antischistosomal agent, was given to renal transplant recipients in addition to azathioprine and prednisolone, as there is experimental evidence that this combination of drugs is highly immunosuppressive. Sera obtained from kidney-graft recipients during the first two weeks after transplantation were examined for their ability to inhibit the one-way mixed lymphocyte reaction (MLR). Sera from seven patients receiving azathioprine, prednisolone, and niridazole (triple-drug treatment), five patients receiving azathioprine and prednisolone, and two other patients treated with niridazole alone for schistosomiasis produced MLR inhibition by comparison with pretreatment (control) sera.A mean of 78% inhibition was observed with sera taken after one day''s treatment with the three-drug combination, whereas this level of in-vitro immunosuppression occurred only after eight days of treatment with azathioprine and prednisolone. Niridazole alone produced an effect similar to azathioprine and prednisolone. Concentrated dialysate of urine from a patient receiving triple-drug treatment not only inhibited the MLR but also significantly prolonged the survival of heterotopic heart allografts in rats, whereas dialysate from the same patient after niridazole had been stopped gave less MLR inhibition and failed to prolong heart allograft survival.Since niridazole thus increased the in-vitro and in-vivo immunosuppressive action of azathioprine and prednisolone, we suggest that this triple-drug combination might be useful for preventing early acute kidney graft rejection.     

Purification of Phosphomannanase and Its Action on the Yeast Cell Wall

An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Expression of fungal phytase on the cell surface ofSaccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast,Saccharomyces cerevisiae by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) ofAspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast α-agglutinin, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced intoS. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.     

Genetically controlled self-aggregation of cell-surface-engineered yeast responding to glucose concentration

We constructed an arming (cell-surface-engineered) yeast displaying two types of agglutinin (modified a-agglutinin and alpha-agglutinin) on the cell surface, with agglutination being independent of both mating type and pheromones. The modified a-agglutinin was artificially prepared by the fusion of the genes encoding Aga1p and Aga2p. The modified a-agglutinin could induce agglutination of cells displaying Agalpha1p (alpha-agglutinin). The upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL), active at a low glucose concentration, was used as the promoter to express the modified a-agglutinin- and alpha-agglutinin-encoding genes. The arming yeast displaying both agglutinins agglutinated and sedimented in response to decreased glucose concentration. When the glucose concentration was high, the arming yeast grew normally. In the late log phase, when the glucose concentration became very low, agglutination occurred suddenly and drastically and yeast cells sedimented completely. Sedimentation was confirmed by weighing the aggregated cells after filtration of the broth. Strains in which aggregation can be genetically controlled can be used in industrial processes in which the separation of yeast cells from the supernatant is necessary.     

Co-expression of human protein disulfide isomerase (hPDI) enhances secretion of bovine follicle-stimulating hormone (bFSH) in Pichia pastoris

Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a superovulation treatment for cows. To increase the production of recombinant bFSH, human protein disulfide isomerase (hPDI) was expressed simultaneously in engineered Pichia strains. The secretion characteristics of bFSH with or without hPDI were examined. The co-expression of bFSH and hPDI is increased to 1.56 mg/l of heterodimer in the culture medium, which is 6-fold higher when compared with the control strain carrying the bFSH gene only. These results may be generally applicable to increase the expression of other glycoprotein hormones in yeast.     

Influence of pH and length of post-treatment incubation on bleomycin-induced DNA damage

The dependence of the extent of DNA damage by anticancer bleomycin on pH and length of post-treatment incubation was studied in yeast. Bleomycin was always removed from cells after 20-min exposures, and cells were washed prior to incubation in non-nutrient buffer. Following exposures of late stationary-phase cells to the very low dose of only 3 micrograms/ml, 1.5 h incubation in non-nutrient buffer, pH 5, had hardly any effect on profiles derived from alkaline sucrose gradient sedimentation of nucleic acids released from spheroplasts. In contrast, after incubation of cells for 1.5 h in buffer, pH 7, DNA was all low molecular weight. Thus, even after extensive washing of cells, pH strongly influences the drug's action on DNA. At pH 5, washed cells were increasingly susceptible to DNA damage up to 26 h in non-nutrient buffer.     

A comparison of liquid-holding recovery and photoreactivation in halophilic and non-halophilic bacteria

The ability of the extreme halophile Halobacterium cutirubrum to recover from the effects of ultraviolet radiation during liquid holding in the dark in non-nutrient medium has been compared with that of (i) a moderately halophilic bacterium (NRC 41227) and (ii) Escherichia coli B. The photoreactivabilities of all three bacteria have also been studied. The extreme halophile was incapable of liquid-holding recovery in these conditions, in marked contrast to both E. coli B and the moderate halophile, and also failed to recover when held in nutrient medium in the dark. These results strongly support the hypothesis that H. cutirubrum lacks DNA excision repair. It was also found that ultraviolet-irradiated H. cutirubrum could be almost completely photoreactivated from any level of survival in the range 10(-4)-80%, provided exposure to visible light was not delayed, whereas the moderate halophile resembled E. coli B and had a comparatively limited capacity for photoreactivation.     

Mutagenicity of styrene on metabolizing D7 strain of saccharomyces cerevisiae

The level of cyt.p-450 in the D7 strain of the yeast S.cerevisiae depended on the substrate supporting the growth, on its concentration, on the starting inoculated number of cells. (1) In the yeast grown on D-mannose where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. It was detected a maximal concentration during the logarithmic phase when in the cultures there are about 50 . 10(6) cell/ml. We use cells harvested at this moment of the growth for mutagenesis tests. The tested substances were dimethylnitrosamine and styrene. DMNA to probe the sensibility of our cells and styrene that has always given contrasting results but from which the formation is known of genetically active metabolite: styrene oxide(6-7). Styrene gave positive results with our metabolizing yeast cells.     

Efficient generation of recessive traits in diploid sake yeast by targeted gene disruption and loss of heterozygosity

Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.     

Identification and purification of immunosuppressive activity in the urine of rats and a human patient treated with niridazole.

Administration of the antischistosomal compound niridazole to mice, guinea pigs, and humans results in the suppression of several manifestations of cell-mediated immunity. Sera from animals treated with niridazole blocked the in vitro production of migration inhibitory factor (MIF) while niridazole itself was inactive, suggesting that these effects are caused by water soluble mediators. We now report that crude extracts prepared from the urine of rats and a patient receiving nirdazole, but not from pretreatment control urine, similarly suppress antigen-induced inhibition of migration of peritoneal exudate cells from sensitized guinea pigs. With immunosuppressive activity monitored by the direct MIF assay, combined solvent extraction and chromatographic techniques were used to fractionate immunosuppressive activity from the urine of niridazole-treated rats and the patient; the most active fractions, purified about 100-to 1000-fold as compared to methanol-water extracts of dried voided urine, inhibited MIF production at 0.1 to 0.01 ng/ml of assay mixture. These purified fractions also showed immunosuppressive activity by an in vivo assay wherein doses as low as 1 mug/kg injected intravenously (i.v.) into mice suppressed cell-mediated granuloma formation around Schistosoma manisoni eggs. Identically purified fractions prepared from urine of rats and the patient before they received niridazole showed no immunosuppressive activity either in the MIF or in the granuloma assay systems.     

Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants.

The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.     

Insertion of transposon Tn9 into the spinal (Escherichia coli-Saccharomyces cerevisiae) plasmids and the expression of the prokaryotic gene of chloramphenicol resistance in yeast cells

Transposon Tn9 carrying camr gene which controls resistance to chloramphenicol has been introduced in vivo (in cells of Escherichia coli) into two chimeric shuttle plasmids pYF91 and YEp13. These plasmids consist of the different parts of the E. coli plasmid pBR322, the yeast 2mkm DNA plasmid and the yeast LEU2 structural gene. The plasmidis able to autonomously replicate in both yeast and bacterial cells. A recipient yeast strain carrying cams and leu2 markers was constructed to study the functional expression of the prokaryotic camr gene in eukaryotic yeast cells. The chimeric plasmids pYF91::Tn9 and YEp13::Tn9 were introduced into the yeast and bacterial recipient strains by transformation. The camr LEU2 yeast transformants were isolated. They were genetically unstable when grown on non-selective medium and they simultaneously lost camr and LEU2 markers with a frequency of 10 to 30%. The E. coli transformants were genetically stable under nonselective conditions and they maintain all plasmid markers. The chimeric plasmid pYF91::Tn9 was isolated from the yeast transformants and reintroduced into the cams leuB bacterial strain by transformation. The camr LEUB transformants were obtained. All these data confirm the possibility of the expression of the prokaryotic camr gene in yeast cells and present evidence for introduction of transposon Tn9 into chimeric plasmids.