Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis. Denaturation mapping by electron microscopy.

Electronmicroscopic observation of the denaturation pattern of 130 partially denaturated linear mitochondrial DNA molecules from Saccharomyces carlsbergensis was used to investigate the distribution of AT-rich sequences within the mitochondrial genome. The molecules were observed after heating to 43 degrees C in the presence of 12% formaldehyde. These conditions resulted in an average denaturation per molecule of 21%. The average length of the molecules was 10 mum, and a few molecules had a length corresponding to the size of the complete genome. The undenaturated regions varied in length from 0.1 to 5.0 mum with denaturated regions of length 0.02 to 0.1 mum in between. A denaturation map was constructed by use of one of the long molecules (28.7 mum) as a master molecule for positioning of all other molecules. This map shows distinct regions corresponding to the position of easily denaturated sequences in the mitochondrial DNA. These sequences which presumably correspond to the very AT-rich regions, known to exist in the yeast mitochondrial DNA, were found at intervals of about 0.5 - 3 mum on the map.     

Heterogeneity of mitochondrial DNA from Saccharomyces cerevisiae and genetic information for tRNA.

Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant were analyzed by hybridization of several tRNAs on DNA fragments of different buoyant density, obtained by sonication and fractionation on a CsCl gradient. The hybridization patterns show that the genes for tRNAser, tRNAphe, tRNAhis, tRNAval, tRNAileu are present on wild-type mitochondrial DNA, while only genes for tRNAser and tRNAhis are present on petite mitochondrial DNA; moreover the hybridization patterns indicate that these genes are not clustered and suggest that more than one gene might exist for tRNAser and tRNAhis.     

Subunit composition of mitochondrial F1-ATPase isolated from Saccharomyces carlsbergensis

1. The subunit stoicheiometry of mitochondrial F1-ATPase from yeast (Saccharomyces carlsbergensis), grown in the presence of [3H]leucine and uniformly labelled [14C]glucose, has been determined. 2. The stoicheiometry on the basis of radioactivity is : alpha: beta: gamma: epsilon = 3 : 3 : 1 : 1. The amount of the smallest subunit, epsilon, could not be measured by this method. 3. The molecular weights of the subunits, determined by urea-SDS gel electrophoresis, are 53 000, 50 000, 33 000, 12 500 and 6500, respectively. The calculated molecular weight of the ATPase is 360 000, assuming the presence of one epsilon subunit per F1. 4. The amino acid composition of the total ATPase and of the individual subunits has been determined. 5. The aurovertin-binding properties of F1 are discussed in relation to the subunit stoicheiometry.     

Electron microscopic and renaturation kinetic analysis of mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Mitochondrial DNA isolated from a series of nine petite yeast strains and from the parent grande strain was characterized by electron microscopic and renaturation kinetic analysis. The mtDNA² from all strains contained a variety of branched molecules which may be intermediates of replication or recombination. Although no circles were observed in the grande mtDNA, all the petites contained circular mtDNA molecules. The size distribution of the circles conformed to an oligomeric series that was characteristic for each strain. In seven petites, the length series could be related to a single circle monomer size, ranging from 0.13 μm to 5.5 μm; and in two petites to two or more circular monomer lengths. In contrast to circular mtDNA, linear molecules showed no unique size distribution. Circle monomer lengths were linearly related to the kinetic complexity (κ₂ or $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of sheared total mtDNA in the seven petite strains that contained a predominant single series of circle lengths. Thus in each of these petite strains the circle monomer length defined the same DNA sequence present in the linear DNA molecules of non-unique length.     

Comparison of the fine structure of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis: electron microscopy of partially denatured molecules.

Denaturation-maps of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis have been derived from electron microscopic observations of partially denatured complete circular molecules and large fragments of these circles. The mitochondrial DNA from the two species differ by 6% in total length, but seems from the maps to contain some regions of apparent close homology. The cleavage pattern of the two DNAs by the restriction endonuclease EcoRI is compared by gel electrophoresis.     

Extrachromosomal circular ribosomal DNA in the yeast Saccharomyces carlsbergensis.

Purified ribosomal DNA from Saccharomyces carlsbergensis contains a small proportion of circular DNA molecules with a contour length of 3 micron or integral multiples thereof. Hybridization of yeast ribosomal DNA with 26 S rRNA, using the R-loop technique, reveals that these circular molecules contain sequences complementary to yeast ribosomal RNA. We suggest that these extrachromosomal rRNA genes may be intermediates in the amplification of rRNA genes in yeast.     

Proteolytic enzymes of Saccharomyces carlsbergensis

1. Of four proteolytic enzymes isolated from autolysing Saccharomyces carlsbergensis, one is inactivated at about 45 degrees C, whereas the others are stable at 50 degrees C. pH optima for activity are from 3.0 to 8.0 but maximum stability is between pH6.0 and 6.5. All appear to be glycoproteins, the carbohydrate moiety containing glucose and mannose residues. 2. Lysed protoplasts of the same yeast release four proteolytic enzymes each of which have two pH optima at pH3.0 and 7.0 approximately. Compared with the enzymes from autolysed yeast, resistance to high temperature is much less, and they are not glycoprotein in nature. 3. The same yeast grown with N-acetyltyrosine ethyl ester as nitrogen source secretes into the medium four proteases believed to be glycoprotein in nature. Generally they resemble the enzymes from lysed protoplasts more than those from autolysing yeast.     

Letter: Electron microscopy of pyruvate kinase crystals from Saccharomyces carlsbergensis

Crystals of pyruvate kinase have been analysed by electron microscopy, optical diffraction and filtering, and the following parameters were obtained: $\text{2a = 93}\text{A}\text{?}$, $\text{2b = 126}\text{A}\text{?}$, $\text{l = 35·2}\text{A}\text{?}$. A comparison of these data with the parameters obtained from small-angle X-ray scattering measurements indicates that two molecules of the tetrameric enzyme are arranged in one packing unit.     

Physico-chemical properties of thiamine kinase from Saccharomyces carlsbergensis yeasts

The amino acid composition and intrinsic fluorescence were studied in thiamine kinase (ES 2.7.6.2) of brewer's yeast. The enzyme molecule is characterized by higher concentrations of amino acids which promote alpha-helix formation of the protein globule, the amount of residues (cysteine, proline) either binding or folding polypeptide chains being considerably high. Amino acids of middle and low hydrophobicities were the most frequent among the amino acid residues with nonpolar R-groups. The value for the protein isoelectric point was 6.21. The eigen pH value and isoionic point were in good agreement with the isoelectric point value and amounted to 6.28. The fluorescence spectrum has a maximum at 328 nm, half-width at 53 nm and a quantum yield at 0.14 nm. The tryptophane residues were located in hydrophobic surroundings, unexposed to anion quenchers and almost unexposed to cation ones. The fluorescence and phosphofluorescence parameters were sensitive to the conformational changes in the molecule. At pH of 5-9 the protein conformation remained unchanged. The temperature rise above 40 degrees C resulted in a disturbance in the nativity of the globule. The elevation of the enzyme concentration from 0.05 to 1 mg/ml increased the polarization degree from 0.115 to 0.194, the quantum yield and the spectrum position remaining unchanged. The results obtained develop knowledge of the equilibrium system of oligomerous forms of thiamine kinase with different catalytic properties.     

The specificity of induction of alpha-galactosidase from Saccharomyces carlsbergensis

A number of sugars and derivatives have been tested for their ability to induce the synthesis of alpha-galactosidase from Saccharomyces carlsbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-alpha-D-galactoside, lactose and isopropyl-beta-D-thiogalactoside were able to act as inducers. Of these, methyl-alpha-D-galactoside, lactose, isopropyl-beta-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10(7) cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10(7) cells at 40 mM), isopropyl-beta-D-thiogalactoside (43 mU/10(7) cells at 60 mM) and methyl-alpha-D-galactoside (25 mU/10(7) cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-beta-D-thiogalactoside and about 5-fold higher for methyl-alpha-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.     

Purification and characterization of an alpha-glucosidase from Saccharomyces carlsbergensis.

alpha-Glucosidase (EC 3.2.1.20) was purified to homogeneity from logarithmically growing cells of Saccharomyces carlsbergensis. The purification involved the following steps: (a) ammonium sulfate fractionation; (b) Sephadex G-100 chromatography; (c) DEAE-cellulose chromatography; and (d) hydroxylapatite chromatography. This procedure gave a preparation judged to be greater than 98% pure by Na-DodSO4-polyacrylamide gel electrophoresis. The enzyme was shown to be a monomer of 63 000 daltons by gel filtration on Sephacryl S-200 under native conditions and by polyacrylamide gel electrophoresis under denaturing conditions. The Km values of the enzyme for the substrates maltose and p-nitrophenyl alpha-D-glucoside were found to be 1.66 X 10(-2) and 3.1 X 10(-4) M, respectively. The corresponding Vmax value for maltose was 44.8 X 10(-6) mol min(-1) mg(-1) and that for p-nitrophenyl alpha-D-glucoside was 134 X 10(-6) mol min-1 mg-1. The pH optimum for the purified enzyme was found to be between pH 6.7 and 6.8. The enzyme has an absolute anomeric specificity for alpha-glycosidic linkages and appears to recognize a glucosyl residue in alpha linkage on the nonreducing end of its substrate. For the strain used in this study, which carries the MAL 6 locus, only a single form of the enzyme was detected.     

Ribonuclease isolated from yeast (Saccharomyces carlsbergensis): Characterization and properties

The homogeneity of a purified ribonuclease from brewers' yeast was determined by velocity sedimentation and polyacrylamide gel electrophoresis techniques. The velocity sedimentation pattern gave a single peak with a S_app 3.46 and polyacrylamide gel electrophoresis showed one major band. The absorption spectrum of the enzyme showed maximum absorption at 277–278 nm and minimum at 252 nm. The enzyme was relatively stable to extreme pH values and high temperature. Both NaCl and KCl increased the enzyme activity whereas enzyme was inhibited by divalent metal ions. The inhibition of the enzyme was increased in the order of Ca²⁺ > Mg²⁺ > Fe²⁺Cu²⁺. Chemical modification studies of the enzyme showed that tryptophan residues and disulfide bonds were required for enzyme activity.     

Analysis of histones from the yeast Saccharomyces carlsbergensis.

Basic chromosomal proteins were isolated from the chromatin of the yeast Saccharomyces carlsbergensis by extraction with H2SO4 and were purified by ion-exchange chromatography. Electrophoresis of the purified fraction on acetic acid/urea gels revealed the presence of four main components. These four proteins were identified as histones H2A, H2B, H3 and H4 on the basis of their amino acid composition, molecular weight and solubility properties, all of which are very similar to the corresponding properties of the various histone proteins from other eukaryotic organisms. A fifth basic protein could be isolated from yeast chromatin by extraction with HClO4. The available evidence indicates this protein to be an H1-type histone. Yeast thus appears to contain a complete set of histone proteins which are strongly homologous to the histones occurring in higher eukaryotes.