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PubMed1997

Yeast artificial chromosome transfer into human renal carcinoma cells by spheroplast fusion.

Successful transfer of yeast artificial chromosomes (YACs) into human cells has been described in only a single study. We here report on the evaluation of YAC transfer strategies into a human renal cell carcinoma cell line by yeast spheroplast fusion and cationic lipids. While the latter approach proved inefficient, significant numbers of clones containing both vector arms were obtained by spheroplast fusion. FISH analyses on such clones revealed the presence of YAC integration and the co-localization of both vector arms with insert sequences. These data demonstrate that under certain experimental conditions efficient YAC transfer into human cells by spheroplast fusion is possible and may be useful for the cloning of human disease-related genes.

Carcinoma, Renal Cell↗
PubMed1987

Transformation of yeast spheroplasts without cell fusion.

The efficiency of genetic transformation of Saccharomyces cerevisiae spheroplasts has been increased 10- to 100-fold over previously published procedures. Optimal transformation frequencies for single-stranded and double-stranded replicating plasmids are 2 X 10(7) and 5 X 10(6) transformants/microgram, respectively. At saturating DNA concentrations, 12 and 3%, respectively, of the viable spheroplasts contain plasmid DNA. The percentage of transformants that have undergone nuclear fusion varies from 0.1 to 3%, indicating that fusion is not required for the uptake of DNA by yeast spheroplasts.

Cell Fusion↗
PubMed1979

Induction of N-acetylglucosamine-catabolic pathway in spheroplasts of Candida albicans.

Synthesis of N-acetylglucosamine-catabolic enzymes, namely permease (high-affinity uptake system), kinase and deaminase was studied in the spheroplasts of the yeast Candida albicans. The presence of N-acetylglucosamine as inducer is essential for the induced synthesis of these enzymes in the spheroplasts, which were active for at least 8--9 h. However, some of the newly synthesized kinase and deaminase leaked out from the spheroplasts into the medium during induction. Experiments with inhibitors of RNA and protein synthesis indicate that the appearance of new enzyme activities is dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level. However, inhibitors of DNA synthesis, e.g. mitomycin-C and hydroxyurea, had no effect on the synthesis of these enzymes.

Acetylglucosamine↗
PubMed1998

Investigation of the yeast mitochondrial unselective channel in intact and permeabilized spheroplasts.

The existence of an activity corresponding to the nucleotide-induced Yeast Mitochondria Unselective Channel (YMUC2) of isolated mitochondria was investigated in permeabilized and intact spheroplasts of the baker's yeast Yeast Foam. In nystatin-permeabilized spheroplasts, ATP and GDP-beta-S induced a decavanadate-sensitive stimulation of the respiration only under conditions equivalent to those previously reported for isolated mitochondria (low phosphate concentration, presence of a salt). On intact spheroplasts parallel measurements of respiration rate, [ATP]/[ADP] ratio and mitochondrial transmembrane potential allowed to show that the addition of the glucose analog 2-deoxyglucose decreased the permeability of the inner mitochondrial membrane owing to cellular ATP depletion. This strongly supports the hypothesis that Yeast Mitochondria Unspecific Channel is active in situ and inhibited by cellular [ATP] depletion.

Adenosine Diphosphate↗
PubMed1964

OBSERVATIONS ON THE FINE STRUCTURE OF SPHEROPLASTS OF RHODOSPIRILLUM RUBRUM.

Spheroplasts of the photosynthetic bacterium Rhodospirillum rubrum were prepared from cultures grown in either the presence or absence of light. Cells were converted into spheroplasts by using lysozyme and Versene and fixed in a sucrose-veronal-acetate buffer mixture containing osmium tetroxide. Some preparations were shadow-cast and examined whole; others were embedded in Epon 812 and sectioned. The action of lysozyme and Versene appears to result in removal of the cell wall in strips. The relationship of the chromatophores to the cytoplasmic membrane is readily visualized in sections of broken spheroplasts, and in areas the chromatophores are seen to be continuous with the membrane. In all preparations examined, no definite connections between individual chromatophores were observed. In some cells large spherical granules were evident which either possessed or lacked a clearly visible limiting membrane. On serial sectioning, all granules appeared bounded by a single membrane 40 A wide. The granule membrane was well defined only if the section came from the center of the granule. Sections at other levels showed either a diffuse membrane or no membrane at all. The reasons for this are discussed.

Cell Biology↗
PubMed1983

Action of chlorophyllase purified from rye seedlings on light-harvesting bacteriochlorophyll of chromatophores and spheroplasts from Rhodospirillum rubrum.

1. The chlorophyllase [EC 3.1.1.14] purified from greened rye seedlings hydrolyzed the bacteriochlorophyll isolated from Rhodospirillum rubrum, but not the pigment bound to the membrane of chromatophores or spheroplasts from the bacterium. 2. Acetone, if added at such concentrations that the bound bacteriochlorophyll would not be solubilized, enabled the enzyme to hydrolyze the bound pigment. The acetone concentrations required for half the maximum hydrolysis rates were 16% with chromatophores and 7% with spheroplasts. 3. The enzymic hydrolysis of the bound bacteriochlorophyll in the presence of acetone removed bacteriochlorophyllide from the membrane, leaving its esterifying alcohol, possibly all-trans-geranylgeraniol, in situ. 4. Washing of chromatophores with 30% acetone removed about 10% of the bound bacteriochlorophyll. The bound pigment remaining after washing was not hydrolyzed by the enzyme unless acetone was added. 5. It seems possible that light-harvesting bacteriochlorophyll was mostly, if not all, bound to the inner surface of chromatophores (the outer surface of spheroplasts), having its esterifying alcohol residue buried in the membrane and its porphyrin residue emerging from the membrane into the inside solution; thus, chlorophyllase could not make contact with the ester linkage between the esterifying alcohol and porphyrin moieties of the pigment unless the esterifying alcohol residue was partly exposed.

Bacterial Chromatophores↗
PubMed1981

Ultrastructural and chemical studies on wall-deficient forms, spheroplasts and membrane vesicles from Mycobacterium aurum.

Wall-deficient forms of Mycobacterium aurum were prepared by agitating the cells during exponential growth with D-cycloserine, glycine, lysozyme, EDTA and LiCl for approximately the time of three cell divisions (18 h). Wall-deficient forms were then converted to spheroplasts by gentle stirring with lysozyme and EDTA in a Tris/HCl buffer containing sucrose until all the cells appeared spherical by phase contrast microscopy. Subsequent lysis by nucleases followed by osmotic shock produced membrane vesicles. Ultrastructural and chemical properties of the spheroplasts and membrane vesicles are described. The spheroplasts were susceptible to lysis by 0.25% (w/v) sodium dodecyl sulphate and were permeable to certain enzyme substrates.

Anti-Bacterial Agents↗
PubMed1983

Spheroplast fusion as a mode of genetic recombination in mycobacteria.

Spheroplasts were prepared from two carotenoid pigment mutants of Mycobacterium aurum named NgR9 and A11, which were obtained by the chemical mutagenesis of the wild type strain A+ with N-methyl-N'-nitro-N-nitrosoguanidine. The carotenoid pigments and the alpha- and beta-mycolic acids were taken as genetic markers and the recombinants were selected on the basis of their colour on Löwenstein-Jensen medium. Spheroplasts of the two mutants were mixed in a 1:1 ratio and were treated with 40% (w/v) polyethylene glycol 6000 for 5 min at 37 degrees C. The frequency of NgR9 X A11 recombination in optimal conditions was about 2.5 X 10(-3). The recombinants selected on the basis of their carotenoid pigment profile were also tested for their alpha- and beta-mycolic acids as a second genetic marker. The results were further confirmed by electron microscopy. The optimal conditions for spheroplast fusion as a mode of genetic recombination in M. aurum are described.

Carotenoids↗
PubMed2006

Pathophysiology of in-vitro induced filaments, spheroplasts and rod-shaped bacteria in neutropenic mice.

This study compared the in-vitro properties and in-vivo effects of Escherichia coli filaments, spheroplasts and normal cells in a murine thigh infection model. E. coli was exposed to ceftazidime, meropenem or saline to obtain filaments, spheroplasts or normal bacilli, which were then injected into neutropenic mice. After 24 h, morphology, CFUs, local and circulating endotoxin levels, cytokine levels and mortality were recorded, and correlations between bacterial and host parameters of infection were investigated. Filaments and spheroplasts contained more endotoxin/CFU than controls. Histological studies showed that morphologically altered bacteria changed into rod-shaped cells in the absence of antibiotics. Bacterial spread to the liver was significantly higher in mice challenged with rod-shaped cells, compared with antibiotic-exposed bacteria (p 0.007). Muscle endotoxin levels correlated significantly with circulating interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha, and both pro-inflammatory cytokines were correlated significantly (p 0.011). Despite a tendency toward higher local and systemic concentrations of endotoxin in the filament group, inflammatory responses and survival did not differ between groups. It was concluded that morphologically altered bacteria contain more endotoxin and can regain a rod shape after withdrawal of antibiotics, while non-antibiotic-exposed bacteria show greater spread to the liver. There was a clear intra-individual relationship between local endotoxin, systemic endotoxin, TNF-alpha and IL-6 production, but these parameters did not differ among groups.

Animals↗
PubMed1985

Lack of accumulation of exogenous adenylyl dihydrostreptomycin by whole cells or spheroplasts of Escherichia coli.

Accumulation of purified adenylylated dihydrostreptomycin (DHS-AMP) was examined in two strains of Escherichia coli. E. coli JSRO-N was plasmid free and aminoglycoside (AG) susceptible; E. coli JSRO-N(pSAD1) contained a plasmid-encoded AG adenylyltransferase which modifies DHS and streptomycin and confers resistance to both of these drugs. Although both whole cells and spheroplasts of JSRO-N accumulated free DHS, we were not able to demonstrate uptake of DHS-AMP by this strain. Whole cells and spheroplasts of JSRO-N(pSAD1) accumulated DHS at a much slower rate than that observed in JSRO-N. This was presumably due to the activity of the adenylyltransferase in JSRO-N(pSAD1). However, this low rate of accumulation of DHS was still higher than the uptake of DHS-AMP by either JSRO-N or JSRO-N(pSAD1). Thus, the rate of accumulation of DHS-AMP was even lower than that of DHS during the slow, initial, energy-dependent phase of AG uptake seen in JSRO-N(pSAD1). We also found that when either JSRO-N or JSRO-N(pSAD1) was incubated with barely inhibitory or subinhibitory concentrations of DHS, rapid uptake of DHS could be stimulated by the addition of an inhibitory concentration of another AG, such as amikacin. Uptake of DHS-AMP could not be similarly enhanced by the addition of amikacin. Our results indicate that DHS-AMP is not accumulated by whole cells or spheroplasts of E. coli. These results are consistent with the postulated intracellular location of AG-modifying enzymes.

Chromatography, Thin Layer↗
PubMed1973

Action of yeast killer factor: a resistant mutant with sensitive spheroplasts.

Yeast killer factor proteins bind to cells of both sensitive and killer-producing strains, although the latter are immune to killer action. Spheroplasts prepared from sensitive cells bind less than 1% of the killer bound to whole cells, but remain fully sensitive to killer. This finding and those obtained from binding studies of partially purified, radioactive killer protein suggest that most of the toxins remain bound to the yeast cell wall and do not function further in the killing process. A killer-resistant mutant R(18) was isolated from a sensitive strain. Whole cells of the mutant were unable to bind killer and were fully resistant. In contrast, spheroplasts of R(18) were fully sensitive to killer. These data suggest that the sites exposed to killer in spheroplasts are distinct from those on the cell wall. These wall sites appear to be necessary for killer action in whole cells.

Binding Sites↗
PubMed1978

Penicillin-induced formation of osmotically stable spheroplasts in nongrowing Bdellovibrio bacteriovorus.

Bdellovibrio peptidoglycan is of typical gram-negative composition. The molar ratios of alanine:glutamic acid:diaminopimelic acid:muramic acid:glucosamine were about 2:1:1:1:1. Nascent, nongrowing Bdellovibrio bacteriovorus 109J were converted from highly motile vibrios to highly motile spheres when shaken in dilute buffer plus penicillin, cephalothin, bacitracin, or D-cycloserine. The spherical forms contained essentially no sedimentable peptidoglycan; i.e., they were spheroplasts. Spheroplasts induced by penicillin, D-cycloserine, and lysozyme were stable in dilute buffer and did not lyse when subjected to osmotic shock. Normal Bdellovibrio suspended in buffer turned over their peptidoglycan at a rate of approximately 30% h during the initial 120 min of starvation. Chloramphenicol and sodium azide strongly inhibited Bdellovibrio peptidoglycan turnover and the induction of spheroplasts by penicillin. The data indicate that nongrowing B. bacteriovorus are sensitive to penicillin and other antibiotics affecting cell walls because of their high rate of peptidoglycan turnover. It is also concluded that an intact peptidoglycan layer is required for maintaining cell shape, but is not required for osmotic stability of B. bacteriovorus.

Bacterial Proteins↗
PubMed1978

Spheroplast fusion and heterokaryon formation in Mucor racemosus.

Heterokaryons of Mucor racemosus were produced by fusion of spheroplasts from two auxotrophic strains of the fungus. Germinated sporangiospores were converted to spheroplasts by using commercial chitinase and Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed in a buffered Ca(NO3)2 solution and fusion occurred. After cell wall regeneration, prototrophs were isolated. The frequency of heterokaryon formation was 1.45 X 10(-4). Prototrophic isolates segregated parental nuclei at a high frequency, indicating that heterokaryons had formed.

Adenine↗
PubMed1979

Scanning electron microscope study of Saccharomyces cerevisiae spheroplast formation.

A suspension of Saccharomyces cerevisiae NCY366 in buffered 1.2 M sorbitol containing Zymolyase-5000 (a beta-glucanase-containing preparation/showed maximum osmotic sensitivity after 30 min of incubation at 30 degrees C. A scanning electron microscope study of spheroplast formation, using a very high resolution (4-nm) machine, revealed several new morphological features. The surface of the plug in bud scars on intact cells appeared warty. The wall, which assumed a beady appearance as digestion proceded, ultimately sloughed off to reveal the furrowed surface of the plasma membrane. Bud scars were resistant to digestion and. as incubation proceeded, they became surrounded by an outer annulus, which may be the seconary septum. Wall material was completely removed from the majority of cells only after 60 min of digestion. The surface of spheroplasts was studded with particles, about 25 to 30 nm in diameter. Many spheroplasts had a single large indentation, which may be in that part of the plasma membrane originally underlying the birth scar.

Cell Membrane↗
PubMed1961

Spheroplasts of the yeast Candida utilis.

Svihla, G. (Argonne National Laboratory, Argonne, Ill.), F. Schlenk, and J. L. Dainko. Spheroplasts of the yeast Candida utilis. J. Bacteriol. 82:808-814. 1961.-The formation and properties of spheroplasts of the yeast Candida utilis, produced by digestion of the cell walls with snail gut juice (Helix pomatia) in isotonic medium, were studied by phase, interference, and ultraviolet microscopy, and tracer techniques. Cells cultivated in the presence of l-methionine, S-methyl-l-methionine, and l-homocysteine (5 mumoles/ml) were more susceptible to enzyme action than ordinary cells. These cells contain increased amounts of S-adenosylmethionine in the vacuole which facilitates ultraviolet microscopy. Spheroplasts are stable in 0.6 m KCl containing 0.04 m phosphate, but are disintegrated by water, sonic vibration, or ultraviolet irradiation. Exposure in isotonic medium to 6 x 10(4) erg/mm(2) energy at 253 mmu breaks the cytoplasmic membrane and liberates the vacuole with membrane intact. The vacuolar membrane may be broken with about three times the initial dose of ultraviolet.

Candida↗
PubMed1964

ISOLATION AND PROPERTIES OF INTACT MITOCHONDRIA FROM SPHEROPLASTS OF YEAST.

Duell, E. A. (Western Reserve University, Cleveland, Ohio), Sakae Inoue, and Merton F. Utter. Isolation and properties of intact mitochondria from spheroplasts of yeast. J. Bacteriol. 88:1762-1773. 1964.-Functionally intact mitochondria can be obtained in good yields by osmotic disruption of spheroplasts formed from yeast by treatment with an enzyme mixture from the snail digestive tract. The useful range of this method is extended greatly by pretreatment of the yeast cells with 2-mercaptoethylamine and ethylene-diaminetetraacetate. The concentration of the yeast suspension can be increased greatly, the incubation period can be shortened considerably, and the requirement for log-phase cells is obviated. Mitochondria prepared by this method have been compared with those obtained by mechanical disruption in terms of respiratory control, specific activity on a wide range of oxidizable substrates, heterogeneity during centrifugation, and structures observed by electron microscopy. In all cases, the mitochondria obtained from spheroplasts appear to be much less damaged by the preparative procedures.

Centrifugation↗
PubMed1988

Progress in culture and subculture of spheroplasts and fastidious acid-fast bacilli isolated from intestinal tissues.

The efficiency of culture media was compared for the culture and subculture of very slowly growing acid-fast bacilli and spheroplast forms obtained from intestinal tissues of patients with Crohn's disease and ulcerative colitis and from controls without inflammatory bowel disease. Media were developed by modifying a nutrient broth medium based on veal infusion broth and yeast extract. We evaluated the effects of pH and the addition of Tween 80, Dubo oleic albumin complex, an extract from intestinal tissue from a patient with Crohn's disease, horse serum, sucrose, magnesium sulfate, ferrous ammonium sulfate, and sodium citrate. All media contained mycobactin J (2 micrograms/ml). We developed a medium (MG3) which was highly successful in promoting the growth of very fastidious organisms and promoted reversion of spheroplasts to acid-fast rods. MG3 contained veal infusion broth, 1% yeast extract, 10% horse serum, 0.3 M sucrose, 0.2% MgSO4, 0.1% ferrous ammonium sulfate, 0.1% sodium citrate, and 2 mg of mycobactin J per liter. We were able to obtain quantities of organisms sufficient for examination of the organisms by molecular techniques. Successful cultivation of all isolates and reversion of spheroplasts to acid-fast forms encourage further studies of the possibility of a complex association of mycobacteria and Crohn's disease.

Crohn Disease↗
PubMedDate not supplied

[Method for producing spheroplasts from yeast cells].

A method for producing spheroplasts from yeast cells has been developed. The method involves: 1) prefreezing and thawing of cells in the minimal nutrient medium; 2) treatment with the mixture of 86 mM 2-mercaptoethanol, 5% sodium dodecylsulphate and 6% Triton X-100; 3) enzymic digestion by the digestive juice of grape helix. The formation of spheroplasts was controlled by microscopy and measurement of the optic density of the spheroplast suspension in the incubation medium and in 1% sodium dodecylsulphate. The method has been successfully tested on some bakery yeast: Saccharomyces cerevisiae, Saccharomyces paradoxus, Schizosaccharomyces pombe, Saccharomyces globosus, Saccharomyces carlsbergensis.

Ascomycota↗