Preparation of RNA from unspheroplasted yeast cells (Saccharomyces cerevisiae).

High-quality RNA can be prepared from up to 100-ml culture volumes of unspheroplasted yeast cells (Saccharomyces cerevisiae) via homogenization in high-temperature phenol:chloroform mixtures. The yield of RNA from this preparative method is equivalent to those of other methods requiring preliminary spheroplasting of cells. Quality and quantity of recovered RNA are independent of yeast strain and cell growth medium used, and the method works equally well on cells in either log phase growth or in stationary phase. Mitochondrial RNAs recovered as part of whole cell RNA mixtures may be slightly degraded. Analyses of individual transcripts in the recovered RNA mixtures suggest that there is no selection for or against any specific single transcript or any group of transcripts when RNA is prepared by this method.

Lipids of dermatophytes. III. Sterol-induced changes in the lipid composition and functional properties of Epidermophyton floccosum.

Sterol supplementation, alone or in the presence of cerulenin, resulted in an increase in the total sterol content of Epidermophyton floccosum. While the total phospholipid levels of E. floccosum exhibited only marginal changes with sterol supplementation, the fatty acid profiles of these phospholipids were highly varied. In the presence or absence of cerulenin, the oleic acid content of phospholipids were increased significantly by cholesterol supplementation, whereas linoleic acid levels were enhanced by ergosterol supplementation. These variations resulted in higher unsaturated/saturated phospholipid fatty acid ratios in sterol-supplemented cells. The uptake of labeled amino acids (aspartic acid, lysine, glycine) was influenced by sterol supplementation. Alterations in the number of binding sites for the membrane probe, 1-anilino-naphthalene-8-sulfonate (ANS), were seen based on Scatchard plot calculations. The results indicate a correlation between sterol-induced changes in membrane lipid composition and function.

Methods for DNA extraction from Candida albicans.

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.

Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica.

Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.

Isolation and structure of glucan from regenerating spheroplasts of Candida albicans.

Regenerating spheroplasts of Candida albicans formed organized glucan nets in liquid culture. The nets consisted of interwoven microfibrils about 50 nm wide, but of an undetermined length. Partial acid hydrolysis of the polysaccharide showed the presence of chains of beta(1----3)- and beta(1----6)-linked glucose residues, but no intrachain beta(1----3) and beta(1----6) linkages. Periodate oxidation and GLC of the methylated glucan indicated a highly branched polymer (9.5% branch points). Sequential enzymic degradation of the isolated nets confirmed the presence of chains of beta(1----3)- and beta(1----6)-linked glucose residues. Degradation by (1----3)-beta- and (1----6)-beta-glucanase released 23% (w/w) and 30% (w/w) respectively of the carbohydrate as glucose equivalents. The residual material was degraded by chitinase. Equal amounts of N-acetylglucosamine and glucose equivalents were detected in the chitinase hydrolysate, suggesting a possible linkage between glucan and chitin. Our data indicate that the cell wall of C. albicans contains at least two highly branched glucans with predominantly beta(1----3) or beta(1----6) linkages.

Characteristics of chromosomal DNA isolated from Escherichia coli spheroplasts.

The chromosomal DNA of Escherichia coli spheroplasts induced by penicillin G was studied biochemically and electron microscopically. Although the spheroplasts were unable to divide, they continued to synthesize chromosomal DNA for several hours even in the presence of penicillin G. Some differences were observed between the chromosomal DNA of the parent cells and that of the spheroplasts in sucrose gradient centrifugation and electron microscopy; two types of chromosomal DNA, a slower sedimenting form and a faster sedimenting form, were released from the gently lysed parent cells. The former was membrane-free folded chromosome and the latter was membrane-associated chromosome. In contrast, the chromosome from the spheroplast showed a single intermediate value of sedimentation coefficient between those of the chromosomal DNA from the parent cell. Cytochrome spreading for electron microscopy showed that the spheroplast chromosomal DNA formed an aggregated mass consisting of several chromosome-molecules of the parent cell.

Isolation and characterization of yeast ring chromosome III by a method applicable to other circular DNAs.

A method is described for the isolation and purification of covalently closed circular (ccc) DNA from yeast (Saccharomyces cerevisiae). Spheroplasts are lysed at pH 12.45 which denatures linear but not ccc DNA. Next, the lysate is taken through a gentle high-salt-phenol extraction to remove single-stranded DNA. The ccc DNA, recovered by ethanol precipitation, can be further studied by agarose gel electrophoresis, can be cut with restriction endonucleases and can be used to transform Escherichia coli. This method efficiently purifies large (approx. 190 kb) and small (approx. 1.5 kb, TRP1-RI Circle) circular DNAs and thus has general applicability for isolation and purification of plasmids from yeast.

Ribosome structure, maturation of ribosomal RNA and drug sensitivity in temperature-sensitive mutants of Saccharomyces cerevisiae.

Selected strains of Saccharomyces cerevisiae were mutagenized with nitrosoguanidine and temperature-sensitive mutants isolated. These mutants were screened by two-dimensional gel-electrophoresis for the presence of ribosomal proteins with altered mobility relative to parental preparations. Electrophoretic changes were detected in three mutants designated ts205, ts212 and ts417, with the alterations apparently the same in the three cases. All three mutants were more sensitive than were their parents to the antibiotics G418, hygromycin B and MDMP. Mutant ts212 has an abnormal distribution of native ribosomal subunits and appears to be defective in its assembly of the smaller subparticle.

The orientation of the major coat protein of bacteriophage f1 in the cytoplasmic membrane of Escherichia coli.

The orientation of the major coat (B) protein of the bacteriophage f1, an integral membrane protein in the cytoplasmic membrane of infected Escherichia coli, was examined. Pyridoxal 5'-phosphate and [3H]NaBH4 were used to label the cytoplasmic membrane proteins in spheroplasts and membrane vesicles of E. coli infected with bacteriophage f1. Under the conditions described, tritium incorporation was almost completely dependent on the presence of pyridoxal 5'-phosphate and little if any of the cytoplasmic proteins were labeled when the reaction was applied to intact spheroplasts. The major coat protein was isolated from the cytoplasmic membranes labeled in this manner and the chymotryptic peptides were analyzed for the presence of tritium in the pyridoxamine 5'-phosphate conjugate. When the proteins were labeled in the intact spheroplast, only the NH2-terminal chymotryptic peptide of the coat protein was labeled. If the proteins were labeled during osmotic lysis of the spheroplasts or in isolated vesicles, the chymotryptic peptide containing the COOH terminus of the coat protein as well as the NH2-terminal peptide was labeled. The NH2-terminal peptide was labeled to approximately the same extent as occurred in the intact spheroplast. These results are consistent with the hypothesis that the mature f1 coat protein asymmetrically spans the cytoplasmic membrane of the infected host with its NH2 terminus exposed on the outside and COOH terminus exposed on the cytoplasmic surface.

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.

[Structural organization of the bacterial nucleoid using endonucleolysis]. / Issledovanie struktyrnoi organizatsii bakterial'nogo nukleoida pospedstvom endonukleoliza.

The fragmentation of bacterial deoxyribonucleoprotein (bDNP) in the spheroplasts of Escherichia coli, Serratia marcescens, Pseudomonas fluorescens and Micrococcus luteus by bacterial intracellular Ca2+ or Ca2+, Mg2+-dependent endonucleases in situ was studied. An electrophoresis of the extracted nuclease-split bDNP revealed the presence of high molecular weight (nuclease-resistant) and low molecular weight multiple fragments (100--120 nucleotide pairs). The electrophoretic mobility of the smallest nuclease-split DNA fragments in all bacterial species under study was similar, indicating the orderly structure of bDNP. Two total fractions whose electrophoretic mobility corresponded to that of the histones H2a and H2b from calf thymus were prevalent in the spectrum of acid-soluble bDNP proteins of gram-negative species. The heterogeneity of DNP with respect to its sensitivity to nucleases, is interaction with membranes and protein distribution pattern were revealed by treatment of the bacterial nucleoid with endogenous endonucleases, which probably reflects differences in the functional state of individual sites of the genome.

[Probing the structure of bacterial deoxyribonucleoproteins with exogenous and endogenous nucleases]. / Zondirovanie struktury bakterial'nykh dezoksiribonukleoproteidov ékzogennoi i éndogennoi nukleazami.

During digestion of deoxyribonucleoproteins (DNP) of gram-negative bacteria by micrococcal nuclease and Ca2+, Mg2+-dependent endonuclease in situ regular series fragments-and large nuclease-resistent fragments of DNP were revealed by electrophoresis. The DNP length of the smallest DNP-fragment was tentatively 120-140 base pairs. In investigated bacterial species DNP contained at least two basic proteins which had electrophoretic mobility similar to that of histone H4 of eucaryot. It is suggested that bacterial DNPs have common regular structure.

Polymorphonuclear leukocyte-inhibitory factor of Bordetella pertussis. II. Localization in the outer membrane.

The outer and inner membranes and cytoplasm of spheroplasts of a strain of phase I B. pertussis were fractionated by density gradient centrifugation. The high density vesicles of the outer membranes isolated had the "Pili" characteristic of the bacteria and the same antigenicty as the bacterial surface. Activities for inhibition of polymorphonuclear leukocytes were also almost exclusively localized in this outer membrane fraction. The histamine-sensitizing activity was more dispersed, but its specific activity was also highest in the outer membrane fraction. These results suggest that molecules carrying these activities, which are probably different entities together with the tissue-adhesive pili, form a virulence complex on the surface of phase I organisms of B. pertussis.

Measurement of pyrimidine dimers in spheroplasts of Bacillus subtilis.

A method is described for making spheroplasts of Bacillus subtilis which are permeable to exogenous enzymes. Conditions are described for measuring small numbers of pyrimidine dimers in the DNA of UV-irradiated cells by use of a partially purified Micrococcus luteus extract containing an enzyme specific for pyrimidine dimers. The system will detect as few as 10-12 pyrimidine dimers per genome.

Phycobilisomes in spheroplasts of Anacystis nidulans.

Phycobilisomes in Anacystis nidulans can be seen more readily in spheroplasts than in cells with intact walls.

Properties of a major protein released from Escherichia coli by osmotic shock.

A large fraction of a constitutively synthesized polypeptide, comprising 5% of the total Escherichia coli protein, is released when plasmolysed cells are subjected to osmotic shock into ice-cold water. Since the protein is not liberated by the conversion of cells to spheroplasts, it is not a typical periplasmic protein. A complex pattern of association with the cell envelope indicates that it is bound to this structure in vivo. Its susceptibility to trypsin and its interaction with specific antibodies vary with the type of preparations used. Based on these observations, we postulate a peripheral location at the inner surface of the plasma membrane. The protein has been purified to homogeneity from osmotic shock fluid. It has a mass of 44 000 daltons. Some of its physical and chemical properties have been investigated. Most remarkable are its strongly aggregating and adhesive characteristics and its precipitation by vinblastine and calcium ions. These unusual properties, its presumed location, and the observation that it is present in large amounts (approximately 70 000 molecules per cell) suggest a structural role for this protein.

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.