Showing papers on "Lysis published in 1980"
TL;DR: A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination.
Abstract: Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a beta-1,3-glucanase with a molecular weight of 55,000. The K(m) for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of beta-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity, however. Neither pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination. Lyticase has been applied in spheroplast, membrane, and nucleic acid isolation, and has proved useful in yeast transformation procedures.
286 citations
TL;DR: The alloantiserum-dependent lysis of TLX9 lymphoma cells by peritoneal cells from Bacille Calmette-Guerin (BCG)-treated mice was inhibited 62 percent by depletion of oxygen, suggesting that cytotoxicity required intact contractile elements.
Abstract: The alloantiserum-dependent lysis of TLX9 lymphoma cells by peritoneal cells from Bacille Calmette-Guerin (BCG)-treated mice was inhibited 62 percent by depletion of oxygen. This effect did not appear to be a result of interference with mitochondrial respiration because cyanide, azide, and dinitrophenol did not inhibit cytotoxicity. Preincubating the effector cells for 2 h without glucose, which markedly reduces their ability to release hydrogen peroxide, likewise suppressed antibody-dependent cytolysis by 62 percent. Lysis of sensitized lymphoma cells was virtually abolished by 6 mg/ml of thioglycollate broth, a concentration that also abrogated the detectable release of hydrogen peroxide and the lysis of lymphoma cells by BCG-activated macrophages in response to phorbol myristate acetate (PMA). This concentration of thioglycollate broth was not toxic to the effector cells, as judged by adherence to plastic, binding of opsonized erythrocytes, and phagocytosis of radiolabeled starch granules. Catalase, superoxide dismutase, horseradish peroxidase, mannitol, ethanol, benzoate, and diazabicyclooctane were without consistent effects. Cytochalasin B and dihydrocytochalasin B both markedly suppressed cytolysis, whether induced by antibody or by PMA (ID(50), 0.5 μg/ml). Cytoehalasin B was an equally potent suppressor of glucose uptake and PMA-induced hydrogen peroxide release by BCG-activated macrophages (ID(50), 0.5 μg/ml). However, dihydrocytochalasin B lacked these latter effects, which suggests that cytotoxicity required intact contractile elements. The extracellular lysis of antibody-coated lymphoma cells by BCG-activated macrophages appears to have a predominantly oxidative basis.
193 citations
TL;DR: A modification of Marmur's method was used to isolate DNA, but difficulties in separating DNA from humic substances gave low yields and impure DNA.
Abstract: DNA has been isolated from the bacterial fraction of two soils. Numbers of bacteria, determined by fluorescence microscopy were 1.1 and 2.2·10 10 cells g −1 dry wt. The total amounts of bacterial DNA in these soils were 90 and 187 μg g −1 dry wt respectively. A modification of Marmur's method was used to isolate DNA, but difficulties in separating DNA from humic substances gave low yields and impure DNA. DNA could be partly separated from humic material in the presence of 8 m urea by ion-exchange chromatography on DEAE-Sepharose CL-6B. Final purification was obtained by chromatography on a hydroxyapatite column. When lowering the EDTA concentration in the saline-EDTA solution used for lysis, the amount of humic substances in the cell-free lysate after centrifugation was considerably decreased. The lysate could then be chromatographed directly on hydroxyapatite. Quantities up to 1.5 mg DNA high purity was isolated from 90 g wet soil (37 g dry wt). The isolated DNA was characterized by treatment with DNAse and absorption spectra. No uncommon bases were revealed by thin layer chromatography of the DNA hydrolysates. Melting curves of the isolated DNA showed a relatively broad melting profile, with half maximum hyperchromicity (T m ) near 90°C. Sedimentation coefficients determined by analytical ultracentrifugation showed that the isolated DNA had a high molecular weight, ranging from 2.3 to 10.1·10 5 daltons.
179 citations
TL;DR: The extrapolated zero time intercepts of these reactions suggested that two-thirds of cellular glyceraldehyde-3-P dehydrogenase was membrane bound prior to hemolysis, similar to that calculated for the association of the enzyme with band 3 in the intact red cell when the high ionic strength and enzyme-eluting compounds in the cytoplasm are taken into account.
177 citations
TL;DR: A method developed for the lysis of oral streptococci that employed the action of lysozyme suspended in dilute tris(hydroxymethyl)aminomethane-hydrochloride buffer containing polyethylene glycol has been adapted for use with lactobacilli, actinomycetes, propionibacteria, and pediococci.
Abstract: A method developed for the lysis of oral streptococci that employed the action of lysozyme suspended in dilute tris(hydroxymethyl)aminomethane-hydrochloride buffer containing polyethylene glycol has been adapted for use with lactobacilli, actinomycetes, propionibacteria, and pediococci Most of the cellular deoxyribonucleic acid was liberated from many strains of bacteria usually thought to be lysozyme resistant The major observations were as follows: (i) supplementation of the growth medium with L-threonine, L-lysine, or both frequently produced cells that were more susceptible to lysis by lysozyme; (ii) glucose-containing media produced cells that were more easily lysed than those from cultures grown on other substrates; (iii) polyethylene glycol not only served as an osmotic stabilizer, it also enhanced the extent of lysis; and (iv) dilute tris(hydroxymethyl)aminomethane buffer was superior to the buffer systems most commonly employed in published muramidase-based lysis techniques Stationary-phase cells of Lactobacillus casei and Streptococcus mutans were more easily lysed than those isolated from log-phase cultures The method as detailed in this report should be generally applicable for the lysis of gram-positive, asporogenous bacteria
132 citations
Journal Article•
TL;DR: Ring-shaped membrane lesions generally similar to, but larger than, those previously described for complement lysis were observed, in agreement with recent measurements of larger functional pores for ADCC than complement.
Abstract: To test the hypothesis that complement-mediated cell lysis and cell-mediated cytotoxicity operate by analogous mechanisms, cell membranes from two antibody-dependent cytotoxicity systems were examined by electron microscopy after negative staining. Ring-shaped membrane lesions generally similar to, but larger than, those previously described for complement lysis were observed. These findings are in agreement with recent measurements of larger functional pores for ADCC than complement.
125 citations
TL;DR: It is concluded that the substrate and the dye-reactive sites of formate dehydrogenase face the outside, while those of fumarate reduct enzyme face the inside of the cytoplasmic membrane of cells of V. succinogenes.
83 citations
TL;DR: The organisation of the protein components of bovine chromaffin granule membrane polypeptides has been investigated by labelling or digesting intact granules or broken membranes with the following reagents: lactoperoxidase/Na125I as a reagent for tyrosine residues, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid as aReagent for cysteine residues
83 citations
TL;DR: The effects of eleven heavy metals on the morphology of several marine diatoms and a silicoflagellate were compared and Hg, Cd, and Pb seemed to interfere with cell division.
Abstract: The effects of eleven heavy metals on the morphology of several marine diatoms and a silicoflagellate were compared. With Thalassiosira aestivalis the main effects of metals that were toxic were granular, yellowed cytoplasm; a disruption of chloroplast integrity and dispersion; and more delicate spines extruded from the marginal processes. The metals could be divided into three groups on the basis of other toxic effects. Cu, Zn, Ge, and a metal mixture increased the chain length and did not allow the normal separation of cells, although complete frustules were formed. With these metals there was an inability to form the central chitinous threads that separate normal cells. With Hg, Cd, and Pb, there was some disruption of cell separation, so that cell clumps were formed, but the main effect was the formation of elongated, bent, out-of-column cells. These metals seemed to interfere with cell division. Cr, Ni, Se, Sb, and AsIII were without effect (except for lysis of cells exposed to AsIII) at a c...
76 citations
TL;DR: It is proposed that inhibition of protein synthesis causes a rapid modification of murein structure at the cell wall growth zone in such a manner that attachment of muresin hydrolase molecules is inhibited.
Abstract: We have studied the mechanisms by which amino acid starvation of Escherichia coli induces resistance against the lytic and bactericidal effects of penicillin. Starvation of E. coli strain W7 of the amino acids lysine or methionine resulted in the rapid development of resistance to autolytic cell wall degradation, which may be effectively triggered in growing bacteria by a number of chemical or physical treatments. The mechanism of this effect in the amino acid-starved cells involved the production of a murein relatively resistant to the hydrolytic action of crude murein hydrolase extracts prepared from normally growing E. coli. Resistance to the autolysins was not due to the covalently linked lipoprotein. Resistance to murein hydrolase developed most rapidly and most extensively in the portion of cell wall synthesized after the onset of amino acid starvation. Lysozymes digests of the autolysin-resistant murein synthesized during the first 10 min of lysine starvation yielded (in addition to the characteristic degradation products) a high-molecular-weight material that was absent from the lysozyme-digests of control cell wall preparations. It is proposed that inhibition of protein synthesis causes a rapid modification of murein structure at the cell wall growth zone in such a manner that attachment of murein hydrolase molecules is inhibited. The mechanism may involve some aspects of the relaxed control system since protection against penicillin-induced lysis developed much slower in amino acid-starved relaxed controlled (relA) cells than in isogenic stringently controlled (relA+) bacteria.
76 citations
TL;DR: It is speculated that the physiological role of normal gp5 functions in the initiation of infection by catalyzing local cell wall digestion to facilitate penetration of the tail tube through the cell envelope and may also be responsible for the well-known phenomenon of lysis from without observed with T4.
Abstract: Analyses of a new bacteriophage T4 mutant that permits lysis of infected cells in the absence of e lysozyme showed that the strain carried a suppressor mutation in gene 5, a gene whose polypeptide product (gp5) is an integral component of the virion baseplate. Indirect experiments indicated that cell lysis was caused by the lytic action of mutant gp5. With regard to the physiological role of normal gp5, we speculate that it functions in the initiation of infection by catalyzing local cell wall digestion to facilitate penetration of the tail tube through the cell envelope. The proposed lytic activity of gp5 may also be responsible for the well-known phenomenon of lysis from without observed with T4.
TL;DR: PtK1 cells lysed late in cell division in a medium containing the nonionic detergent Brij 58 and polyethylene glycol with continue to undergo cleavage after lysis is the first cell model system for studying cleavage since the pioneering studies of Hoffman- Berling in 1954.
Abstract: PtK1 cells lysed late in cell division in a medium containing the nonionic detergent Brij 58 and polyethylene glycol with continue to undergo cleavage after lysis. Maintenance of cleavage after lysis is dependent on the composition of the lysis medium; the pH must be around neutrality, MgATP must be present, and the free Ca++ concentration should be 1 microM for optimal constriction to occur. Cleavage can be stopped and reinitiated by raising and lowering the Ca++ levels in the lysis medium. Cleavage in the permeabilized cell is blocked by addition of phalloidin, cytochalasin B, and N-ethylmaleimide-modified myosin subfragment-1 to the lysis medium. This represents the first cell model system for studying cleavage since the pioneering studies of Hoffman-Berling in 1954.
TL;DR: Cyclic AMP levels increased 4- and 3-fold following incubation of intact vascular endothelial cells with 1 mM 3-isobutyl-1-methylxanthine, and isoproterenol or PGI 2, and adenylate cyclase activity of cell lysates was selectively inhibited by the β-adrenergic blocking agent propranolol.
TL;DR: A close correlation was established between autolysis and the degradation of peptidoglycan and both phenomena were induced by the same shock treatment, followed similar kinetics, and were efficiently blocked by addition of divalent cations.
Abstract: Autolysis of unwashed exponential-phase Escherichia coli cells was efficiently promoted by first submitting them to a quick downshock with distilled water before an upshock with 0.5 M sodium acetate, pH 6.5. The association of these two osmotic shocks had a remarkable synergistic effect and led to significant decreases in turbidity and viability. Different factors influencing the rate of cell lysis were examined. A close correlation was established between autolysis and the degradation of peptidoglycan. Both phenomena were induced by the same shock treatment, followed similar kinetics, and were efficiently blocked by addition of divalent cations. Cell lysis was also inducible by a shock treatment with 10(-3) M ethylenediaminetetraacetic acid or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid and blocked by the addition of divalent cations.
TL;DR: The effect of penicillins and cephalosporins on the rate of growth of individual cells of Escherichia coli on agar was determined by measurement of cell length using photomicrographs taken at frequent intervals, and the mode of action of beta-lactam antibiotics was discussed.
Abstract: Summary: The effect of penicillins and cephalosporins on the rate of growth of individual cells of Escherichia coli on agar was determined by measurement of cell length using photomicrographs taken at frequent intervals. When exposed to cephalexin or carbenicillin at twice the minimum inhibitory concentration cell division was inhibited but growth in length proceeded at a uniform exponential rate until abruptly terminated by lysis. The rate of growth of such filamentous cells did not differ significantly from that of normal cells grown in the absence of antibiotic. In contrast, exposure to cephaloridine and amoxycillin at twice the minimum inhibitory concentration resulted in a marked diminution in the rate of cell growth, and lysis occurred very much sooner. At concentrations greatly in excess of the minimum inhibitory concentration (> 100-fold) cephalexin also resulted in a diminution in the rate of cell growth and this was accompanied by earlier lysis. The significance of these findings is discussed in relation to the mode of action of β-lactam antibiotics.
TL;DR: The results indicate that activation of the alternative pathway by Raji cells leads to formation and binding of the membrane attack complex which in turn produces functional membrane lesions.
TL;DR: Group B streptococci, refractory to previously tested muralysins under physiological conditions, were successfully converted to protoplasts by use of a recently describede N-acetyl muramidase, mutanolysin, derived from a streptomycete.
Abstract: Group B streptococci, refractory to previously tested muralysins under physiological conditions, were successfully converted to protoplasts by use of a recently describede N-acetyl muramidase, mutanolysin, derived from a streptomycete. Purified enzyme was effective, but crude preparations, although degrading cell walls, simultaneously produced peculiar effects of cytoplasmic coagulation, retention of cell shape, loss of some intracellular enzymes, and a rise in optical density. Addition of purified mutanolysin to the array of muralysins (group C streptococcal phage-associated lysin, lysozyme), previously successful in preparing protoplasts of different streptococci, now makes possible enzymatic preparation of protoplasts of streptococci of groups A, B, C. D. G, and H.
TL;DR: It is proposed that the secondary inhibitions of cellular RNA or protein synthesis, or both, are involved in the tolerance of these organisms to lysis and killing by Pen G and other inhibitors of insoluble peptidoglycan assembly.
Abstract: Exposure of exponentially growing cultures of Streptococcus mutans strains FA-1 and GS-5 to various concentrations of benzylpenicillin (Pen G) resulted in inhibition of turbidity increases at low concentrations (0.02 to 0.04 mug/ml). However, in contrast to some other streptococcal species, growth inhibition was not accompanied by cellular lysis or by a rapid loss of viability. In both strains, synthesis of insoluble cell wall peptidoglycan was very sensitive to Pen G inhibition and responded in a dose-dependent manner to concentrations of about 0.2 and 0.5 mug/ml for strains GS-5 and FA-1, respectively. Higher Pen G concentrations failed to inhibit further either growth or insoluble peptidoglycan assembly. Somewhat surprisingly, Pen G also inhibited both ribonucleic acid (RNA) and protein syntheses, each in a dose-dependent manner. Compared with inhibition of peptidoglycan synthesis, inhibition of RNA and protein syntheses by Pen G was less rapid and less extensive. Maximum amounts of radiolabeled Pen G were specifically bound to intact cells upon exposure to about 0.2 and 0.5 mug/ml of Pen G for strains GS-5 and FA-1, respectively, concentrations consistent with those that resulted in maximum or near-maximum inhibitions of the synthesis of cellular peptidoglycan, RNA, and protein. Five polypeptide bands that had a very high affinity for [(14)C]Pen G were detected in a crude cell envelope preparation of strain FA-1. After exposure of cultures of strain FA-1 to the effects of saturating concentrations of the drug for up to 3 h, addition of penicillinase was followed by recovery of growth after a lag. The length of the lag before regrowth depended on both Pen G concentration and time of exposure. On the basis of these and other observations, it is proposed that the secondary inhibitions of cellular RNA or protein synthesis, or both, are involved in the tolerance of these organisms to lysis and killing by Pen G and other inhibitors of insoluble peptidoglycan assembly.
TL;DR: Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification.
Abstract: Vacuoles from beetroot (Beta vulgaris L var esculenta Gurke) isolated by a mechanical procedure were osmotically lysed to separate the membrane and sap components for analysis Approximately 62% of the vacuole proteins, 70% of the nondialyzable carbohydrates and almost all of the phospholipids and sterols were recovered in the membrane fraction The vacuole membrane had a phospholipid protein ratio of 068 and a sterol:phospholipid ratio of 021 17 complex polar lipids including phosphatides and glycolipids have been tentatively identified Phosphatidylcholine (54%) and phosphatidylethanolamine (24%) were the most prominent phosphoglycerides besides phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid (1, 4, 5, and 12%, respectively) A putative sulfoglycoside and two major ceramide glycoside-like lipids, resembling those of animal lysosomes, were identified by thin-layer chromatography High-resolution SDS-acrylamide gel electrophoresis of the polypeptides from the vacuole revealed 15 major bands with apparent molecular weights ranging from 91,000 to 12,000 Selective elution experiments delineated those polypeptides that were peripheral membrane proteins or sap proteins adsorbed to the membrane, and those that exhibited hydrophobic interactions with the lipid core Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification
TL;DR: Iradiation of protoporphyrin‐sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis.
Abstract: — Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).
Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3s-hydroxy-5α-hydroperoxy-△6-cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.
TL;DR: It is proposed that gps normally prevents functional expression of wild-type gp5 activity from either side of the cell wall, whereas the 5ts1 form of gp5 is insensitive to the gps barrier and induces lysis from either Side of the Cell wall.
Abstract: Previous studies indicated that (i) T4 gene s product (gps) protects infected cells from superinfection lysis from without, (ii) the absence of gps in infected cells also leads to lysis from within even when T4 e lysozyme is absent, (iii) T4 gene 5 product (gp5), a polypeptide of the virion baseplate, may be responsible for inducing lysis from without, and (iv) altered gp5 of the T4 mutant 5ts1 can replace e lysozyme to cause lysis from within. Results of this study showed that (i) wild-type gp5 in infected cells lacking e lysozyme was responsible for lysis from within in the absence of gps, and (ii) gps did not protect infected cells from superinfection lysis from without by 5ts1 phage. We prpose that gps normally prevents functional expression of wild-type gp5 activity from either side of the cell wall, whereas the 5ts1 form of gp5 is insensitive to the gps barrier and induces lysis from either side of the cell wall.
TL;DR: This is the first demonstration of liposome lysis by streptolysin O and demonstrates the cholesterol specificity which has previously been shown by inhibition studies.
TL;DR: It is concluded that unsaturated fatty acids induce cell and protoplast lysis by acting as more potent membrane destabilizers than saturated fatty acids.
Abstract: Palmitic, stearic, oleic, and linoleic acids at concentrations of 200 nmol/ml all inhibited autolysin activity 80% or more in whole cells or cell-free extracts. This concentration of the saturated fatty acids palmitic acid and stearic acid had little or no effect on the growth of whole cells or protoplasts. However, the unsaturated fatty acids oleic acid and linoleic acid induced lysis in both situations. This lytic effect is apparently not related to any uncoupling activity or inhibition of energy catabolism by unsaturated fatty acids. It is concluded that unsaturated fatty acids induce cell and protoplast lysis by acting as more potent membrane destabilizers than saturated fatty acids.
TL;DR: Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large-scale purification of the neutrophil plasma membrane, suitable for biochemical studies.
Abstract: 1. Large amounts of granulocytes can be isolated from bovine blood by differential centrifugation and hypotonic lysis of erythrocytes, followed by separation of neutrophils and eosinophils by centrifugation through a gradient of colloidal silica. 2. Careful homogenization of the purified neutrophils and subfractionation of the postnuclear supernatant by centrifugation through a discontinuous sucrose gradient provides a membrane fraction (at the 20/32%, w/w, sucrose interface), which collects about 20--35% of the activity of plasma membrane marker enzymes. 3. Treatment of the plasma membrane fraction with 0.5 M KCl removes some protein and activity of granule enzymes, leading to an about 20--35-fold enrichment in specific activity of plasma membrane marker enzymes. In particular, there is a 25-fold enrichment in a Ca2+-dependent ATPase, whose half-maximal reaction velocity is reached at 2.2 X 10(-7) M Ca2+. 4. High-resolution sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals a complex composition of the neutrophil plasma membrane, with about 40 polypeptides stained by Coomassie blue. Ten of these peptides are more intensely stained by the dye; their apparent molecular weight ranges from 81 000 to 34 000. All these ten polypeptides but one (which is likely to be actin) are markedly enriched in the plasma membrane fraction over the original homogenate. 5. Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large-scale purification of the neutrophil plasma membrane, suitable for biochemical studies.
Journal Article•
TL;DR: The physiological and morphological properties of this strain were similar to those of "deep rough" and mlp or lpo mutants of Escherichia coli K-12, respectively.
Abstract: The protein compositions of the membrane preparations from Selenomonas ruminantium grown in glucose or lactate medium were determined by sodium dodecyl sulfate- and two-dimensional (first, isoelectric focusing; second, sodium dodecyl sulfate) polyacrylamide slab gel electrophoresis. The outer membrane from both glucose- and lactate-grown cells contained two major proteins with apparent molecular weights of 42,000 and 40,000. These proteins existed as peptidoglycan-associated proteins in the outer membrane. The critical temperature at which they were dissociated completely into the monomeric subunits of 42,000 and 40,000 daltons was found to be 85 degrees C. The amount of each protein varied considerably depending upon the cultural conditions. The absence of the lipoprotein of Braun in S. ruminantium was suggested in our preceding paper (Y. Kamio, and H. Takahashi, J. Bacteriol. 141:888--898, 1980), and the possible absence of the protein components corresponding to the Braun lipoprotein in this strain was confirmed by electrophoretic analysis of the outer membrane and the lysozyme-treated peptidoglycan fractions. Examination of the cell surface of S. ruminantium by electron microscopy showed that the outer membrane formed a wrinkled surface with irregular blebs, some of which pinched off forming vesicles of various sizes. Rapid cell lysis occurred with the addition of a low level of lysozyme to the cell suspension. These findings led us to conclude that the physiological and morphological properties of this strain were similar to those of "deep rough" and mlp or lpo mutants of Escherichia coli K-12, respectively.
TL;DR: A rapid and simple purification of covalently closed circular (supercoiled) DNA from both bacterial clones (plasmids) and African green monkey cells (SV40) is presented, which is virtually independent of the molecular size and gives good yields of supercoiled DNA.
TL;DR: The use of synchronous-culture systems distinguished cell elongation from cell division and revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis.
Abstract: Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.
TL;DR: A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells and it is possible that gene 10 may code for a regulator of late gene expression for P1.
Abstract: A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.