Immunoprecipitation distinguishes non-overlapping groups of snRNPs in Schizosaccharomyces pombe.

The large number of snRNAs in the fission yeast Schizosaccharomyces pombe can be divided into four non-overlapping groups by immunoprecipitation with antibodies directed against mammalian snRNP proteins. 1) Of the abundant snRNAs, anti-Sm sera precipitate only the spliceosomal snRNAs U1, U2, U4, U5 and U6. Surprisingly, three Sm-sera tested distinguish between U2, U4 and U5 and U1 from S.pombe; one precipitating only U1 and two precipitating U2, U4 and U5 but not U1. 2) A group of 11 moderately abundant snRNAs are not detectably precipitated by human anti-Sm sera, but are specifically precipitated by monoclonal antibody H57 specific for the human B/B' polypeptides. From Aspergillus nidulans this antibody also precipitates at least 12 snRNAs. 3) Anti-(U3)RNP sera do not precipitate the above snRNAs, but precipitate at least 6 further snRNAs, including the homologues of U3. Both the anti-(U3)RNP sera and H57 also efficiently precipitate a number of discrete non-capped RNAs. 4) A small number of additional snRNAs are not detectably precipitated by any anti-serum tested to date, further analysis may identify antisera specific for these snRNPs. Western blots of purified snRNP proteins were used to identify the S.pombe proteins responsible for these immunoprecipitations. Several Sm-sera decorate a 16.3kD protein which may be a D protein homologue, monoclonal H57 decorates a further protein of 16kD and an anti-(U3)RNP serum decorates the homologue of the 36kD U3-specific protein, fibrillarin.

Occurrence of peroxisomal membrane proteins in methylotrophic yeasts grown under different conditions.

We have studied the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained indicated that no significant ultrastructural differences existed between the membranes of variously grown cells. The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate-polyacrylamide gels of purified microbody membranes isolated from methanol-grown Hansenula polymorpha, prominent protein bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph, Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D-alanine instead of ammonium sulphate as the nitrogen source.

Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris.

The secreted glycoproteins of Pichia pastoris contain more than 35% of their N-linked oligosaccharides as structures smaller than Man14GlcNAc2 (Man = mannose; GlcNAc = N-acetylglucosamine). On heterologous invertase produced in P. pastoris, approximately 85% of the oligosaccharides are in the size range Man8-14GlcNAc2. The structures appear to contain alpha-linked mannose. In addition, one-third of the structures contain net negative charge and can be radio-labelled in vivo with 32P. The largest oligosaccharides isolated from P. pastoris are significantly shorter than the hypermannosylated structures typical of S. cerevisiae, indicating that the factors which influence the processing of N-linked oligosaccharides in P. pastoris are different from those which influence processing in S. cerevisiae. The smaller N-linked oligosaccharides synthesized by P. pastoris resemble high-mannose oligosaccharides synthesized by animal cells, and this finding increases the utility of P. pastoris as a host for the production of heterologous glycoproteins.

Influencia de la ingestión de biomasa de Kluyveromyces fragilis sobre la actividad de las disacaridasas intestinales en ratas / Influence of Kluyveromyces fragilis biomasa intake on the intestinal dissacharidase activity in rats

Se evaluó la influencia de la ingestión de Kluyveromyces fragilis en dietas para ratas con 20 y 40 % de la levadura como fuente de proteinas sobre la actividad de las disacaridasas intestinales: lactasa, maltasa, sacarosa y trealasa en 4 niveles sucesivos de ubicación de la microvellosidad intestinal: luminal, membrana, enterocito y actividad total, para lo cual se utilizó una técnica de lavados y homogeneizados en fragmentos de intestinos tomados en la zona del ángulo de Treittz, en un total de 67 ratas agrupadas según la dieta recibida en a) grupo con pienso para roedores, b) grupo con caseina como fuente de proteinas, c)grupo con levadura al 40% durante un período experimental de 90 dias. Al comparar los resultados de las actividades enzimáticas entre los grupos estudiados, no se encontraron diferencias significativas para ninguna de las enzimas analizadas ni en ninguno de los niveles de localización en el enterocito

Cu(I) binding to the Schizosaccharomyces pombe gamma-glutamyl peptides varying in chain lengths.

The metal-gamma-glutamyl peptide complex of Schizosaccharomyces pombe is an oligomer of peptides of the general structure (gamma-Glu-Cys)n-Gly with n defining the number of dipeptide repeats. The complexes induced with either cadmium or copper salts are heterogeneous with respect to the number of repeat units or n. Peptides isolated from two preparations of the Cd-gamma-Glu complex by reverse-phase HPLC at low pH were of an n range of 2 to 6 with n3 and n4 peptides being predominant. In addition to peptides of the mentioned structure, peptides of n3 and n4 without the terminal Gly were isolated. These n3 and n4 desGly peptides were present in an abundance of about 10-20% of the concentration of the parent peptide. Peptides of unique n were studied in Cu(I) reconstitution experiments in an attempt to understand the significance of the peptide length heterogeneity in the oligomeric metal-thiolate cluster. Cu-gamma-Glu complexes were formed with each peptide as determined by the characteristic 260-nm shoulder in the ultraviolet absorption spectrum and luminescence indicative of Cu(I)-thiolate coordination in a solvent-inaccessible environment. Cluster formation also occurs with desGly peptides, so the carboxyl-terminal Gly is not critical for cluster formation. Maximal Cu binding stoichiometry with n3 and n4 peptides was markedly less than the maximal Cu(I) stoichiometry of a peptide mixture or the native complex. Cu ions in complexes formed with unique n peptides were more reactive with bathocuproine than Cu ions in complexes with a peptide n mixture. The results suggest that metal-peptide complexes consisting of peptides differing in n probably exist and not all metal-peptide complexes have the same n peptide constituents.

[Variation of the sterols from Kluyveromyces lactis and from a resistant mutant by culture in the presence of sublethal doses of amphotericin B]. / Variation des stérols chez Kluyveromyces lactis et chez un mutant résistant par culture en présence de doses sublétales d'amphotéricine B.

A mutant strain of Kluyveromyces lactis resistant to amphotericin B and weakly to nystatin has been isolated from subcultures of the wild strain grown in the presence of sublethal doses of amphotericin B. The mutant and the wild strain were equally sensitive to pimaricin, filipin, and candicidin. The efficacy of fungizone was very low. In comparison with the wild strain the level of sterols was two times lower in the resistant strain but the composition of these sterols was about the same in the two strains. The action of sublethal doses of amphotericin B on the composition of the sterols was the same in these two yeasts and brought a 40% decrease of the total sterol level and a modification in their distribution. This variation cannot fully explain the resistance of the yeast but it may be associated to other changes of the membranes.

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.

Studies on the gamma-glutamyl Cu-binding peptide from Schizosaccharomyces pombe.

The gamma-glutamyl peptide induced in Schizosaccharomyces pombe in response to metal stress has been purified following exposure of the organism to cadmium and copper salts. Induction of the peptide enables S. pombe to proliferate in media containing high concentrations of cadmium and copper. Two Cd-gamma-Glu peptide complexes are produced which differ in the content of acid-labile sulfur. One Cu-gamma-Glu peptide complex is induced, and it lacks acid-labile sulfur in the metal-binding cluster. The peptides are composed of repeating dipeptide units of gamma-Glu-Cys with a carboxyl-terminal glycine with heterogeneity observed in the repeat unit n. The number of repeats averages 3.2 and 3.8 for the Cd-peptides I and II and 3.6 for the Cu-peptide, in the case of the Cu-complex peptides with n values from 2 to 4 were separated by reverse phase high pressure liquid chromatography. The Cu-gamma-Glu peptide complex is oligomeric, but the exact number of peptide units per complex is not known. The copper binding stoichiometry averages 2.3 g atoms of Cu/mol of peptide, whereas Cd-peptides I and II average 1.8 and 2.7 mol eq of Cd(II)/peptide unit. The pH of half-dissociation of Cu ions from the gamma-Glu peptide is near 1.3, whereas pH values of 4 and 5.4 are sufficient for half-displacement of Cd ions from the sulfide-containing and -lacking peptides II and I, respectively. In the Cu-peptide complex copper is bound as Cu(I) as the complex exhibits luminescence characteristic of Cu(I)-S chelation. The luminescence emission peaks at 619 nm with a corrected excitation peak centered at 290 nm. The luminescence of the Cu-complex indicates the clustering of Cu(I) ions within a solvent-inaccessible complex. The complex is air-labile as the luminescence emission is gradually lost upon air exposure.

[A method for S-adenosylmethionine determination in extracts of microbial cells]. / Metod opredeleniia S-adenozilmetionina v ékstraktakh kletok mikroorganizmov.

A technique for quantification of S-adenosylmethionine in microbial cell-free extracts is proposed that involves dilution of S-adenosyl-L-(methyl-3H)methionine with non-labelled S-adenosylmethionine followed by DNA-cytosine-methyltransferase reaction. The content of S-adenosylmethionine and the activity of S-adenosylmethionine synthetase in yeasts and E. coli MRE-600 are in good agreement with the results obtained with labelled L-methionine and consistent with literature data. The sensitivity of the technique is about 0.1 nmol/0.1 ml of the reaction mixture (10(-6) M). The error was about 5% in every series of experiments. However, the combined use of different DNA-methyltransferase preparations resulted in a higher experimental error (up to 15%), which should be taken into consideration.

Method for estimating activity of killer toxin from Kluyveromyces lactis.

At the exponential growth phase, nystatin-treated Saccharomyces cerevisiae cells stain with Rhodamine B if they have not previously been exposed to killer toxin produced by Kluyveromyces lactis. This finding constitutes the principle of a method for estimating the activity of this toxin.

Identification and isolation of core histones from Schizosaccharomyces pombe.

Core histones have been isolated from Schizosaccharomyces pombe and compared electrophoretically to core histones from Saccharomyces cerevisiae and rat liver. The molecular masses of all cognate histones examined were found to be very similar as determined by SDS gel electrophoresis. Histones H3, H2A and H2B from Sch. pombe migrated almost identically to their respective counterparts from S. cerevisiae as determined by acid/urea gel electrophoresis. Two-dimensional gel electrophoresis with a Triton X-100 acid/urea gel in the first dimension followed by an SDS gel in the second dimension was used to separate Sch. pombe histones from contaminating ribosomal proteins.

Structural study of phosphomannan-protein complex of Citeromyces matritensis containing beta-1,2 linkage. Application of partial acid degradation and acetolysis techniques under mild conditions.

The phosphomannan-protein complex of Citeromyces matritensis IFO 0651 strain was investigated for its chemical structure by a sequential degradation procedure, partial acid degradation followed by acetolysis under mild conditions. Upon treatment with 10 mM HCl at 100 degrees C for 1 h, this complex released mannotriose and mannotetraose consisting solely of 1,2-linked beta-D-mannopyranosyl residues, ca. 20% on weight basis of the parent complex. The acid-degraded complex was then subjected to acetolysis using an acetolysis medium of low sulfuric acid concentration, a 100:100:1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 36 h. A phosphate-containing manno-oligosaccharide fraction eluted in the void-volume region of a Bio-Gel P-2 column was found to consist of Manp beta 1----2Manp beta 1----2Manp alpha 1----2Man to which 1 mol of phosphate group was attached, while a manno-oligosaccharide fraction eluted in the diffusable region was a mixture of Manp beta 1----2Manp beta 1----2Manp beta 1----2Manp alpha 1----2Man, Manp beta 1----2Manp beta 1----2Manp alpha 1----2Man, Manp beta 1----2Manp alpha 1----2Man, Manp alpha 1----2Man, and mannose in the molar ratio of 0.08:0.33:0.19:0.32:1.00. Therefore, the structural analysis of the polysaccharide moiety of a beta-1,2 linkage-containing phosphomannan-protein complex of fungal origin can be achieved by means of a sequential degradation procedure, partial acid degradation followed by acetolysis under mild conditions.

Pulse radiolysis study of a yeast cytochrome c from Hansenula anomala.

The reduction of Hansenula anomala yeast cytochrome c by e-aq and CO-.2 was investigated by pulse radiolysis, at a high reductant to protein concentration ratio. The reactivity of the radicals was studied by observing absorbance changes in the cytochrome c spectrum over the wavelength range 280-600 nm. At pH 7, over the time scale of the radical decays (i.e. 0-4 microseconds for e-aq; 0-40 microseconds for CO-.2s) and beyond, the hemoprotein was reduced without any spectrally detected intermediate between ferri-and ferro-forms. This conclusion was reached by simulation studies based on the direct reduction of the yeast cytochrome c from the ferri- to the ferro-form, yielding a correct fit between experimental and calculated absorbance curves. The reduction rate constants were determined to be 1.0 +/- 01 X 10(10) M-1 S-1 for e-aq and 0.7 +/- 0.05 X 10(9) M-1 S-1 for CO-.2 at 0.16 M ionic strength, pH 7.0 and 20 degrees C, thus not significantly different from other values reported for horse heart cytochrome c. However, in the 360-390 nm region the generation of an additional radical species was noticed. The present experimental data were compared with previously published reports.

Acetolysis of Pichia pastoris IFO 0948 strain mannan containing alpha-1,2 and beta-1,2 linkages using acetolysis medium of low sulfuric acid concentration.

To obtain manno-oligosaccharides containing beta-1,2-linked nonreducing terminal groups from the mannan of Pichia pastoris IFO 0948 strain by acetolysis, an attempt was made to establish the reaction conditions under which cleavage of the alpha-1,6 linkage took place preferentially leaving manno-oligosaccharides composed largely of beta-1,2 linkages. By the action of an ordinary acetolysis medium, a 10/10/1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 13 h or at 25 degrees C for 120 h, the O-acetyl derivative of this mannan gave mannose, mannobiose, mannotriose, and mannopentaose. However, treatment of the same O-acetyl mannan with a 50/50/1 (v/v) acetolysis medium at 40 degrees C for 15 h gave a mannotetraose in addition to mannose, mannobiose, mannotriose, and mannopentaose. Use of a 100/100/1 (v/v) acetolysis medium at 40 degrees C for 36 h gave a more satisfactory result, a mixture of oligosaccharides, from mannose to mannopentaose, which contained more mannotetraose than mannopentaose. Because both mannotetraose and mannopentaose contained alpha-1,2 and beta-1,2 linkages, it was concluded that an acetolysis medium containing a low concentration of sulfuric acid, up to 0.5% (v/v), facilitates the preferential cleavage of the alpha-1,6 linkage, leaving manno-oligosaccharides containing the beta-1,2 linkage which was found to be labile to the action of the 10/10/1 (v/v) acetolysis medium.

Long-chain fatty acid composition of selected genera of yeasts belonging to the Endomycetales.

The cellular long-chain fatty acids of 36 strains representing 18 genera of the Saccharomycetace, Endomycetaceae, Metchnikowiaceae, Saccharomycodaceae, Schizosaccharomycetaceae and Dipodascaceae were extracted and analyzed as methyl esters by gas chromatography. On the basis of their fatty acid content the set of strains was divided into 6 groups, coinciding with the above families. The members of the Saccharomycetaceae (group I) had a high percentage of oleic acid while the strains classified under the Endomycetaceae (group II) and Metchnikowiaceae (group III) were characterized by oleic acid and linoleic acid as major fatty acids. The Saccharomycodaceae (group IV) had the highest percentage of palmitoleic acid. The Schizosaccharomycetaceae (group V) had the highest percentage of oleic acid, while the Dipodascacea (group VI) were characterized by a high percentage of linoleic acid.

Primary structures of ribosomal protein YS25 from Saccharomyces cerevisiae and its counterparts from Schizosaccharomyces pombe and rat liver.

Protein YS25 and its counterparts, SP-S28 and rat S21 [nomenclature according to Sherton, C. C., & Wool, I. G. (1972) J. Biol. Chem. 247, 4460-4467], from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and rat liver cytoplasmic ribosomes, respectively, were sequenced by a combination of various enzymatic digestions and/or chemical cleavage. Proteins YS25 and SP-S28 consist of 87 amino acid residues, and rat S21 consists of 83. The amino termini are all N alpha-acetylated. The amino-terminal halves of the protein molecules are highly conserved (73-85% homologies) in contrast to the carboxy-terminal parts. Overall, rat S21 is 54% homologous to YS25 and 57% to SP-S28, despite a 76% homology between YS25 and SP-S28. Direct comparison with the available prokaryotic ribosomal protein sequences did not reveal any significant homology.

Kluyveromyces lactis killer toxin inhibits adenylate cyclase of sensitive yeast cells.

K1 killer toxin secreted by the K1 strain of Saccharomyces cerevisiae, has been well characterized. It is a simple protein of molecular weight (MW) 11,470 (ref. 3), encoded by a double-stranded, linear RNA plasmid, called M RNA, of MW 1.1-1.7 x 10(6) (refs 4-6). It is lethal to sensitive Saccharomyces cerevisiae which does not carry M RNA. Leakage of K+ and ATP is the first distinct response in sensitive cells, and the toxic action is thought to be due to its action as a protonophore or K+ ionophore. Recently, a further killer toxin has been found in Kluyveromyces lactis IFO 1267, and it is associated with the presence of the double-stranded linear DNA plasmids, pGK1-1 (MW 5.4 x 10(6)) and pGK1-2 (MW 8.4 x 10(6)). It has been shown, by curing pGK1-1 or deletion mapping, that the structural gene for the killer toxin and immunity-determining gene reside on the smaller plasmid. Moreover, the plasmids could be transferred from K. lactis to S. cerevisiae by protoplast fusion and protoplast transformation. As the K. lactis toxin is encoded by a DNA plasmid and has a relatively wider action spectrum than K1 killer toxin, the mode of action of the toxin is highly interesting. Here we report that K. lactis toxin inhibits adenylate cyclase in sensitive yeast cells and brings about arrest of the cells at the G1 stage.

Intracellular pH topography: determination by a fluorescent probe.

The distribution of pH inside the yeast Endomyces magnusii was measured at 1 micron resolution using different external pH values. In a neutral buffer the pH of the cytoplasm was 6.7-7.2 in the center, decreasing to 6.0 toward the periphery of the cell. A decrease of external pH was followed by a gradual uniform decrease of internal pH. Using a comparison of the 'pH map' with phase-contrast picture of the same cell, the pH of the vacuoles was estimated to be 5.5-5.6.

[Characteristics of microorganisms subjected to the action of a vacuum]. / O nekotorykh osobennostiakh mikroorganizmov, podvergnutykh deistviiu vakuuma.

The object of this work was to study the effect of vacuum on Endomyces magnusii, Serratia marcescens, Escherichia coli and Mycobacterium luteum. The zone of tolerance to the water activity was determined for the intact cells of E. magnusii and for the cells subjected to vacuum. Suspensions of the above cells were studied by UV spectroscopy with the aim of detecting changes in the permeability of cell membrane after the action of vacuum.