Infect Immun, 1977 Mar, 15(3), 789 - 95 Adoptive transfer of immunity from mice immunized with ribosomes or live yeast cells of Histoplasma capsulatum; Tewari RP et al.; This investigation was designed to compare the role of lymphoid cells and immune serum in protective immunity induced by immunization with ribosomes or live yeast cells of Histoplasma capsulatum . Spleen cells, peritoneal cells, and serum from C3H mice immunized with Histoplasma ribosomes or live cells were transferred intravenously to separate groups of syngeneic recipients . All recipients along with a set of immunized and control mice were challenged intravenously with 4 x 10(6) yeast cells of H . capsulatum, and protection was assessed . Immunization with ribosomes or live cells provided 90 to 100% protection . Mice receiving filtered spleen cells or peritoneal cells from donors immunnized with live cells showed 90 to 100% protection; 80 to 90% protection was observed for mice receiving cells from ribosome-immunized donors . In contrast, no evidence of protection was seen in mice receiving serum from either live-cell- or ribosome-immunized mice . Peritoneal cells were far more efficient than spleen cells in adoptive transfer of immunity . The adoptive immunity in recipients persisted for at least 3 weeks after transfer, the longest period tested in the present study . These results indicate that the immunity elicited by immunization with Histoplasma ribosomes or live cells is mediated by a cellular mechanism. Biometrics, 1977 Mar, 33(1), 113 - 20 Fitting a model to the growth of yeast colonies; Gani J et al.; When yeast cells reproduce, scars are left on the parent cell where the offspring has budded . Using a branching process model, it is possible to obtain the expectations of numbers of cells with 0, 1, 2, .. . offspring . In this paper, theoretical results are tested against empirical data for three types of yeast cells . We examine the hypothesis that birth and death rates of cells with no previus offspring may differ from those of cells with one or more offspring . It is suggested that the oscillatory empricial results for the proportions of cells with 0, 1, 2, 3 and 4 offspring may be due to different mean budding times for these cells. Eur J Biochem, 1977 Mar 1, 73(2), 553 - 6 Studies on a carboxypeptidase Y mutant of yeast and evidence for a second carboxypeptidase Activity; Wolf DH et al.; Immunological studies on the carboxypeptidase Y mutant prcl-l of Saccharomyces cerevisiae revealed the origin of mutation in the structural gene of carboxypeptidase Y . The absence of carboxypeptidase Y has no effect on growth, even after drastic changes of growth conditions . A double mutant (prc 1- leu2-) lacking carboxypeptidase Y and auxotrophic for leucine is able to grow on the peptide benzyloxycarbonylglycylleucine (Cbz-Gly-Leu) as sole nitrogen source, indicating the existence of a second carboxypeptidase . Using a new peptidase test, the existence of this second enzyme, called carboxypeptidase S, was confirmed biochemically. Cell, 1977 Mar, 10(3), 453 - 62 Number and distribution of polyadenylated RNA sequences in yeast; Hereford LM et al.; The poly(A)-containing RNA, isolated from the budding yeast Saccharomyces cerevisiae, has been characterized with regard to the number and distribution of sequences by a kinetic analysis of RNA-cDNA hybridization . In agreement with results previously obtained on metazoan eucaryotes (Bishop et al., 1974), discrete complexity classes were observed . There exist low, medium, and high complexity classes which contain approximately 20, 400, and 2400 sequences, respectively . This measurements of the number of sequences has been verified by hybridization with single copy DNA . 20% of the single copy fraction of the yeast genome is rendered double-stranded by poly(A)-containing RNA . Assuming asymmetric transcription, this is equivalent to approximately 4000 poly(A)-containing sequences, verifying the results obtained with RNA-cDNA hybridization . In addition, the first-order kinetics of the hybridization with single copy DNA verified the notion that most of the sequence complexity is present at the same intracellular concentration . The same number and distribution of sequences were found in poly(A)-containing polysomal RNA and in total RNA, suggesting that most or all of the sequence complexity is on polysomes and is adenylated . The results indicate that RNA-cDNA hybridization is an accurate method for determining sequence complexity values and that yeast, grown under vegetative conditions, has 3000-4000 different mRNA sequences. Biophys J, 1977 Mar, 17(3), 205 - 12 Inositol-less death in yeast results in a simultaneous increase in intracellular viscosity; Keith AD et al.; Inositol auxotrophs of yeast developing on isositol-deficient medium continue protein synthesis for 4-6 h, lose viability rapidly after 6 h, and show an increase in cytoplasmic viscosity as measured by spin label rotational motion . Cycloheximide prevents the rapid loss of cell viability, stops protein synthesis, and simultaneously prevents an increase in cytoplasmic viscosity . From these observations, we infer that intracellular translational diffusion is upset as a consequence of inositol starvation . Cell death may be caused by a modified intracellular diffusion environment. Biotechnol Bioeng, 1977 Mar, 19(3), 365 - 75 Characteristics of yeast invertase immobilized on porous cellulose beads; Dickensheets PA et al.; Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond . The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1 . Both temperature profiles were fairly similar up to 55 degrees C . However, above this temperature the immobilized enzyme was more stable than the free enzyme . From the temperature data, the activation energies were found to be 7,322 and 4,052 cal/g mol for the free and the immobilized enzyme, respectively . Candida invertase shows characteristics of substrate inhibition . Both the Km and Ki for the free and the immobilized enzymes were determined . The apparent Ki for the immobilized invertase was much higher than the Ki of the free enzyme, suggesting a diffusion effect . Immobilized invertase molecules deep in the pores only see sucrose concentrations much less than the bulk concentrations . Immobilization, thus, offers certain processing advantages in this regard. J Biol Chem, 1977 Feb 25, 252(4), 1471 - 5 Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes; Skogerson L et al.; Factor requirements for yeast and rat liver ribosomes were determined in several different reactions using either yeast or liver factors . In polymerization assays yeast ribosomes required a factor in addition to elongation factor 1 (EF-1) and elongation factor 2 (EP-2) . The third factor (EF-3) requirement was observed with EFs from either yeast or liver for both poly(U)-directed polyphenylalanine synthesis and elongation of endogenous peptidyl-tRNA . No significant effect of EF-3 was observed with liver risomes in either assay . In contrast to results with polypeptide synthesis EF-3 was not required for EF-1 dependent binding of {3H}Phe-tRNA or the translocation-dependent formation of N-acetylphenylalanylpuromycin . Up to 2-fold stimulation of the binding reaction was observed with saturating levels of either yeast or liver EF-1 . No effect of EF-3 was observed on ribosome-EF-2-GDP-fusidic acid complex formation . The data suggest that the yeast EF-3 may be a loosely bound ribosomal protein which is not required for a specific step in the elongation cycle but is involved in the coordination of the partial reactions required for polymerization. J Biol Chem, 1977 Feb 25, 252(4), 1344 - 9 Noncoordinated transcription in the absence of protein synthesis in yeast; Shulman RW et al.; In the yeast Saccharomyces cerevisiae we have carried out a detailed study of the response of transcription to the inhibition of translation . Measurement of the incorporation of labeled bases and nucleosides into the nucleoside triphosphate pools revealed that amino acid deprivation brings about a 10- to 50-fold inhibition of such labeling . Therefore accurate comparisons of RNA synthesis using such precursors are difficult to obtain . To overcome this problem we have turned to the use of L-{methyl-3H} methionine as a precursor, because the labeling of the S-adenosylmethionine pool is relatively unaffected by the rate of protein synthesis . Using this precursor, we have observed that in the absence of protein synthesis the transcription of ribosomal RNA is reduced by 80%, the transcription of messenger RNA is reduced by about 25%, and the transcription of transfer RNA is reduced by less than 20% . These results are obtained when protein synthesis is inhibited either by deprivation of an amino acid or by the addition of cycloheximide . Ribosomal precursor RNA synthesized in the absence of protein synthesis is fully methylated . We conclude that the transcription of rRNA is the primary target of stringent control . Furthermore the inhibition of protein synthesis, itself, may be the trigger for this response. Eur J Biochem, 1977 Feb 15, 73(1), 131 - 40 Structural studies of yeast flavocytochrome b2: cooperative roles of the alpha and beta globules in the formation of the flavin-binding sites; Mevel-Ninio M et al.; The purpose of the study reported here was the localization of the heme binding sites on the two globular fragments, alpha and beta, of the 'cleaved' form of the flavocytochrome b2 chain . These fragments were partially resolved by means of molecular sieving under denaturing conditions (3 M or 6 M guanidine in the presence of 2-mercaptoethanol) . They were then renatured in the presence of excesses of FMN and protoheme . The protoheme was found to be quantitatively bound to the alpha subunit, confirming previous findings . The flavin binds neither to alpha alone nor to beta alone, but only to the reassociated alphabeta protomer . the results are discussed in terms of the possible occurrence of gene fusion in the formation of the complex flavocytochrome chain of this very particular L-lactate cytochrome c reductase found specifically in yeasts. Mol Gen Genet, 1977 Feb 15, 150(3), 265 - 70 The extrachromosomal control of nonsense suppression in yeast: an analysis of the elimination of {psi+} in the presence of a nuclear gene PNM; McCready SJ et al.; When a {psi-} strain of yeast mutates to {psi+}, the efficiency of suppression by certain ochre suppressors is increased . The {psi+} phenotype is inherited extrachromosomally . There is a nuclear gene, PNM, which, when mutant, causes loss of the {psi+} phenotype . PNM- is dominant to PNM+ and a heterozygous diploid gradually loses the ability over successive generations, to produce PNM+ {psi+} spores . This paper describes the kinetics of this elimination and the data obtained are discussed in relation to two models of the molecular nature of the {psi} genetic determinant--one considering the {psi} determinant as an autonomous nucleic acid, the other treating the possibility that the {psi} nucleic acid is that which codes for rRNA in the nuclear genome. Eur J Biochem, 1977 Feb 15, 73(1), 7 - 15 Non-equivalence of the sites of yeast phenylalanyl-tRNA synthetase during catalysis; Fasiolo F et al.; Yeast phenylalanyl-tRNA synthetase, an enzyme with an alpha2beta2 structure, has two active sites for phenylalanine, tRNAphe, phenylalanyladenylate and phenylalanyl-tRNAphe . Determination of phenylalanine binding properties to the free enzyme by equilibrium dialysis shows that only one mole of amino acid binds per mole of enzyme, i.e . absolute negative cooperativity . Binding of the amino acid in the presence of tRNA or of ATP and PPi unmasks the second phenylalanine binding site . The difference between the affinities at the tight and loose binding sites under such conditions is about 10--15 . Titration of phenylalanyladenylate sites by the burst of ATP consumption shows the formation of a (enzyme-phenylalanyladenylate)2 complex in the presence of pyrophosphatase; however, the two sites differ widely in their affinity as shown by dialysis experiments . Measurements of hydrolysis rates of enzyme-bound phenylalanyladenylate suggests that when only the high-affinity adenylate site is occupied, the other protomer can still bind phenylalanine and ATP (in the presence of phenylalanine) . Two moles of Phe-tRNAphe bind to the enzyme with a very high affinity (Kd less than 48 nM) . The presence of millimolar concentrations of ATP, phenylalanine and pyrophosphate triggers negative cooperativity and under these conditions only one mole of Phe-tRNAphe is bound per mole of enzyme with a Kd value of 0.15 muM . The present results give support to interprotomer catalytic cooperativity in the mechanism of action of yeast phenylalanyl-tRNA synthetase. J Biol Chem, 1977 Feb 10, 252(3), 919 - 26 Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase; Kang CH et al.; 1 . The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions . The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c . On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range . 2 . The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions . 3 . Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics . Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site . At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values . At ionic strengths below optimal, binding becomes too strong and above optimal, too weak . 4 . Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site . The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site . 5 . The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively . Both oxidizing equivalents may be discharged at either site . Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c. Biochemistry, 1977 Feb 8, 16(3), 436 - 45 Energetics of the cooperative and noncooperative binding of nicotinamide adenine dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase at pH 6.5 and pH 8.5 . Equilibrium and calorimetric analysis over a range of temperature; Niekamp CW et al.; The binding of nicotinamide adenine dinucleotide (NAD+) to yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) has been studied at pH 6.5 and 8.5, at 5,25, and 40 degrees C, by calorimetry, fluorometry, spectrophotometry, equilibrium dialysis, and flow dialysis . As reported earlier for pH 7.3 (Velick S.F., Baggott, J.P., and Sturtevant, J.M . (1971), Biochemistry 10, 779), the binding is accompanied by enthalpy changes which become rapidly more negative as the temperature increases, with delta Cp = -500 to -750 cal deg-1 (mole of NAD+ bound)-1, and by entropy changes which also, as required by the large negative delta Cp, become rapidly more negative with increasing temperature . The binding data at pH 6.5 can be fitted on the basis of either four identical noninteracting sites, or of four sites showing a small degree of negative cooperativity . The data at pH 8.5, particularly at 40 degrees C, require the introduction of positive cooperativity, as was previously shown by Kirschner et al . (Kirschner, K., Eigen, M., Bittman, R., and Voigt, B . (1966), Proc . Natl . Acad . Sci . U.S.A . 56, 1661), and can be equally well fitted on the basis of a sequential model (Adair, G.S . (1925), J . Biol . Chem . 63, 529) or a concerted model (Monod, J., Wyman, J., and Changeux, J.P . (1965), J . Mol . Biol . 12, 88) . It is proposed that the observed thermodynamic changes are largely the result of a hydrophobic effect due to a decrease in the exposure of nonpolar groups to the solvent, and of a tightening of the protein structure when the coenzyme is bound with concomitant decrease in the number of easily excitable internal degrees of freedom. Biochim Biophys Acta, 1977 Feb 7, 459(2), 290 - 9 Transport of pyruvate and lactate in yeast mitochondria; Briquet M; Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented . 1 . The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited . 2 . The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate . 3 . The {14C}pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate . 4 . Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate . These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA. Mol Cell Biochem, 1977 Feb 4, 14(1-3), 87 - 9 Intramitochondrial synthesis of membrane proteins in yeast: differential inhibition by ethidium; Rogers P et al.; Yeast cells (Saccharomyces cerevisiae) were grown in the presence of {14C}phenylalanine and pulse-labelled with {3H}phenylalanine in the presence of cycloheximide . The proteins extractable into chroloform: methanol (2:1) were isolated from mitochondria and analysed by SDS gel filtration . Four protein fractions varying in molecular weight were separated . In order to identify the transcriptional origin and the site of protein synthesis ethidium bromide was used . Different sensitivity of protein syntheses to various concentrations of ethidium was shown . These data are discussed in relation to the possible presence of two classes of membrane-bound polyribosomes in mitochondria. Mol Cell Biochem, 1977 Feb 4, 14(1-3), 67 - 79 Integration and regulation of mitochondrial assembly in yeast; Mahler HR et al.; The interactions between the mitochondrial and nucleocytoplasmic systems required for mitochondriogenesis have been investigated at several different levels . Those involved in the formation of functional enzyme complexes have been studied using cytochrome oxidase: this multimeric (2 X 7 and 2 X 6 subunits for enzymes from yeast and beef heart respectively) has been resolved, and the mitochondrial contribution has been shown to be dispensible for catalytic function proper . Using novel mutants, with a mitochondrial mode of inheritance, a mitochondrial gene product localized in the oligomycin-sensitive ATPase has been implicated in the assembly not only of this complex, but of cytochrome oxidase as well . Interactions required for the genetic competence of the mitochondrial system have become apparent as a result of studies in the mechanism of action of the highly effective mitochondrial mutagen ethidium bromide . This agent first becomes covalently inserted into mitochondrial DNA and, after its excision, eventually results in extensive degradation of the macromolecule . The excision reaction has now been shown to be performed by a complex between the oligomycin-sensitive ATPase and a DNA-binding protein presumably involved in recognizing the damage . On the level of replication and expression of the mitochondrial genome studies using thermolabile mutants have demonstrated that these processes appear independent of the replication of nuclear DNA but not of its expression. Appl Environ Microbiol, 1977 Feb, 33(2), 231 - 9 Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture; Gill CO et al.; Lipid accumulation of Candida 107, grown at dilution rates from 0.03 to the maximum of 0.21/h, with carbon, nitrogen, phosphate, and magnesium limitations in a chemostat, was maximal at about 40% (wt/wt) with nitrogen-limited medium at a dilution rate of 0.06/h, giving an efficiency of substrate conversion of 22 g of lipid per g of glucose consumed . At higher dilution rates the lipid content decreased . With carbon-limited growth, the highest lipid content (14%, wt/wt) was at the maximum dilution rate . High lipid contents also occurred with phosphate + nitrogen as double limitations of growth, with the lipid content of the yeast (about 35%, wt/wt) continuing to be near maximum at dilution rates also near maximum (0.17/h), thus giving the highest specific rate of lipid formation of any growth conditions (0.59 g of lipid/g of yeast per h) . However, the efficiency of substrate utilization was only 5.2 g of lipid formed per 100 g of glucose consumed . The composition of the fatty acyl residues within the lipid remained constant over many weeks if the steady-state conditions remained unchanged . With carbon-limited growth, the degree of unsaturation of the fatty acids markedly decreased as the dilution rate was increased, but with nitrogen limitation the reverse trend was seen . In all cases, linoleic and oleic acids were the principal fatty acyl residues affected, and their relative proportions always varied in opposite directions . When magnesium was a limiting nutrient, there was a considerable increase in the proportion of myristic acid produced within the lipid . Neutral lipids (predominantly triglycerides) varied from 66 to 92% of the total lipid from carbon- and nitrogen-limited growth; phospholipids (varying from 2 to 25%) were highest in nitrogen-limited growth . The fatty acyl residues within each lipid fraction showed the same variations with changing growth rates. Genetics, 1977 Feb, 85(2), 225 - 47 Kinetics of mutation induction by ultraviolet light in excision-deficient yeast; Eckardt F et al.; We have measured the frequency of UV-induced reversions (locus plus suppressor) for the ochre alleles ade2-1 and lys2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad2-20) . For very low UV doses, both mutational systems exhibit linear induction kinetics . However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm2 (25% survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm2 (4.4% survival) and then declines . This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data . The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems . We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a "single event" Poisson process, and that mutation and killing are not necessarily independent of one another . In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25% less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29% greater than the population average . We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth. Int J Radiat Biol Relat Stud Phys Chem Med, 1977 Feb, 31(2), 121 - 9 Unscheduled DNA synthesis in diploid yeast after X-irradiation in diffent phases of the cell-cycle; Holtz GW et al.; The uptake of radioactive 5'-dTMP into the DNA of diploid yeast cells was measured in the G1 and S-phase of the cell-cycle . In control cells, the uptake is zero in G1 and increases with time in the S-phase . Cells irradiated in early G1 show an uptake (unscheduled DNA synthesis) which is higher than if irradiation is performed later in G1 . An analysis which takes into consideration the incomplete synchronization of the cell population shows that, at the end of G1, no uptake would be present in an ideally-synchronous population . At the end of G1 the shoulder in the dose-effect curve for cell survival also disappears . This provides additional evidence that the shoulder in a dose-effect curve might be due to repair reactions in living cells. Int J Radiat Biol Relat Stud Phys Chem Med, 1977 Feb, 31(2), 113 - 9 Photodynamic action of thiopyronine on polyribosomes and cell-free protein synthesis in yeast; Nishiyama-Watanabe S et al.; The treatment of yeast with thiopyronine (TP) caused a degradation of polyribosomes, even if the cells were not illuminated . In contrast, no differences in the polyribosome profiles of illuminated and unilluminated cells could be seen . Likewise, in in vitro experiments, there was no degradation of polyribosomes caused by dark effect or photodynamic action . Cell-free protein synthesis was inhibited up to 75 per cent by the photodynamic effect when the complete system was treated, but only up to 40 per cent when the polyribosomes or enzyme fraction were treated . Since the enzyme fraction contained aminoacyltRNA-synthetases and tRNA, it was necessary to investigate separately the effect of TP on the enzyme and the tTNA . It was shown that the aminoacyl-tRNA-synthetase and not tRNA was effected by the photodynamic action in its biological activity. J Virol, 1977 Feb, 21(2), 516 - 21 Homology between double-stranded RNA and nuclear DNA of yeast; Vodkin M; The relationship between mycoviral double-stranded (ds) RNA and host cell DNA was investigated . Radiolabeled ds RNA was denatured and reannealed in the presence and absence of denatured DNA . RNA from killer strains of the yeast Saccharomyces cerevisiae and from nonkiller derivatives was utilized . The above-mentioned strains, as well as one that lacks all ds RNA, were sources for extracted DNA . Net hybridization of ds RNA to DNA occurred regardless of the strains from which the respective nucleic acids were prepared. J Bacteriol, 1977 Feb, 129(2), 640 - 50 Killer toxin for sake yeast: properties and effects of adenosine 5'-diphosphate and calcium ion on killing action; Kotani H et al.; The killer character of strain isolated from the main mash of sake brewing which produces a killer substance for sake yeast was transmitted to hybrids of the strain and a standard strain of Saccharomyces cerevisiae through a cytoplasmic determinant . The character was eliminated at 41 degrees C by incubation followed by growth at 30 degrees C . The killer strain produced the killer toxin in a growth-associated manner . A preparation of crude killer toxin extract showed first-order inactivation and a linear Arrhenius plot between 25 and 40 degrees C, with an activation of energy of 55.0 kcal/mol . Addition of 1% of synthetic polymer protected the toxin from inactivation by agitation but not by heat . Enhancement of the killer action toward sensitive yeast cells by only the nucleotide adenosine 5'-diphosphate (ADP) was observed after plating on agar medium as well as after incubation in liquid medium . The addition of CaCl2 reversed the enhancing effect of ADP on killing activity . This action of CaCl2 was inhibited by cycloheximide, suggesting that protein synthesis is required for recovery of toxin-induced cells in the presence of CaCl2 . Further, CaCl2 overcame the decrease in the intracellular level of adenosine 5'-triphosphate (ATP) enhanced by ADP in killer-treated cells and also inhibited leakage of ATP from the cells with immediate response . The mode of killing action is discussed in terms of a transient state of the cells and the action of ADP and CaCl2. J Biol Chem, 1977 Jan 25, 252(2), 504 - 7 Purification and properties of acetyl coenzyme A synthetase from bakers' yeast; Frenkel EP et al.; Acetyl-CoA synthetase, utilized in a coupled reaction system, has been shown to be applicable to the spectrophotometric determination of propionic and methylmalonic acids in biological fluids . The isolation of acetyl-CoA synthetase from yeast is simpler than the purification from mammalian sources . This study also presents some properties of the yeast enzyme and compares it to the more extensively studied enzyme isolated from ammmalian tissue . Isolation and purification yielded a preparation with a specific activity of 44 units/mg at 25 degrees . The purified acetyl-CoA synthetase was apparently homogeneous by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis with an estimated subunit molecular weight of 78,000 . Polyacrylamide gel electrophoresis in the presence of ATP revealed a single protein band which contained all of the enzyme activity . Analytical ultra-centrifuge studies indicated the presence of a single protein with a molecular wright of 151,000 and sedimentation velocity analysis revealed a single peak with a sedimentation coefficient of 8.65 So20,w . Similar to the enzyme from mammalian sources, yeast acetyl-CoA synthetase has a high degree of substrate specificity and is active only on acetate and propionate . In addition, the reaction mechanism, as demonstrated by initial velocity patterns obtained from substrate pairs, appeared to be identical to the enzyme from bovine heart . However, the apparent Michaelis constants for the substrates were significantly different from the mammalian enzyme . The yeast-derived enzyme also differed from the mammalian in terms of molecular weight, amino acid composition, pH optimum, effect of monovalent cations, and stability characteristics . Thus, yeast acetyl-CoA synthetase is more easily purified than the mammalian enzyme and provides an excellent preparation for the assay of propionic and methylmalonic acids. Biochim Biophys Acta, 1977 Jan 24, 496(1), 103 - 14 Effects of glucose and nitrogen source on the levels of proteinases, peptidases, and proteinase inhibitors in yeast; Hansen RJ et al.; In Saccharomyces cerevisiae harvested from early exponential growth on glucose-containing media, the specifc activities of proteinases A and B, carboxypeptidase Y, and the inhibitors IA, IB, IC of these three proteinases, respectively, are found to be 10-30% of the specific activities observed in media without glucose, containing acetate as a carbon source; the activities of two aminopeptidases in glucose-grown cells were 30-50% of those in acetate-grown cells . In contrast to fructose-biphosphatase, phosoenolpyruvate carboxykinase, and cytoplasmic malate dehydrogenase, which are inactivated after the addition of glucose to derepressed cells, the proteinases and inhibitors are not inactivated after glucose addition, but appear to be repressed . Growth of the yeast on poor nitrogen sources or starvation for nitrogen results in 2-3 fold increases in the levels of most proteinases and peptidases, but this effect is not observed with glucose as the carbon source. Biochim Biophys Acta, 1977 Jan 20, 474(2), 188 - 98 The specificity of the chemical modification of N6-delta 2-(isopentenyl)adenosine in purified yeast tRNA Ser; Horvath P et al.; The specific modification of N6-delta 2-(isopentenyl)adenosine in purified tRNA Ser yeast by mild treatment with KMnO4 and I2 was studied . N6-delta 2-(isopentenyl)adenosine in tRNA SER is specifically modified by iodination, providing us with a suitable method for the quantitative determination of N6-delta 2-(isopentenyl)adenosine in tRNA was found to contain 114 +/- 8 pmol/A260nm unit of N6-delta 2-(isopentenyl)adenosine and gave three labelled fractions on an RPC-5 column . The product obtained after KMnO4 treatment of tRNA Ser was not homogeneous . The enzymatic "reisopentenylation" of KMnO4-treated tRNA Ser resulted in the regeneration of only traces of the original molecule(s) . Most of them had been damaged either by the KMnO4 treatment or in the incubation mixture used for "reisopentenylation". Biochemistry, 1977 Jan 11, 16(1), 117 - 21 Relaxation spectra of yeast hexokinases . Isomerization of the enzyme; Jentoft JE et al.; Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8 . The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red . The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6 . The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization . The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1 . The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes . The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity . The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed . These data imply that isomerizing and ionizing forms are sensitive to events at the active site . These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme. Eur J Biochem, 1977 Jan 3, 72(1), 79 - 86 Calorimetric studies on melting of tRNA Phe (yeast); Hinz HJ et al.; The heat effects involved in thermal unfolding of tRNAPhe from yeast have been determined in various buffer systems by direct differential scanning calorimetry . Perfect reversibility of the melting process has been demonstrated for measurements in the absence of Mg2+ ions . The overall molar transition enthalpy, delta Ht = 298 +/- 15 kcal mol-1 (1247 +/- 63 kJ mol-1), has been shown to be independent of the NaCl concentration and the nature of the buffers used in this study . Delta Ht is identical in the presence and in the absence of Mg2+ ions within the margin of experimental error . This experimental result implies a vanishing or very small heat capacity change to be associated with melting . Decomposition of the calorimetrically determined complex transition curves, on the assumption that the experimental melting profile represents the sum of independent two-state transitions, results in five transitions which have been assigned to melting of different structural domains of the tRNA. Folia Microbiol (Praha), 1977, 22(5), 363 - 72 Transport of ribitol and D-glucose in the yeast Candida guillermondii; Miersch J; The uptakes of the linear polyol ribitol and of D-glucose by Candida guillermondii were found to be carrier-mediated and to require metabolic energy . In glucose-grown cells ribitol possibly enters by simple diffusion but after an induction period a specific transport system is synthesized, inhibitable by higher concentrations of arabinitols, xylitol, mannitol and sorbitol . Actidione blocks the synthesis of the inducible ribitol transport system . Two systems of different affinity for substrate were found to operate in the uptake of both glucose and of ribitol . Counter-transport experiments with ribitol, D-glucose and 3-O-methyl-D-glucose support the carrier nature of the uptake system. Biochimie, 1977, 59(1), 97 - 104 Lipids of the yeast Hansenula anomala; Ng KH et al.; An analysis of the free lipids of Hansenula anomala was performed . The main fatty acids obtained by saponification of whole cell crude lipids were palmitic, C18:1, C18:2 and C18:3 acids . In mitochondrial lipids the tri-unsaturated acid was present as traces . Fatty acid composition of each class of lipids was also determined . Phosphatidylcholine and phosphatidylethanolamine were the main phospholipids; phosphatidylserine, phosphatidylinositol and cardiolipin were also characterized . The most abundant sterols were ergosterol and lanosterol . An acetate of a 24-ethyl cholesterol was also isolated . Two glycolipids, a galactosyl diglyceride and a glucosyl ceramide were identified; concerning the galactosyl diglyceride, the content of C18:3 acid was higher than in other lipid classes . In the glucosyl ceramide, the main fatty acid was alpha-hydroxy C18:0 acid; C16:0, C18:1, C18:2 and C18:3 acids were present too . The long chain base was shown to be C18-phytosphingosine (4-hydroxy C18 sphinganine) . Some similarities and differences with Saccharomyces cerevisiae are discussed. Z Allg Mikrobiol, 1977, 17(2), 109 - 15 {Protein crystals and tubuli bundles in yeast cells . VI . Light-and electron microscopy studies as well as biochemical studies on alcohol dehydrogenase activity of isolated crystals}; Kunkel W; Isolated crystals from Saccharomyces carlsbergensis, stabilized by Cd2+ or Zn2+, retain their enzymatic activity as shown by topochemical reactions . During these reactions the crystals disintegrate characteristically . Stages of this disintegration and deposition of reaction products have been demonstrated by light and electron microscopy . Ground plasmatic and mitochondrial alcoholdhydrogenase has been solubilized by means of 0.01 M EDTA from Zn2+ stabilized crystals separated by gel electrophoresis and proved to be active. Bioinorg Chem, 1977, 7(2), 141 - 50 Multiple roles of metal ions in the reaction catalyzed by yeast inorganic pyrophosphatase; Butler LG et al.; Yeast inorganic pyrophosphatase has three roles for metal ions in its reaction: activator, substrate and structural . Out of a wide variety of metal ions tested, only Mg2+, Zn2+, mn2+ and Co2+ can fulfill both the activator and substrate roles . Several other metal ions inhibit the Mg2+-stimulated activity; the strong inhibition by Ca2+ (and probably Cd2+) is due to interference with both activator and substrate roles, while the weaker inhibition by Sr2+ (and possibly Cu2+ and Ni2+) is due to interference with only the substrate role . Rare earth ions strongly stimulate nonenzymic PPi hydrolysis but do not activate the enzyme . Despite its ability to fulfill both the activator and substrate roles . Zn2+ causes inactivation of the enzyme, probably by interference with the "structural" Mg2+ . The results suggest that the three roles for metal ions are independent (an individual metal ion can satisfy only one at a time) and that the metal ion specificity for the three roles declines in the order: structural greater than substrate greater than activator. Z Allg Mikrobiol, 1977, 17(5), 353 - 8 {Comparative morphological studies of yeast cells using automated image analysis}; Muhlig P et al.; The ultrastructural alterations in cells of Candida utilis caused by the influence of copper ions have been studied by means of quantitative image analysis . A model has been proposed which presents the following informations: The main effect of the copper ions is represented by an increase of the volume of the whole cell and of that part of the cell which consists of nucleus, vacuoles, and inclusions (particles and globules) . Nevertheless, neither the absolute volume of mitochondria, nor the density of mitochondria are influenced by high concentrations of copper ions in the culture medium. Antonie Van Leeuwenhoek, 1977, 43(2), 129 - 42 Biosynthesis of the yeast cell wall: selective assays and regulation of some mannosyl transferase activities; Elorza MV et al.; Assays have been developed for some transfer reactions involved in the synthesis of Saccharomyces cerevisiae wall mannoproteins, both in a particulate preparation in the presence of EDTA or Triton X-100, and after lipid extraction with chloroform-methanol at -20 C . The mannosyl transferase activities were also studied in cells made permeable to GDP-mannose by toluene-ethanol treatment ("in situ") . In these permeabilized cells, the glycosylating reactions dependent on lipid carriers (dolichol derivatives) did not function, but those independent of them were unaffected . The lipid-independent mannosyl transferase activities were partially inhibited by nucleotide diphosphates probably in a competitive manner . Increase of the nucleotide diphosphate pool "in vivo" might slow down the speed of the transfer reactions carried out by the mannan synthetase system. Biochimie, 1977, 59(4), 381 - 91 {Primary structure of tRNA Thr 1a and b from brewer's yeast}; Weissenbach J et al.; One of the two major species of brewer's yeast tRNA threonine (tRNA Thr 1) has been purified by countercurrent distribution followed by two chromatographic steps (respectively on a Sepharose 4B and a BD-cellulose column) . Complete digestion with pancreatic and T1 RNases and a partial hydrolysis with T1 RNase followed by the isolation and determination of the nucleotide sequences of the resulting fragments permitted the derivation of its primary structure . tRNA Thr 1 is in fact a mixture of two subspecies differing only by a A49-U65 base pair in 50 per cent of the molecules which is replaced by a G49-C65 pair in the other 50 per cent . These two subspecies consist of 76 nucleotide residues including 14 minor nucleotides . They show a characteristic m3C at the 3'terminal end of the anticodon loop, an anticodon I-G-U followed by t6A and C48, uncompletely modified (50 per cent) to m5C within the 5 nucleotides long extra-arm . The minor nucleotides m2G m2 2G are located at positions in which they generally occur in the tRNA structures as does m1A within the T-psi-C loop. Acta Biol Med Ger, 1977, 36(11-12), 1523 - 4 Characterization and function of intracellular proteinases and proteinase inhibitors from yeast; Holzer H; Data on the proteinase inhibitors IA, IB and IC from yeast and their possible intracellular interaction with the proteinases A and B and carboxypeptidase Y are presented . A role of proteolysis in "catabolite inactivation" is discussed. Acta Biol, 1977, 28(1), 49 - 58 Optical polarization reveals different ultrastructural molecular arrangement of polysaccharides in the yeast cell walls; Fischer J; The topo-optical aldehyde bisulfite-toluidine blue (ABT) reaction of vicinal OH and amino-OH groups offers new ways to study the ultrastructure of polysaccharides in different biological substrates . Through oriented dye binding on the reacting groups, the ABT reaction induces strong birefringence on the linearly ordered polysaccharides, which is negative with respect to their chain length . Using this method, two types of molecular order of the polysaccharides could be distinguished in the cell walls and capsules of yeasts . (1) The optically negative spherulitic character of the yeasts after the ABT reaction indicated that the toluidine blue molecules were bound tangentially (in a surface-parallel pattern) while the polysaccharide chains of the cell walls and capsules were oriented mainly radially . This structural pattern may be explained as resulting from a helicoid conformation of the polysaccharide component . (2) Acid or alkali hydrolysis removed the radially oriented polysaccharide component of the cell wall . The remaining, resistant polysaccharides showed up in the form of optically positive spherulites indicating radially oriented dye molecules on a circularly ordered, micellar polysaccharide texture. Nucleic Acids Res, 1977, 4(5), 1609 - 31 Complementary addressed modification of yeast tRNA Val 1 with alkylating derivative of d(pC-G)-A . The positions of the alkylated nucleotides and the course of the alkylation in the complex; Grineva NI et al.; Yeast tRNA Val 1 alkylation with 2', 3'-O-4-(N-2-chloroethyl-N-methylamino) benzylidene d(pC-G)-A proceeds at 20 degrees - 30 degrees C in the complementary complexes which are formed by d(pC-G)-A greater than RC1 binding to 3 sequences of tRNA Val 1 : psi-C-G58 in the T loop, C-G40 at the 3'-side of the anticodon loop and C-G18 in the D loop . The reaction in the complexes results in A53, I35, and psi 13 alkylation to form beta-/N-methyl-N-(formylphenyl 17 amino/ethyl-tRNA Val 1 with the relative rate constants of the alkylation that are 3 or 2 orders of magnitude higher than that for the alkylation without a complex formation . It is the third nucleotide from the 5'-terminus of the binding site of the modifying agent that is subjected to alkylation in the t RNA Val 1 . The course of the alkylation does not depend on the possible base pairing of the 3'-terminal nucleotide of the reagent . The extent of the reagent binding and the relative rate constants of the alkalytion in the complexes indicate the following order of the complex stability: (psi-C-G58) greater than (CO-G40) approximately (C-G18) at 20 degrees and (psi-C-G58) greater than (C-G40) greater than (C-G18) at 30 degrees. Nucleic Acids Res, 1977, 4(5), 1429 - 48 Analysis of chromosomal integration and deletions of yeast plasmids; Cameron JR et al.; Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared . Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA . DNA from each of the plasmids was inserted into a lambda vector DNA . Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques . All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences . Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region . The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector . Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA. Nucleic Acids Res, 1977 Jan, 4(1), 207 - 21 The iminoproton NMR spectrum of yeast tRNA-Phe predicted from crystal coordinates; Geerdes HA et al.; The ring current effects on the base paired iminoprotons in yeast tRNA-Phe have been calculated from crystal coordinates . The results in conjunction with independently determined intrinsic positions of the iminoprotons in various base pairs enable us to predict the low field NMR spectrum of yeast tRNA-Phe . It turns out that the calculated NMR spectra are very sensitive to slight changes in structure . Moreover the crystal and solution structure are identical as far as the present methods go. Mikrobiologiia, 1977 Jan-Feb, 46(1), 101 - 3 {Study of preliminary irradiation with wave-lengths 366 and 405 nm on lethal UV-damage to yeast cells}; Buturlakin MS; The protective action of irradiation with wavelengths of 366 and 405 nm on the cells of Saccharomyces cerevisiae, XI race, against lethal lesions caused by short-wavelength UV was studied . Among the total number of the cells with lethal lesions caused by UV, 25--30% of the cells were protected by light with the wavelength of 405 nm, and 50--55% of the cells were protected by light with the wavelength of 366 nm . Lethal UV lesions of the cells are supposed to be of different nature. Biochem J, 1977 Jan 1, 161(1), 181 - 3 The catalytic activity of monomeric yeast hexokinase A; Yip BP et al.; It has been suggested {Williams, D.C . & Jones, J.G . (1976) Biochem . J . 155, 661-667} that monomeric hexokinase isoenzyme A is not catalytically active . We here present data from reacting-enzyme sedimentation, dissociation experiments and from previous studies which are consistent with the monomeric form possessing catalytic activity. Proc Natl Acad Sci U S A, 1977 Jan, 74(1), 305 - 9 Nonsense suppressors of yeast cause osmotic-sensitive growth; Singh A; Many nonsense suppressors of Saccharomyces cerevisiae cause growth inhibition on hypertonic media . Eight tyrosine-inserting UAA (ochre) suppressors, eight tyrosine-inserting UAG (amber) suppressors, a leucine-inserting UAG suppressor, and a serine-inserting recessive lethal UAG suppressor cause osmotic sensitivity, whereas a serine-inserting UAA suppressor does not cause sensitivity . Although the mechanism is not understood, the growth inhibition of specific suppressors on hypertonic media is correlated with their efficiencies of suppression . This heretofore unknown property of nonsense suppressors is useful for mitotic mapping, selecting tRNA mutants, selecting antisuppressors, and scoring nonsense suppressors. J Gen Microbiol, 1977 Jan, 98(1), 215 - 21 Synthesis of ribosomal and transfer ribonucleic acids in yeast during a nutritional shift-up; Waldron C; The growth rate of Saccharomyces cerevisiae was increased by adding a mixture of amino acids to cultures containing proline as the sole nitrogen source . The transition from balanced growth in the basal medium (doubling time 4 h) to balanced growth in the enriched medium (doubling time 2 h) took about 2-5 h . The rate of RNA accumulation increased soon after the enrichment to almost its final value . This increase began after a short lag of 10 to 15 min, therefore synthesis of new RNA polymerase molecules may be required before stable RNA production can increase . The different stable RNA species were not stimulated at different times after the enrichment, but all increased continuosly throughout the transition . The rRNA species accumulated in a co-ordinate fashion at a rate faster than the rate of tRNA accumulation. J Bacteriol, 1977 Jan, 129(1), 472 - 81 Deoxyribonucleic acid sequence organization of a yeast plasmid; Livingston DM et al.; Two-micrometer deoxyribonucleic acid (DNA), a circular plasmid of Saccharomyces cerevisiae, contains two nontandem repeated sequences which are inverted with respect to one another . These repeated sequences together account for 21% of the molecule length . Restriction endonuclease analysis and electron microscopy demonstrated the existence of two forms of 2-mum DNA differing in the orientation of the interstitial segments bounded by the inverted repeated sequences . The two forms of 2-mum DNA could result from an intramolecular reciprocal recombination between inverted repeat elements . A map containing the restriction endonuclease sites and the units of the inverted repeat has been constructed. Biochimie, 1977, 59(10), 805 - 11 {Inhibition of the yeast respiratory system by Zn-protoporphyrin and effect of photolysis of this substance}; Djavadi FH et al.; We have shown earlier that yeast cells grown in synthetic mediums supplemented with Zn++ accumulate large amounts of Zn-protoporphyrin within their mitochondria . This accumulation is accompanied by an inhibition of respiration (3) . This study deals with the effect of light on the respiratory inhibition and the release of respiratory control which are observed if Zn-protoporphyrin is added to isolated mitochondria which are initially devoid of this pigment . In addition, we have studied the effect of light on the respiratory inhibition exerted by Zn-protoporphyrin accumulated in vivo . The following results were obtained: 1) The light-induced destruction of Zn-protoporphrin which had been added in vitro to Zn-protoporphyrin-free mitochondria significantly inhibits respiration and phosphorylation . Under these conditions, the extent of the inhibitions increases with the concentration of the added Zn-protoporphyrin and the duration of illumination . 2) Accumulation of Zn-protoporphyrin within the cells causes an inhibition of the respiratory activities and the activities of succinate-cytochrome c reductase and NADH-cytochrome c reductase of the mitochondria . Illumination of the isolated mitochondria from Zn-protoporphyrin-containing cells enhances the inhibition of these activities . No light-induced inhibition of these activities is observed with mitochondria from cells devoid of Zn-protoporphyrin. Z Allg Mikrobiol, 1977, 17(4), 293 - 7 Electron microscopic studies on the formation of vesicular bodies during cell wall degradation and regeneration in yeast; Dmitriev VV et al.; Vesicular bodies are observed during enzymatic degradation of the yeast cell wall and in the course of wall regeneration . In both cases various types of vesicles are formed and exocytated by different mechanisms . They are characterized cytochemically and their possible role in cell wall regeneration is discussed. Biokhimiia, 1977 Jan, 42(1), 79 - 84 {Interaction of thiamine pyrophosphokinase from brewer's yeast with thiamine and adenosine-5'-triphosphate}; Voskoboev AI et al.; The interaction of thiamine pyrophosphokinase (Thiaminkinase EC 2.7.6.2) with thiamine and ATP was studied . It was shown that the mechanism of the thiaminokinase reactions is Rapid Equilibrium Random Bi -- Bi . The enzyme binds 8 moles ATP and 1 mole thiamine per mole protein Ks = 0,8-10(-3) and 4.10(-6) M for ATP and thiamine respectively. Folia Microbiol (Praha), 1977, 22(1), 74 - 8 Dependence of yeast sporulation on mitochondrial protein synthesis; Jayachandran S et al.; Sporulation in diploid Saccharomyces cerevisiae is not dependent on continued protein synthesis in the mitochondria . Using chloramphenicol, it is shown that proteins essential for respiration and sporulation are synthesized in mitochondria early during growth in a presporulation medium. Genetics, 1977 Jan, 85(1), 1 - 22 Role of DNA sequences in genetic recombination in the iso-1-cytochrome c gene of yeast . II . Comparison of mutants altered at the same and nearby base pairs; Moore CW et al.; X-ray-induced mitotic recombination rates and spontaneous meiotic recombination rates have been determined in two-point crosses of various defined cyc1 mutants of the yeast Saccharomyces cerevisiae . All but one of the 17 cyc1 mutants chosen for this study contained either the addition, deletion or substitution of single base-pairs located within a defined segment of the gene that corresponds to the 11 amino acid residues at the amino terminus of iso-1-cytochrome c; approximately half of these mutants had alterations of the AUG initiation codon, some at the same base pair . Up to 66-fold differences in X-ray-induced recombination rates were observed when the same cyc1 mutant was crossed to cyc1 mutants having different alterations in the AUG initiation codon; over a ten-fold difference was observed in series of homologous crosses involving mutants with different changes at the same base-pair . Recombination rates that were associated with specific cyc1 mutants co-segregated with the particular alleles following meiosis, and comparable recombination patterns were also observed for independently isolated, identical mutations . With the mutants used in this study, the frequencies of meiotic recombination did not differ as markedly, suggesting a dissimilar dependence on specific DNA sequences for these two modes of recombination . These disproportionalities of recombination rates suggest that the nature of the mismatched bases influences the recombination process, but not in a way that can be simply interpreted. Eur J Biochem, 1977 Jan, 72(2), 361 - 9 Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast; De Jonge P et al.; The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed . Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures . The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed . 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA . However, the 3'-terminal sequences of the two types of RNA appeared to be different . The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH . In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence . These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule . The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed. Proc Natl Acad Sci U S A, 1977 Jan, 74(1), 96 - 100 Enzymatic synthesis of oligonucleotides of defined sequence: synthesis of a segment of yeast iso-1-cytochrome c gene; Gillam S et al.; The deoxyribooligonucleotide, d(pT-T-A-G-C-A-G-A-A-C-C-G-G), constituting a segment of yeast iso-1-cytochrome c gene, has been synthesized by a combination of chemical and primarily enzymatic methods . The starting primer, d(pT-T-A-G1, was chemically synthesized by the phosphodiester method and was extended stepwise, by reactions catalyzed by polynucleotide phosphorylase. Acta Biol Med Ger, 1977, 36(7-8), 1027 - 33 Binding of MgATP to yeast phosphofructokinase; Nissler K et al.; Binding of MgATP to yeast phosphofructokinase was investigated by the gel filtration equilibrium dialysis technique . Per subunit of yeast phosphofructokinase two molecules of MgATP are bound in the absence of fructose-6-phosphate, one to a high-affinity and one to a low-affinity site . The experimental data were compared with a kinetic model of yeast phosphofructokinase as described by Freyer et al . {3}. Biochimie, 1977, 59(11-12), 869 - 75 Several classes of binding sites for metals and nucleotides on yeast mitochondrial oligomycin sensitive ATPase; Jallon JM et al.; The binding properties of Mg2+, Mn2+ and Co2+ to yeast mitochondrial oligomycin sensitive ATPase complex are studied, as reflected by their catalytic effect (hydrolysis of ATP or pNPP, a pseudo substrate) or by a physical parameter (atomic absorption, electron paramagnetic reasonance of Mn2+, enhanced fluorescence of chelating chlorotetracyclin) . At least two classes of sites with very different affinities respectively around 10(-5) M and 10(-4) M are demonstrated: high affinity sites for cations which participate in pNPP hydrolysis and can bind ADP or ATP, although they have a poor efficiency for ATP hydrolysis, and low affinity sites for cations which participate efficiently in both pNPP and ATP hydrolysis . The possibility that the tight site class has itself two sub-classes is also discussed. Adv Exp Med Biol, 1977, 95, 271 - 89 Characteristics and functions of proteinase A and its inhibitors in yeast; Holzer H et al.; A purification and some properties of proteinase A from yeast are described . A specific macromolecular inhibitor of proteinase A from yeast cytosol has been isolated and shown to be a protein (molecular weight 7,700) consisting of a majority of polar amino acids . Proline, arginine, cysteine and tryptophan were not detected in the inhibitor . Possible biological functions of proteinase A and the proteinase A-inhibitor (and of other yeast proteinases and their inhibitors) in the following processes are discussed: general protein turnover, catabolite inactivation of enzymes, enzyme degradation at starvation and at transition to spore formation, and activation of pre-enzymes and precursor proteins by limited proteolysis. Biochem J, 1977 Jan 1, 161(1), 73 - 82 A study of the ionic properties of the essential histidine residue of yeast alcohol dehydrogenase in complexes of the enzyme with its coenzymes and substrates; Dickenson CJ et al.; 1 . Initial-rate studies of the reduction of acetaldehyde by NADH, catalysed by yeast alcohol dehydrogenase, were performed at pH 4.9 and 9.9, in various buffers, at 25 degrees C . The results are discussed in terms of the mechanism previously proposed for the pH range 5.9-8.9 {Dickenson & Dickinson (1975) Biochem . J . 147, 303-311} . 2 . Acetaldehyde forms a u.v.-absorbing complex with glycine . This was shown not to affect the results of kinetic experiments under the conditions used in this and earlier work . 3 . The variation with pH of the dissociation constant for the enzyme-NADH complex, calculated from the initial-rate data, indicates that the enzyme possesses a group with pK7.1 in the free enzyme and pK8.7 in the complex . 4 . The pH-dependences of the second-order rate constants for inactivation of the enzyme by diethyl pyrocarbonate were determined for the free enzymes (pK7.1), the enzyme-NAD+ complex (pK approx . 7.1) and the enzyme-NADH complex (pK approx . 8.4) . The essential histidine residue may therefore be the group involved in formation and dissociation of the enzyme-NADH complex . 5 . Estimates of the rate constant for reaction of acetaldehyde with the enzyme-NADH complex indicate that acetaldehyde may combine only when the essential histidine residue is protonated . The dissociation constants for butan-1-ol and propan-2-ol, calculated on the basis of earlier kinetic data, are, however, independent of pH . 6 . The results obtained are discussed in relation to the role of the essential histidine residue in the mechanism of formation of binary and ternary complexes of the enzyme with its coenzymes and substrates. J Biochem (Tokyo), 1977 Jan, 81(1), 197 - 205 Studies on the microsomal electron-transport system of anaerobically grown yeast . V . Purification and characterization of NADPH-cytochrome c reductase; Kubota S et al.; A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically . The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme . The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5 . The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione) . The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0 . It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase {EC 1.6.2.4}. J Biochem (Tokyo), 1977 Jan, 81(1), 187 - 95 Studies on the microsomal electron-transport system of anaerobically grown yeast . IV . Purification and characterization of NADH-cytochrome b5 reductase; Kubota S et al.; The presence of NADH-cytochrome b5 reductase {EC 1.6.2.2} in microsomes from anaerobically grown yeast was confirmed by its isolation and purification . The purified preparation of the reductase showed an apparent molecular weight of 27,000 daltons . The reductase appeared to contain loosely-bound FAD as a prosthetic group . The reductase required NADH as a specific electron donor, and could reduce some redox dyes as well as cytochrom b5 . The reductase, however, could not reduce cytochrome c . Michaelis constants of the reductase for NADH and calf liver cytochrome b5 were 6.3 and 1.5 micron M, respectively, and optimal pH for cytochrome b5 reduction was 5.6 . Although some differences exist between the properties of NADH-cytochrome b5 reductase from yeast and from mammalia, the results indicate a functional similarity of the present enzyme to mammalian NADH-cytochrome b5 reductase in the microsomal electron-transport system. Scand J Dent Res, 1977 Jan-Feb, 85(2), 130 - 4 Denture stomatitis-yeast occurrence and the pH of saliva and denture plaque; Olsen I et al.; The study comprised 30 denture wearers with generalized simple or granular inflammation in the palate and 30 without (controls) . )easts, mostly Candida species, were cultivated from the maxillary dentures of all subjects with inflammation and of 23 controls . Hyphae were found in the maxillary denture smears from 28 subjects with inflammation and from 18 controls . Thus it seems unjustified to consider the occurrence of hyphae pathognomonic of denture stomatitis . The pH of whole saliva did not differ in the two groups (inflammation:mean pH 6.5, control:6.6) . There was no clear relation between the pH of resting saliva and the amount of fungal cultures . Thirty minutes after a mouthrinse with 10 ml of a 25% sucrose solution, the mean saliva pH had dropped equally in both groups . With regard to the denture plaque, samples taken 40 min after the rinse indicated a more pronounced acid production in the plaque associated with inflammation . The pH of "resting" plaque was also lower in the inflammation than in the control group (mean maxillary pH 5.7 and 6.3 respectively, a=0.002) . No association was found between the pH and the occurrence of hyphae in "resting" denture plaque . This supports the view that the pH is of no major importance for filamentation in vivo. Biochim Biophys Acta, 1976 Dec 22, 453(2), 453 - 8 End group analysis of yeast fatty acid synthetase; Schwietz H et al.; The purified yeast fatty acid synthetase complex has been subjected to amino and carboxyl terminal amino acid end group analysis . Amino end groups were studied by Edman degradation and by dansylation of the sodium dodecyl sulfate- or urea-denatured complex . No N-terminal amino acid could be identified by either method . C-terminal amino acids were investigated by tritium labeling and by digestion of the complex with carboxypeptidases A and B . By both methods, the two amino acids valine and lysine were consistently identified as the C-termini of two different polypeptide chains . After separation of the fatty acid synthetase subunits A and B by sodium dodecyl sulfate polyacrylamide gel electrophoresis lysine was identified as the C-terminus of subunit A and valine as that of subunit B . The results are interpreted as evidence that the yeast fatty acid synthetase complex is basically composed of two nonidentical and multifunctional polypeptide chains. Eur J Biochem, 1976 Dec 11, 71(2), 425 - 36 Stopped-flow studies of changes in fluorescence of 8-anilino-1-naphthalene sulfonic acid caused by magnesium and salt binding to yeast enolase; Brewer JM; Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1-naphthalenesulfonic acid fluorescence changes . The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements . These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates . From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation. J Biol Chem, 1976 Dec 10, 251(23), 7521 - 5 pH-dependent effects of Cr(NH3)2ATP on kinetics of yeast hexokinase PII . Relationship to the slow transition mechanism; Peters BA et al.; The effect of Cr(NH3)2ATP, a virtually inert, inner sphere metal-ligand complex, on the kinetics of purified yeast hexokinase PII has been studied at pH 6.5 and pH 7.5 . At pH 6.5, where the normal assays exhibit a slow burst-type transient, low concentrations of Cr(NH3)2ATP were found to activate both phii, the initial velocity, and phiII, the steady state velocity . At higher concentrations, Cr(NH3)2ATP was found to be a competitive inhibitor versus MgATP for both phii and phiII . The apparent Ki values for both velocities were the same . The inhibition by Cr(NH3)2ATP at pH 6.5 was found to be a slow process with half-times similar to those found for the normal burst-type transient at this pH value . At pH 7.5, where normal assays exhibit linear progress curves, Cr(NH3)2ATP behaved similarly to that observed before at pH 7 (Danenberg, D . D., and Cleland, W . W . (1975) Biochemistry 14, 28-39), i.e . it was a competitive inhibitor versus MgATP and it caused a slowing of the reaction rate over the first several minutes . The apparent Ki for the initial velocity was 8-fold higher than the apparent Ki for the steady state velocity, suggesting tighter binding of Cr(NH3)2ATP with time . Preincubation experiments indicated that the normal pH 6.5 burst-type transient could be eliminated by appropriate preincubation with Cr(NH3)2ATP and a sugar . In agreement with Danenberg and Cleland (1975), similar preincubations have been shown to produce linear assays at pH 7.5 in the presence of Cr(NH3)2ATP . Similar results were seen with MgITP as the nucleotide substrate, where a burst-type transient is not seen at either pH value under normal assay conditions . At pH 7.5, a slow decrease in the reaction rate is seen over the first several minutes in the presence of Cr(NH3)2ATP . The apparent Ki for phii was 7-fold higher than the apparent Ki value for phiII, again suggesting a tighter binding of Cr(NH3)2ATP with time . A similar observation was made at pH 6.5, but the Ki values for phii and phiII were the same, suggesting no tightening of the binding of Cr(NH3)2ATP with time at this pH value . These results suggested that both slow processes reflect the same basic molecular change, but the consequences are different at the two pH values, presumably because of the difference in the charge of the enzyme . The Cr(NH3)2ATP kinetics at pH 6.5 have been interpreted in terms of a modification of the slow transition mechanism for hexokinase (Shill, J . P., and Neet, K . E . (1975) J . Biol . Chem . 250, 2259-2268) . It is postulated that glucose and Cr(NH3)2ATP induce the same slow conformational change at pH 6.5 as that induced by glucose and MgATP, which gives rise to the normal burst-type transient . This suggests that Cr(NH3)2ATP may be a useful tool for physical studies to determine the cause of the slow transition of yeast hexokinase . Activation by low concentrations of Cr(NH3)2ATP was interpreted as binding of the nucleotide to an activator site on the enzyme, causing a shift in the distribution of enzyme towards the more active form. Biochim Biophys Acta, 1976 Dec 8, 452(2), 452 - 7 External yeast beta-fructosidase . Affinity labeling of the active site; Braun H; Conduritol-B-epoxide, a compound structurally related to the substrates of external yeast beta-fructosidase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26), is an active-site directed inhibitor of this enzyme . The inactivation is irreversible and first-order with respect to time and inhibitor concentration . From the kinetic data obtained, it is concluded that one molecule of inhibitor reacts with one molecule of the enzyme causing inactivation . The inactivation is prevented by the presence of substrates . The pH-dependence of inactivation shows two dissociating groups in the enzyme with pKa values 3.05 and 6.8 being involved in the inactivation process . A carboxylate at the active site with pKa 3.05 is suggested to be the reactive group with conduritol-B-epoxide. Arch Microbiol, 1976 Dec 1, 111(1-2), 193 - 4 A note on the kinetics of uptake of D-glucose by the food yeast, Candida utilis; Barnett JA et al.; Unlike other yeasts so far investigated, the D-glucose carrier of Candida utilis (strain NCYC 737) appears to change affinity for D-glucose according to its exogenous concentration . When the concentration of D-glucose was less than 0.4 mM, the apparent Km approximately 0.2 mM; at greater than 0.4 mM, the Km approximately 10 mM. J Bacteriol, 1976 Dec, 128(3), 851 - 2 Detection, with the dye phloxine B, of yeast mutants unable to utilize nitrogenous substances as the sole nitrogen source; Middelhoven WJ et al.; Yeast mutants unable to degrade certain nitrogen compounds produce characteristic small red colonies on an agar medium containing the red dye phloxine B, galactose, the test nitrogen compound, and a small amount of ammonium chloride. Proc Natl Acad Sci U S A, 1976 Dec, 73(12), 4334 - 8 Interaction of integral and peripheral membrane proteins: affinity labeling of yeast cytochrome oxidase by modified yeast cytochrome c; Birchmeier W et al.; To identify possible substrate-binding subunit(s) of yeast cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1-9-3-1), the purified enzyme was reacted with yeast iso-1-cytochrome c whose single free sulfhydryl group at position 107 had been activated with 5,5'-dithiobis(2-nitrobenzoate) . The resulting cytochrome c derivative appeared to function as an "affinity-label" of cytochrome oxidase, since it rapidly inactivated the enzyme . Inactivation was competitively prevented by underivatized cytochrome c . When the "affinity-labeled" oxidase was analyzed by two-dimensional polyacrylamide electrophoresis in dodecyl sulfate (separation in the second dimension being carried out in the presence of excess sulfhydryl compound), it was found that the derivatized cytochrome c had specifically formed a mixed disulfide with the mitochondrially made subunit III (apparent molecular weight 24,000) of the oxidase . Similar results were obtained when underivatized iso-I-cytochrome c was crosslinked to the oxidase by oxidative disulfide bridge formation in the presence of ortho-phenanthroline and Cu++ . These data indicate that the hydrophobic mitochondrially made subunit III of yeast cytochrome c oxidase is in close proximity to the cytochrome c binding site on the enzyme . Since cytochrome c and the mitochondrially made cytochrome oxidase subunit III are typical peripheral and integral membrane proteins, respectively, the present study suggests a useful approach for analyzing specific interactions between these different classes of membrane proteins. Biokhimiia, 1976 Dec, 41(12), 2173 - 8 {Cytosolic alcohol dehydrogenases from yeast Torulopsis candida}; Sudovtsov VE; A comparative study of cell cytosol alcohol dehydrogenase (ADH) from yeast Torulopsis candida IBFM-Y-127 grown on glucose and hexadecane which were the only source of carbon, was made . In both cases ADH had a pH optimum within the range of 7.0--10.0, when various normal primary alcohols (C2--C16) were used . The enzyme was active only in the presence of NAD, which cannot be substituted by NADP . The total activity of ADH decreased approximately 8-fold when the length of hydrocarbon radicals was changed from C2 up to C16 . When the cells were grown on hexadecane, only ethyl, n-buthyl, n-amyl and n-hexyl alcohols were active as substrates . The dehydration rate of each alcohol was far lower than that for the cytosol of glucose-grown cells . In the latter case the enzyme activity also decreased with an increase in the alcohol radical from C2 to C6 . In all cases studied methyl alcohol and cyclic (cinnamyl alcohol--C8) alcohol were not dehydrated at all . Disc-electrophoresis in polyacrylamide gel, involving gel colouration for the assay of enzyme activity showed that glucose--grown cell cytosol contained three forms of ADH . One of those forms was highly active when short--chain normal primary alcohols were used; this form may be probably regarded as "classical" ADH (EC 1.1.1.1) . The two other forms caused intensive dehydration of long-chain alcohols (the best substrates were C7--C10 alcohols for one form and C10--C14 for the others) . The two forms of ADH are probably isoenzymes of octanol dehydrogenase (EC 1.1.1.73) . Cytosol of cells grown on n-alcane, had a reduced number of ADH forms . The data obtained are discussed in terms of the regulatory role of carbon and energy source (glucose or hexadecane) in the redistribution of alcohol dehydrogenases between structural components of cells (mitochondria) and cytosol. Hoppe Seylers Z Physiol Chem, 1976 Dec, 357(12), 1771 - 7 Effects of yeast proteinase A, proteinase B and carboxypeptidase Y on yeast phosphofructokinase; Afting EG et al.; Incubation of a crude yeast extract containing phosphofructokinase with proteinase A, proteinase B or carboxypeptidase Y gave the following results: Proteinase B and carboxypeptidase Y did not change the activity of phosphofructokinase during incubation . On the other hand, incubation with proteinase A resulted in a 40-100% activation; continued incubation, however, led to an inactivation of the enzyme . Addition of allosteric effectors did not change the activation or inactivation process . The activated phosphofructokinase was not changed with respect to pH optimum and ATP inhibition . Molecular weight determination of phosphofructokinase in crude extracts in the presence of inhibitors of proteinase A indicated a molecular weight of 700000 . Without inhibitors of proteinase A, the molecular weight was determined to be 600 000, while after 40-100% activation by proteinase A, a molecular weight of 500 000 was obtained . The activity profile of proteinase A in density gradients indicated that this enzyme is bound to variety of cellular proteins. Mol Cell Biochem, 1976 Nov 30, 13(2), 73 - 8 Determination of intermediary metabolites in yeast . Critical examination of the effect of sampling conditions and recommendations for obtaining true levels; Saez MJ et al.; The effect of sampling conditions on the levels of adenine nucleotides, pyridine nucleotides, glycolytic intermediates and related metabolites in yeast has been studied . A systematic examination of the conditions for harvesting has shown that it can be best accomplished by rapid filtration . Delays in the handling for removal of the medium, as is usual in the process of obtaining a number of data reported in the literature, lead to important changes in some of the metabolites examined . It is also shown that when a washing is imperative it can be carried out with a methanol-water mixture (50/50, v/v) cooled at -40 degrees without loss of intracellular concentrations of non-readily diffusible metabolites . On the basis of this experience the outline of a generally applicable procedure is presented. J Biol Chem, 1976 Nov 25, 251(22), 7157 - 61 Interaction of tetraiodofluorescein with yeast hexokinase; Yip BP et al.; The dye tetraiodofluorescein (TIF) was found to be an effective inhibitor of yeast hexokinase . It is a competitive inhibitor relative to MgATP2- and a noncompetitive inhibitor of glucose binding, a kinetic pattern consistent with the previously proposed random kinetic mechanism . TIF interacts directly with the native dimeric protein to give a difference spectrum with a maximum at 543 nm . Monomeric protein (produced by addition of 0.6 M NaCl) interacts with TIF to give a slightly altered difference spectrum, with the gammamax at 545 nm . The difference spectrum of the dimeric form is not perturbed by the addition of substrates but the absorbance with the monomer is lowered by MgATP2- . The Kd for MgATP2- was estimated to be 7 nM for monomeric hexokinase . These results suggest that results of previous binding studies with hexokinase at high concentrations which have been interpreted as being at variance with kinetic studies are due likely to different conformations of the protein under different experimental conditions. Eur J Biochem, 1976 Nov 15, 70(2), 385 - 95 Protein degradation during yeast sporulation . Enzyme and cytochrome patterns; Betz H et al.; The levels of several enzymes have been studied during sporulation of Saccharomyces cerevisia . The specific activities of ribonuclease and aminopeptidase I raised several-fold after transfer of the cells to sporulation medium, whereas the specific activities of phosphofructokinase, glucose-6-phosphate dehydrogenase, tryptophan synthase and pyruvate decarboxylase were not significantly altered . The specific activities of NAD-dependent glutamate dehydrogenase, isocitrate lyase, malate dehydrogenase and fructose bisphosphatase all decreased from the onset of sporulation . The inactivation of these latter enzymes was inhibited by cycloheximide and by inhibitors of energy metabolism . Hexokinase, alcohol dehydrogenase and glutamate oxaloacetate transaminase were partially lost from the cells during the period of ascus maturation . None of the enzyme changes observed proved to be 'sporulation-specific' in that it occurred exclusively in sporulating diploid yeast cells . Therefore it is postulated that the meiotic events and the metabolic changes required for ascospore formation are under separate genetic control in this organism . During sporulation, the cellular content of cytochromes b, c, and aa3 was reduced to 20% or less of that present in vegetative derepressed cells . Since the relative percentage of total to cycloheximide-insensitive mitochondrial protein synthesis was not significantly altered throughout sporulation, and the pattern of mitochondrially synthesized polypeptides was rather similar both in vegetative and in sporulating cells, it appeared that not only degradation but also synthesis and therefore turnover of the mitochondrially coded polypeptides of cytochromes b and aa3 took place during sporulation . The activity ratio of cytochrome c oxidase to F1-ATPase in submitochondrial particles isolated from vegetative cells and from purified asci was almost identical . This indicates that the loss of membrane-bound mitochondrial cytochromes during sporulation is probably due to a nonselective degradation of inner mitochondrial membrane proteins. Arch Microbiol, 1976 Nov 2, 110(23), 313 - 8 Mating reaction in yeast protoplasts; Svoboda A; Protoplasts prepared from complementary haploid strains of Saccharomyces cervisiae were studied with regard to their ability of conjugating . Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion . However, they were capable of inducing sexual activation in normal cells of opposite mating type . After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex . The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration . From the results the following conclusions may be drawn: 1 . The initiation of mating is dependent on the integrity of the cell wall . 2 . The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls . 3 . The lysis of cell walls does not occur until the walls come into close contact . 4 . The fusion of plasma membranes in sex-activated protoplasts cannot be induced by arteficial agglutination. Biochemistry, 1976 Nov 2, 15(22), 4760 - 4 A cycloheximide sensitivity factor from yeast required for N-acetylphenylalanylpuromycin formation; Somasundaran U et al.; A protein (factor P) has been isolated from yeast, which was required for sensitivity to cycloheximide of a partially purified polyphenylalanine synthesis system . In the absence of factor P, 10(-3) M cycloheximide was required for 50% inhibition of polyphenylalanine synthesis, while in its presence, 10(-6) M gave 50% inhibition . Coincident with cycloheximide sensitivity was an activity required for EF-2 dependent N-acetylphenylalanylpuromycin (N-AcPhePuro) formation . Transfer of N-AcPhe to puromycin from the tRNA bound in the presence of 26 mM MgCl2 required factor P, as well as EF-2 . Studies with antibody against EF-2 demonstrated that P factor was not required during the EF-2 translocation step but for some subsequent step. Biochemistry, 1976 Nov 2, 15(22), 4881 - 5 Reaction of yeast carboxypeptidase C1 with group-specific reagents; Kuhn RW et al.; The reactions between yeast carboxypeptidase C and the group-specific reagents, phenylglyoxal and iodoacetamide, have been studied in detail and the reactions of residue at the active site with N-tosyl-L-phenylalanine chloromethyl ketone and diisopropyl phosphorofluoridate have been confirmed . Modification of the enzyme by either phenylglyoxal or iodoacetamide results in the loss of peptidase activity, while esterase activity remains unchanged . Inactivation by phenylglyoxal appears to be the result of the modification of a single arginine residue, whereas inhibition by iodoacetamide can be correlated with the modification of a single methionine residue . Inactivation of the enzyme by either N-tosyl-L-phenylalanine chloromethyl ketone or diisopropyl phosphorofluoridate is the result of the modification of a single histidine and a single serine residue, respectively . The pattern of inhibition indicates certain analogies in the mechanism of yeast carboxypeptidase C to pancreatic chymotrypsin, on the one hand, and to carboxypeptidase A, on the other. Mikrobiologiia, 1976 Nov-Dec, 45(6), 1040 - 4 {Biological productivity of hybrids and strains of yeast cultures of different ploidy}; Kosikov KV et al.; Ploidy and productivity of yeast strains and hybrids obtained by crossing various industrial races were studied . Biological productivity of yeast cultures increases with ploidy as was found by comparing biological productivity (biomass accumulation) in haploids, diploids, triploids, and tetraploids . Possible factors causing an insignificant decrease in productivity of some tetraploids as compared to triploids are discussed. Mikrobiologiia, 1976 Nov-Dec, 45(6), 1018 - 22 {Studies of the primary distribution kinetics of n-alkane oxidation products according to morphological components of the yeast cell}; Davidova EG et al.; Incorporation of 14C into various classes of lipids localized in the cell walls, large membranes, mitochondria and microsomes of yeast cells was studied during their incubation with 1-14C-octadecane . Cell structures were found to differ considerably by the kinetics and level of accumulation of 14C-components of the lipid fraction . Waxes and triglycerides prevail during primary accumulation of 14C-lipids in the large membranes and cell walls . Synthesis of these compounds is presumed to play an important role in the course of primary transformation of n-octadecane. Br J Nutr, 1976 Nov, 36(3), 471 - 8 The digestion of yeast cell wall polysaccharides in veal calves; Gaillard BD et al.; 1 . The digestibility of the cell wall polysaccharides of an alkane-grown yeast in different parts of the digestive tract of two veal calves fitted with re-entrant cannulas at the end of the ileum was studied by replacing part of the skim-milk powder of their 'normal', milk-substitute (all-milk-protein) diet by yeast (yeast diet) . 2 . The lactose and glucose of both the all-milk-protein diet and the yeast diet were almost completely digested before the end of the ileum . During this digestion a small amount of oligosaccharides composed of galactose and glucose was synthesized . These oligosaccharides were digested again in the large intestine . 3 . The constituent sugars of the water-soluble fraction of the yeast cell wall carbohydrates were glucose and mannose . The 0-5 M-sulphuric acid-hydrolysate of the water-insoluble fraction contained glucose and mannose and the 12 M-H2SO4-hydrolysate only glucose . 4 . Digestibilities measured at the end of the ileum varied considerably between the two animals and averaged only about 0-40 . 5 . These findings suggest that the cell wall polysaccharides of yeast are digested very little by the normal digestive enzymes of the calf's small intestine, but are used as a substrate by the bacterial flora which are mainly concentrated in the large intestine. Clin Chem, 1976 Nov, 22(11), 1802 - 5 Spectrophotometric and electrochemical determinations of L(+)-lactate in blood by use of lactate dehydrogenase from yeast; Durliat H et al.; Lactate can be determined rapidly in blood by spectrophotometric and amperometric (enzyme electrode) procedures based on its oxidation by ferricyanide, the reaction being catalyzed with yeast L(+)-lactate dehydrogenase (cytochrome b2) (EC 1.1.2.3) . In the photometric method lactate can be measured in a few minutes, but blood samples must first be deproteinized . In the amperometric procedure no treatment of blood is needed except ferricyanide addition . The enzyme electrode we used has a response time shorter than 1 min when its critical variables are optimized . Preliminary standardization is reduced to minimum operation, because electrode response is proportional to lactate concentration over a wide range (0.1 to 8.0 mol/liter) and many determinations can be done with little cost in enzyme . A simple electrical device ("two-electrode device") is described that is well suited for furture micro-cell construction . Lactate determinations on a series of normal blood samples show no deviation between results by these new methods and the usual ultraviolet spectrophotometric lactate tests. Mol Biol (Mosk), 1976 Nov-Dec, 10(6), 1260 - 71 {Complementarily addressed alkylation of yeast tRNA 1 Val with chloroethylmethylaminobenzylidene d(pC-G)-A . Proof of the modification of the third nucleotide located at the 5'-terminus of the complete binding site of the reagent}; Grineva NI et al.; Yeast tRNAlVal alkylation with 2',3'-0-{4-(N-2-chloroethyl-N-methylaminobenzylidene}dpCdpGrpA proceeds within complementary complexes that are formed due to attachment of the reagent to three sequences of tRNAlVal . The acetal bond of the initial product of alkylation has been hydrolyzed to yield beta-{N=methyl-N-(4-formylphenyl) amino}ethyl-tRNAlVal (R-tRNA) that contains from 1 to 3 residues of the specifically modified nucleosides: alkyl adenosine (R-A), R-I and probably R-psi . Individual alkylated oligonucleotides were isolated from R-tRNAlVal pyrimidyl-RNAse digest . The alkylated oligonucleotides correspond to 93% of all modified products . The major component is beta-{N-methyl-N(4-formylphenyl)aminoA1ethyl-A53-G-Tp . This indicates that the reagent is attached to complementary psi-C-G-sequence in the T-loop of tRNAlVal and that alkylation of the third nucleotide counting from the 5'-terminus of the sequence which binds the reagent completely takes place within the complementary complexes . This site of the tRNAlVal is modified quantitatively at 20 degrees and 19-fold excess of the reagent . The alkylation of two other sites of tRNA reaches 50% under these conditions. Eur J Biochem, 1976 Nov 1, 70(1), 25 - 31 Calorimetric investigations on thermal stability of tRNAIle (yeast) and tRNASer (yeast); Filimonov VV et al.; Variation with temperature of the partial heat capacities of tRNAIle (yeast) and tRNASer (yeast) has been determined in two buffers at various salt conditions by scanning microcalorimetry . The overall molar transition enthalpy, deltaHt = 320 +/- 20 kcal mol-1 (1339 +/- 84 kJ mol-1) is identical for the two tRNA species within the limits of experimental error . deltaHt does not show any dependence on the nature of the buffer, nor does it vary on addition of 1 mM MgCl2 or 150 mM NaCl . Thermal unfolding of the native structure to the random coil cannot adequately be described by a two-state, concerted transition under the experimental conditions applied in this study, but exhibits a multistep mechanism characterized by sequential unfolding of separable cooperative domains. Mutat Res, 1976 Nov, 37(2-3), 193 - 200 Effect of dimethyl sulfoxide (DMSO) on radiation-induced heteroallelic reversion in diploid yeast; Singh DR et al.; Dimethyl sulfoxide has cryoprotective and radioprotective properties . It is also an efficient scavenger of radicals produced by raiolysis of water . Gamma-induced reversions of diploid yeast in the presence of this chemical during irradiation have been studied . The dose-modifying factor was in the same range as for survival . When the yeast was irradiated in the frozen state the observed protection by DMSO disappeared . The results are discussed in terms of direct and indirect actions of radiations and the radical-scavenging ability of this chemical. Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 3933 - 6 Acitivity of yeast extracts in cell-free stimulation of DNA replication; Jazwinski SM et al.; Extracts of the cytoplasm of disrupted spheroplasts of Saccharomyces cerevisiae (bakers' yeast) stimulated DNA synthesis in a cell-free system consisting of nuclei from spleen cells of the frog Xenopus laevis, The stimulation required Mg++8 ATP, and the deoxynucleoside triphosphates, was saturated by an excess of nuclei or extract, and had kinetics resembling those obtained previously with extracts from mammalian and avian cells . After addition of the yeast extract, replication "eyes" were formed in the DNA from the nucleochromatin of the frog, suggesting that the extract stimulated the initiation of DNA replication . The activity was susceptible to heat, was nondialyzable, and was abrogated by tryptic digestion . Temperature-sensitive mutants of the cell division cycle (cdcmutants 4,7,8, and 28) grown at permissive temperature (23 degrees) yielded extracts that were capable of stimulating DNA replication . When the cells were incubated for one generation at the nonpermissive temperature (36 degrees), their extracts showed very low or no activity . All of these mutants are deficient in events of the dependent pathway leading to initiation of DNA synthesis in the yeast cell cycle . A ts mutant, cdc10, deficient in the separate pathway for cytokinesis, showed little or no loss of activity at the nonpermissive temperature . These data indicate that the "initiation" activity, as assayed in vitro, is subject to control in the yeast cell cycle, and its appearance may be one of the terminal events in the pathway leading to DNA synthesis . The finding that extracts from yeast cells can stimulate DNA synthesis in nucleochromatin from frog cells, and the fact that the cdc mutants 4,7,8, and 28 describe a dependent pathway terminating in development of "initiation" activity, are in accord with the hypothesis that the function of proteins in the dependent pathways of the cell cycle is conserved during evolution. Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 3928 - 32 Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity; Gennis LS; A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase {D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12} with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+.Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity . The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity . A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested. J Biol Chem, 1976 Oct 25, 251(20), 6320 - 6 Yeast cytochrome c messenger RNA . In vitro translation and specific immunoprecipitation of the CYC1 gene product; Zitomer RS et al.; An assay based upon indirect immunoprecipitation has been developed for yeast cytochrome c and apocytochrome c . The specificity of this assay was demonstrated by its ability to selectively precipitate cytochrome c from an autolysate of yeast cell proteins . Translation of the polypeptide chain of cytochrome c in a wheat germ extract programmed with yeast poly(A) RNA was demonstrated using this immunoprecipitation assay . Translation of poly(A) RNA from yeast strains carrying nonsense mutations in the cyc1 gene yielded in vitro cytochrome c polypeptides which were shorter than the wild type protein by the amount expected for polypeptide chains which had terminated at the nonsense codon . The in vivo rate of cytochrome c synthesis was shown to be 6-fold greater in derepressed cells than in glucose-repressed cells . The 6-fold difference is sufficient to account for the 6-fold higher level of cytochrome c in derepressed than in repressed cells . The level of translatable cytochrome c mRNA is at least 4 times as high in derepressed as in glucose-repressed cells, suggesting that regulation occurs at some step in the synthesis of this messenger. Science, 1976 Oct 22, 194(4263), 433 - 5 Nuclear and mitochondrial DNA replication during zygote formation and maturation in yeast; Sena E et al.; Nuclear and mitochondrial DNA replication were monitered during the development of synchronous yeast zygotes . Purified first zygotic buds were also analyzed . Nuclear DNA replicated discontinuously but coincidently with bud initiation, while mitochondrial DNA replicated throughout the zygotic formation and maturation period . First zygotic buds contained the diploid level of both nuclear and mitochondrial DNA. Biochim Biophys Acta, 1976 Oct 18, 447(3), 294 - 303 Ribosomal subunit entry into polysomes in yeast; Petersen N et al.; The kinetics of entry of newly synthesized 40 S and 60 S ribosomal subunits into yeast polysomes is described . The entry times for 40 S and 60 S subunits were found to be 3 and 8 min, respectively . The kinetics of entry of 40 S subunits into large polysomes is found to be different from the kinetics of entry of 60 S subunits into large polysomes. Biochem J, 1976 Oct 15, 160(1), 37 - 41 Glycosylation in vitro of an asparagine sequon catalysed by preparations of yeast cell membranes; Khalkhali Z et al.; Preparations of yeast cell membranes can catalyse in vitro the N-acetyl-beta-D-glucosaminylation of the asparagine sequon at residues 34--36 of bovine pancreatic ribonuclease A . The relevant glycopeptides were isolated from tryptic hydrolysates of the glycosylated ribonuclease and analysed . The donor used was UDP-N-acetyl-D-glucosamine, although the mechanism of the transfer is unknown . Mn2+ ions at concentrations of 25 mM double the activity of the enzymic transfer. Radiat Environ Biophys, 1976 Oct 7, 13(3), 167 - 75 Liquid holding recovery in stationary and log phase cultures of diploid yeast exposed to gamma and alpha radiations; Reddy NM et al.; The kinetics of liquid-holding recovery (LHR) in diploid yeast after gamma and alpha irradiation is studied . In case of stationary phase culture the rate and extent of LHR is found to be greater for gamma-ray-induced damage than for alpha-ray-induced damage . At 10% survival level, the half-time for recovery is 5.2 h for gamma-ray damage and 12 h for alpha-ray damage . Further, while the recovery factor for alpha damage reaches saturation at 5% survival level, that for gamma damage continues to increase as survival level decreases . Oxygen is required for the recovery process during LH after gamma irradiation . The cells can recover to the same extent from both oxygen-dependent and oxygen-independent components of damage . Log phase cells containing a high per cent of budding cells, however, exhibit negative liquid holding effect after gamma irradiation. J Biochem (Tokyo), 1976 Oct, 80(4), 805 - 9 The aconitase of yeast . V . The reconstitution of yeast aconitase; Suzuki T et al.; The apoenzyme of yeast aconitase {EC 4.2.1.3} was prepared by treatment of yeast aconitase with sodium mersalyl, followed by passage by passage of the reaction mixture through a column of Dowex A-1 and gel filtration on Sephadex G-25 . The apoenzyme had no aconitase activity, but the active enzyme could be reconstituted by treatment of the apoenzyme with ferrous ions and sodium sulfide in the presence of 2-mercapto-ethanol . The reconstituted active enzyme was isolated by DEAE-Sephadex A-50 column chromatography and Sephadex G-100 gel filtration from the reaction mixture . The reconstituted enzyme was identical with the original untreated enzyme in terms of specific activity, iron content and spectral characteristics, but not in terms of labile sulfur content . A significant difference in visible spectra between the holo- and apoenzymes appeared to be due to the difference in iron and labile sulfur contents between the two proteins. J Biochem (Tokyo), 1976 Oct, 80(4), 799 - 804 The aconitase of yeast . IV . Studies on iron and sulfur in yeast aconitase; Suzuki T et al.; Chemical analyses were carried out to determine the active components of the crystalline aconitase {EC 4.2.1.3} of Candida lipolytica . The enzyme contained 2 atoms of non-heme iron, 1 atom of labile sulfur, and 6 sulfhydryl groups per molecule . One atom of the non-heme iron was released by the addition of metal-chelating agents such as sodium citrate, sodium nitrilotriacetate (NTA) or sodium ethylenediaminetetraacetate (EDTA) without loss of the enzyme activity . The non-heme iron and labile sulfur were released by the addition of sulfhydryl reagents such as rho-chloromercuribenzoate (PCMB), sodium mersalyl or urea with loss of the enzyme activity . o-Phenanthroline reacted with the iron atoms in the enzyme at pH 6.0 with loss of the activity . These results show that yeast aconitase is an iron-sulfur protein and that only one of the two non-heme iron atoms is essential for enzyme activity. Nucleic Acids Res, 1976 Oct, 3(10), 2697 - 707 Topographical analysis of yeast ribosomal DNA by cleavage with restriction endonucleases . II . The physical map of EcoRI fragments; Meyerink JH et al.; Yeast ribosomal DNA (rDNA) was digested with the restriction endonuclease EcoRI . Eight distinct fragments were obtained with a molecular weight of 4.35 (1), 1.75 (2), 1.45 (3), 1.07 (4), 0.42 (5), 0.37 (6), 0.26 (7) and 0.22 x 10(6) (8) daltons, respectively . Except for fragment 1 with a molecular weight of 4.35 x 10(6) daltons, all fragments are derived from the multiple ribosomal transcription units . The 'spacer' sequences, on the other hand, gave rise to digestion products which are very heterogeneous in size . By analysis of the partial digestion products which are very heterogeneous in size . By analysis of the partial digestion products, together with the data obtained by digestion with a combination of two restriction enzymes (EcoRI and Hind II or Hind III) and redigestion of the Hind II-and Hind III-fragments with EcoRI, the physical map of the EcoRI cleavage sites in the ribosomal transcription unit could be established. Biotechnol Bioeng, 1976 Oct, 18(10), 1337 - 49 Growth kinetics of a yeast grown on glucose or hexadecane; Mason TJ et al.; The kinetics of growth of a Torulopsis sp . was investigated in a continuous culture with glucose or hexadecane as the carbon source; growth was limited by either carbon or nitrogen . The relationship between the concentration of the limited substrate and the steady-state growth rate of the organism was examined and tested against various models of growth . No existing model was found to describe the growth accurately and a new model has been proposed: (see article) . It is postulated that this behavior would result from a simple first order reaction between the reactants of the rate-limiting enzymic reaction of the organism's metabolism. J Biochem (Tokyo), 1976 Oct, 80(4), 787 - 97 O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast . Subunit structure; Yamagata S; The molecular weight of O-acetylserine (OAS)-O-acetylhomoserine (OAH) sulfhydrylase purified from yeast was estimated to be about 200,000 by Sephadex G-200 gel chromatography in various buffers . The S20, w value of this protein was determined to be about 9.0 by sucrose density gradient centrifugation . The calculated molecular weight based on this value was similar to that estimated by gel chromatography . Treatment with 1% sodium dodesylsulfate (SDS) or 6 M urea dissociated the enzyme into 4 subunits; these had a molecular weight estimated to be 51,000 by SDS-poly-acrylamide gel electrophoresis and to be 57,000 by Sephadex G-100 gel chromatography in the presence of 6 M urea and 0.5% beta-mercaptoethanol . The 4 subunits appeared to be identical, based on the symmetric subunit elution pattern from a Sephadex column, a single peptide band on SDS-polyacrylamide gel, and the detection of histidine as the sole N-terminal amino acid in the native enzyme . Since dissociation into the subunits occurred without the use of reducing agents, the association of the subunits seems to require no disulfide linkage . One mole of the subunit contained one mole of sulfhydryl group which appeared to be buried inside the molecule . Partial restoration of the catalytic activity was observed when the urea-denatured enzyme was dialyzed to remove urea, especially in the presence of reducing agents such as dithiothreitol . The urea-denatured enzyme showed a tendency in the absence of reducing agents to form a subunit dimer linked by a disulfide bond between the cystine residues exposed by denaturation . The amino acid composition of the enzyme was determined; it contained one half-cystine residue per subunit, and the content of acidic residues was much higher than that of basic residues . Based on these findings, the subunit structure of the enzyme is discussed. J Biochem (Tokyo), 1976 Oct, 80(4), 777 - 85 O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast . Further purification and characterization as a pyridoxal enzyme; Yamagata S et al.; O-Acetylserine-O-acetylhomoserine sulfhydrylase {EC class 4.2.99}, catalyzing the sulfhydrylation of both O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) (O-acetyl-L-serine(O-acetyl-L-homoserine) + H2S leads to L-cysteine (L-homocysteine) + acetate), was extracted and purified from bakers' yeast by an improved method . The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis both in the absence and presence of sodium dodecylsulfate and by ultracentrifugal analysis . The apo-enzyme was protected by pyridoxal phosphate (PALP) from inactivation by heat, urea, and trypsin {EC 3.4.21.4}, suggesting that the binding of PALP to the apo-enzyme rendered the conformation of the protein more stable . The holo-enzyme showed absorption peaks at 420 and 330 nm due to bound PALP, in addition to a peak at 280 nm . Upon reduction with borohydride, the 420-nm peak disappeared and an increase in the 330-nm peak occurred concomitant with loss of the catalytic activity . Lysine appeared to be the pyridoxal binding site, based on identification of pyridoxyl-lysine in the hydrolyzate of the holo-enzyme . It was shown by both spectral and chemical determinations that 4 moles of PALP could bind to 200,000 g of apo-protein . The apo-enzyme showed a lower association constant with PALP than some other enzymes . Pyridoxal inhibited the activity competitively with respect to PALP . Based on these findings, it appears that the reaction mechanism