Eur J Biochem, 1981 Apr, 115(3), 635 - 41 Conformational changes of yeast tRNAphe as monitored by 31P NMR; Salemink PJ et al.; The 31P NMR spectra of tRNAs contain approximately 17 resonances resolved from the main resonance which consists of about 80% of the total resonance intensity arising from the sugar phosphate backbone . In the present paper we study the behavior of the 31P resonances of yeast tRNAPhe as a function of temperature and of solution conditions . By comparison with other melting experiments we show that three resonances (called c, e and j2) belong to phosphates in the anticodon loop, while the remaining resolved 31P resonances come from phosphates in specific conformations in the central part of the molecule imposed by the tertiary structure . These conformations are different from the normal g-,g- conformation found in A-RNA double helices . The assignments are in good agreement with those previously made on the basis of chemical and enzymatic modification experiments {P . J . M . Salemink, T . Swarthof & C . W . Hilbers (1979) Biochemistry, 18, 3477-3485} . AT high Mg2+ concentrations the anticodon loop is found to be present in two different conformations . For all solution conditions studied loss of the anticodon loop structure takes place before the tertiary structure is melted out . The melting of the tertiary structure is not strictly an all- or-none process . The lifetimes of phosphate conformations involved in the tertiary structure may differ by at least a factor of two . It can also be concluded that the range of chemical shifts observed for phosphodiesters cannot at the moment be accounted for by theoretical calculations. Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2244 - 8 Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated; Hashimoto C et al.; Carboxypeptidase Y, a vacuolar enzyme from Saccharomyces cerevisiae, was digested with endo-beta-N-acetyl-D-glucosaminidase H to release the four oligosaccharide chains that are linked to asparagine in the glycoprotein . The oligosaccharides were fractionated into a neutral and acidic component, and the latter proved to phosphorylated . From its gel filtration pattern, the neutral fraction was shown to be a mixture of at least four homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with eight mannoses and one N-acetylglucosamine {Cohen, R . E . & Ballou, C . E . (1980) Biochemistry 19, 4345--4358} . The yeast oligosaccharide has one additional mannose unit in an alpha 1 leads to 3 or alpha 1 leads to 6 linkage, whereas the larger homologs appear to have two, three, and four more mannose units . One phosphorylated oligosaccharides with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and then subjected to mild acid hydrolysis . This released mannose and mannobiose that were glycosidically linked to the phosphate group, whereas complete acid hydrolysis yielded D-mannose 6-phosphate . The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with alpha-mannosidase, and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2 . These results suggest that the mannosidase-resistant phosphorylated oligosaccharide has the structure Man leads to P leads to 6 alpha Man leads to alpha Man leads to 6 beta Man leads to 4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead of mannose . A second phosphorylated oligosaccharide with a mannose/phosphate ratio of 6.5 probably contains two phosphodiester groups, but its structure has not been investigated in detail. Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2082 - 5 Molecular rulers for measuring RNA structure: sites of crosslinking in chlorambucilyl-phenylalanyl-tRNAPhe (yeast) and chlorambucilyl-pentadecaprolyl-phenylalanyl-tRNAPhe (yeast) intramolecularly crosslinked in aqueous solution; Wickstrom E et al.; Intramolecular crosslinking of yeast phenylalanine tRNA in aqueous solution with rigid, variable-length crosslinking reagents, which we call "molecular rulers," has given results in reasonable agreement with the crystal structure . Chlorambucilyl-{3H}phenylalanyl-tRNAPhe crosslinked intramolecularly at G-71 and A-73, whereas chlorambucilyl-pentadecaprolyl-{3H}phenylalanyl-tRNAPhe crosslinked at G-20 and Y-37 . The pentadecaprolyl reagent was predicted to be 62 A long, including chlorambucil and phenylalanine; the sites that it reached are 60 A distant from the 3' OH (in the case of G-20) or 80 A distant (in the case of Y-37) in the crystal structure of tRNAPhe . The close agreement between the length of the reagent and the distance of G-20 from the 3' OH in the crystal structure illustrates the rigidity of the tRNAPhe molecule in the dihydrouridine loop region at the corner of the molecule . The apparent ability of the 62-A-long reagent to crosslink to a site, Y-37, that is 80 A distant from the 3' OH in the crystal structure appears to illustrate the flexibility of both the 3' A-C-C-A terminus and the anticodon stem and loop, with respect to the tRNA molecule . These observations demonstrate the utility of oligoproline-based crosslinking reagents as rigid, variable-length molecular rulers for biological macromolecules in solution. Biochim Biophys Acta, 1981 Mar 20, 642(1), 173 - 81 Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate; Borst-Pauwels GW et al.; Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme . The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2- . The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7 . KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase . On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude. Biochemistry, 1981 Mar 17, 20(6), 1653 - 9 Changes in the solution structure of yeast phenylalanine transfer ribonucleic acid associated with aminoacylation and magnesium binding; Potts RO et al.; The effect of aminoacylation on the structure of yeast phenylalanine tRNA was evaluated by laser light scattering . In these experiments, the translational diffusion coefficient (D20,w) of phenylalanyl-tRNA was monitored continuously during spontaneous deacylation in a variety of solution conditions . The results reveal that significant changes can occur in the hydrodynamic volume and electric charge as a consequence of aminoacylation but that the effects are magnesium dependent . At neutral pH, 20 degrees C, and 0.1 M salt, the D20,w value increased by 18% when deacylation occurred in 2--10 mM Mg2+ concentrations while no change in diffusivity was observed for tRNA deacylating in 0.5--1.0 mM Mg2+ . The Mg2+ concentration dependence of the D20,w changes behaves in highly cooperative manner . The electric charges of aminoacyl-tRNA and nonacylated tRNA in 1 and 10 mM Mg2+ were estimated from the diffusive virial coefficients . In the higher Mg2+ conditions, aminoacyl-tRNA has a charge of 15 +/- 2e- while that of the nonacylated form is 10 +/- 2e-; both acylated and nonacylated tRNA have a charge of 11 +/- 4e- in 1 mM Mg2+ . Taken together, the results indicate that aminoacylation permits the binding of additional Mg2+, resulting, in turn, in the formation of a more extended conformer of lower diffusivity and greater negative charge . The results also provide a possible explanation for several contradictory results in the literature. Biochemistry, 1981 Mar 17, 20(6), 1513 - 20 Glutathione reductase from yeast . Differential reactivity of the nascent thiols in two-electron reduced enzyme and properties of a monoalkylated derivative; Arscott LD et al.; Two-electron reduced glutathione reductase from yeast reacted with iodoacetamide is alkylated almost exclusively in the nascent thiol nearer the amino terminus of the protein . The charge-transfer absorbance, maximal at 530 nm, characteristic of the two-electron reduced enzyme is not lost as the alkylation proceeds, and the product has a spectrum virtually identical with that of the two-electron reduced enzyme . This observation demonstrates that the thiol alkylated is not the charge-transfer-donor thiolate which interacts with the FAD . The spectrum of the monoalkylated derivative is stable in the presence of oxidized glutathione, indicating that the charge-transfer-donor thiol is not involved in interchange with the substrate in the native enzyme . Thus, the nascent thiols produced upon two-electron reduction of glutathione reductase have distinct functions, interchange with the substrate and interaction with the FAD . Treatment of the monoalkylated derivative with the apolar phenylmercuric acetate eliminates the charge-transfer interaction . The spectrum of the resulting species is similar to that of the oxidized enzyme but less resolved and blue shifted by 10 nm . The dependence on pH of the absorbance associated with the thiolate to FAD charge-transfer interaction in native two-electron reduced glutathione reductase is biphasic, with pK values at approximately 4.8 and 7.4 . By analogy with glyceraldehyde-3-phosphate dehydrogenase and papain, these data indicate that the thiolate is stabilized by an adjacent basic residue . The pK 7.4 is associated with the titration of the base to give the ion pair, and the pK of 4.8 is associated with the titration of the thiolate . Unlike lipoamide dehydrogenase, glutathione reductase is sufficiently stable to allow titration with dithionite at pH 3.7 . The spectrum at this pH is essentially the same as that of the monoalkylated derivative treated with phenylmercuric acetate . The changes with pH are completely reversible. Biochim Biophys Acta, 1981 Mar 12, 635(1), 187 - 93 Hydrophobic photolabelling of the yeast cytochrome c oxidase subunits in contact with lipids; Gutweniger H et al.; The hydrophobic domain of the membrane-bound enzyme yeast cytochrome c oxidase was labelled with photoactivable phosphatidylcholines . Subunits I, II and III were labelled; a minor labelling was also found on subunits V and VII . The labelling of subunit V was located in a small terminal polypeptide sequence. Biochemistry, 1981 Mar 3, 20(5), 1229 - 35 Liberation of the triosephosphate isomerase reaction intermediate and its trapping by isomerase, yeast aldolase, and methylglyoxal synthase; Iyengar R et al.; When a mixture of triosephosphate isomerase (rabbit muscle) and dihydroxyacetone phosphate (DHAP) is quenched with acid, a compound is liberated, presumed to be the cis-enediol 3-phosphate, that decomposes to inorganic phosphate (Pi) and methylglyoxal {Iyengar, R., & Rose, I.A . (1981) Biochemistry (preceding paper is this issue)} . The decomposition can be prevented by rapid neutralization if a catalytic amount of fresh isomerase is present . Varying the time between acidification and rescue gave a half-life of the liberate compound of approximately 12-17 ms . Varying the concentration of enzyme used for rescue gave a minimum second-order rate constant for trapping of 10(9)M(-1)s(-1) . These results add further evidence favoring a stepwise mechanism for the aldose-ketose isomerase reactions in which a chemically defined enzyme-bound intermediate is found . The high rate of trapping over a wide pH range indicates that the enediol phosphate, not the enediolate phosphate, is the intermediate . One property of the enzyme is to stabilize the intermediate with respect to its fragmentation in solution by greater than 1000-fold . Yeast aldolase is also able to rescue all of the isomerase intermediate, though higher concentrations of enzyme are required . Although different enantiotopic protons of DHAP are abstracted by isomerase and aldolase, both enzymes use the same enediol phosphate intermediate . Methylglyoxal synthase at a 50-fold greater concentration was unable to compete with triosephosphate isomerase for cis-enediol phosphate . Either the synthetase has a low V/K for the cis isomer or it uses the trans-enediol phosphate form specifically . A new strategy for the chemical and enzymological characterization of enzyme reaction intermediates is proved here based on the liberation of the intermediate from the reaction equilibrium and its recovery by fresh enzyme or another enzyme species. Biochemistry, 1981 Mar 3, 20(5), 1147 - 56 Nuclear magnetic resonance and nuclear Overhauser effect study of yeast phenylalanine transfer ribonucleic acid imino protons; Johnston PD et al.; Results directed primarily toward spectral assignment and nuclear spin dynamics are described for yeast tRNAPhe in 0.1 M NaCl, pH 7 . Magnesium titrations were performed . Changes in the spectrum occur for Mg2+/tRNA ratios of about 2 and above 10 . Difference spectroscopy between 43 and 29 degrees C in zero Mg2+ concentration, together with prior identification of the GU4 acceptor stem base pair, indicates early acceptor melting and is used to identify acceptor resonances . Transport of spin energy (spin diffusion) is described in tRNA together with a summary of relevant experiments . A survey of nuclear Overhauser effects (NOE's) between imino and aromatic and amino protons is included, together with some recent conclusions based on methyl NOE's and experiments with tRNAs deuterated at the purine C8 position . Assignment of the imino NMR spectrum on the basis of these and previous data is reviewed and discussed in detail . Preliminary distance estimates based on the NOE for AU and GU4 base pairs are in reasonable agreement with the expected distances. Int J Pept Protein Res, 1981 Mar, 17(3), 393 - 400 Structural studies on yeast 3-phosphoglycerate kinase . Linear arrangement of the CNBr fragments, partial amino acid sequence of the inner part of the polypeptide chain, and analyses of the N-terminal domain of the protein; Fattoum A et al.; The purpose of this work was to contribute to the study of the covalent structure of yeast 3-phosphoglycerate kinase . First, we undertook the complete alignment of the four fragments produced by cyanogen-bromide cleavage and which constitute the intact protein; we then established the total amino acid sequence of a 30-residue peptide and the N-terminal sequence of a 65-residue peptide . Second, we analyzed the acetylated state of the protein . The analyses of the acid fraction "P" obtained after digestion of 3-phosphoglycerate kinase by pronase enabled us to determine the N-terminal sequence of this enzyme as N-acetylserylglycine . Third, we isolated, purified and analyzed seven tryptic peptides from a fragment containing 102 amino acids coming from the N-terminal end of the protein . The peptides occupying the N- and C-terminal ends of this fragment were also identified. Mutat Res, 1981 Mar, 81(1), 27 - 36 Light-flash analysis of the photoenzymic repair process in yeast cells . II . Determination of the rate constant for formation of photoreactivating enzymes-pyrimidine dimer complexes and its activation energy term; Fukui A et al.; As reported in the previous paper, the number of deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) molecules per yeast cell was determined by the use of intense light flashes . In the present work, the reaction rate constant for the formation of PRE-substrate complexes, k1, and the activation energy term of k1 were determined by yeast cells in vivo by the use of light flashes . At 30 degrees C, k1 equalled (6.5 +/- 1.1) x 10(-5) (nuclear volume) x (molecule)(-1) sec(-1), which corresponded to 1.1 x 10(5) 1 mole(-1) sec(-1), on the assumption that a nuclear volume is 3 x 10(-15) 1 . k1 showed positive temperature dependence as described by the arrhenius expression with an activation energy of 11.8 +/- 1.6 kcal mole(-1). Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1606 - 8 Conformational activation of the yeast phenylalanyl-tRNA synthetase catalytic site induced by tRNAPhe interaction: triggering of adenosine or CpCpA trinucleoside diphosphate aminoacylation upon binding of tRNAPhe lacking these residues; Renaud M et al.; Adenosine or CpCpA trinucleoside diphosphate can be aminoacylated by phenylalanyl-tRNA synthetase {L-phenylalanine:tRNAPhe ligase (AMP forming), EC 6.1.1.20} when the reaction takes place in the presence of tRNAPhe deprived of its 3' adenosine or pCpCpA terminus . This shows that, upon interaction with tRNA, a structural alteration of the enzyme's active site is achieved . This process may be a determining step in the specificity of the aminoacylation reaction. J Bacteriol, 1981 Mar, 145(3), 1342 - 50 Timing of ribosome synthesis during ascosporogenesis of yeast cells: evidence for early function of haploid daughter genomes; Emanuel JR et al.; During meiosis and sporulation in Saccharomyces cerevisiae, the recessive genetic marker for cycloheximide resistance, believed to be due to an altered ribosomal protein (C . S . McLaughlin, p . 815-827, in M . Nomura et al., ed., Ribosomes, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), is expressed as early as meiosis II . Ribosomal ribonucleic acid synthesis peaks near the time that cycloheximide resistance begins to appear . Less than 25% of the 17S and 25S ribonucleic acid of the vegetative cells persists in spores, but pulse-labeling studies indicate that greater than 90% of the stable ribonucleic acid made after 6 h survives in spores . These results indicate that the haploid daughter genomes begin to function near the time of meiosis II. Int J Radiat Biol Relat Stud Phys Chem Med, 1981 Mar, 39(3), 281 - 90 Responses of yeast cells to heat applied alone or combined with gamma-rays; Petin VG et al.; Experiments were carried out to determine the effects of hyperthermia alone or combined with gamma-rays on the survival of yeast cells of different species . Arrhenius plots for the heat inactivation of yeast cells show inflection points . This suggests that hyperthermic killing above or below these temperatures is mediated by different mechanisms . The synergism between hyperthermia and ionizing radiation was almost linearly dependent on temperature as shown by Arrhenius plots for the combined action of both modalities . The data obtained may imply that different processes are involved in heat inactivation and thermal enhancement of yeast cell radiosensitivity. Mol Cell Biol, 1981 Mar, 1(3), 269 - 80 Ribonuclease "XlaI," an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors; Otsuka A et al.; A ribonuclease (RNase) activity, RNase "XlaI," responsible for the excision of intervening sequences from two yeast transfer ribonucleic acid (tRNA) precursors, pre-tRNA(Tyr) and pre-tRNA(3Leu), has been purified 54-fold from nuclear extracts of Xenopus laevis oocytes . The RNase preparation is essentially free of contaminating RNase . A quantitative assay for RNase XlaI was developed, and the reaction products were characterized . RNase XlaI cleavage sites in the yeast tRNA precursors were identical to those made by yeast extracts (including 3'-phosphate and 5'-hydroxyl termini) . Cleavage of pre-tRNA(3Leu) by RNase XlaI and subsequent ligation of the half-tRNA molecules do not require removal of the 5' leader or 3' trailer sequences. Nucleic Acids Res, 1981 Feb 25, 9(4), 1031 - 44 Conformational states of yeast tRNA Phe in the complex with cognate and non cognate synthetases; Rigler R et al.; The influence of phenylalanyl-tRNA synthetase and seryl-tRNA synthetase on the conformation and structural kinetics of yeast tRNA Phe was investigated . Ethidium substituted for dihydrouracil at position 16 or 17 was used as a structural probe, showing the existence of three conformational states in tRNA . The distribution of states (T1, T2, T3) is changed only by the cognate synthetase towards T3 which probably is related to the X-ray structure . The binding of phenylalanyl-tRNA synthetase leads to an about 10-fold increase in the fast transition T1 in equilibrium or formed from T2 which has been assigned to changes in the anticodon loop conformation and to a 2-3 fold increase in the slow transition which probably extends to other parts of the tRNA molecule . The observed rates for the transition T2 in equilibrium or formed from T3 are close to that observed for the transfer of the activated phenylalanine to tRNA Phe . This raises the possibility that the conformational transition in tRNA is the rate limiting step in the charging reaction. Nucleic Acids Res, 1981 Feb 25, 9(4), 921 - 34 Characterization of the yeast tRNA Ser genomic organization and DNA sequence; Page GS et al.; Purified, isolated yeast tRNA Ser2 was used as a hybridization probe to estimate the number of tRNA Ser2 genes in the yeast genome . Molecular clones of several of the genes were obtained . Three examples were studied in detail with respect to their genomic organization, and DNA sequences were determined for them . There appear to be eleven tRNA Ser2 genes in the yeast genome . They are neither tandemly repeated, nor clustered with other tRNA genes . They contain no intervening sequences. Nucleic Acids Res, 1981 Feb 25, 9(4), 789 - 99 The structure of the yeast ribosomal RNA genes . 3 . Precise mapping of the 18 S and 25 S rRNA genes and structure of the adjacent regions; Bayev A et al.; The 5'-terminal of Saccharomyces cerevisiae 18 S and 25 S rRNA are precisely mapped within the sequence of the rDNA repeating unit . The 3'-terminal of 25 S rRNA and 37 S pre-rRNA are located within a 548 bp segment of the rDNA repeating unit by the use of a DNA polymerase I extension technique . The analysis of the rDNA sequences at the structural gene boundaries reveals the presence of oligonucleotide repeats which may be involved in transcription or processing control mechanisms . The sequence of rDNA in the transcription termination region is determined and possible mechanisms shaping the 3'-end of 25 S rRNA are discussed. Nucleic Acids Res, 1981 Feb 25, 9(4), 1019 - 29 tRNA synthesis: identification of in vivo precursor tRNAs from parental and mutant yeast strains; Hopper AK et al.; In vivo yeast precursor tRNAs have been identified using a modification of the Northern-hybridization procedure . Two species of pre-tRNA Tyr, 1 species of pre-tRNA Ser2 and 2 species of pre-tRNA Serminor have been found in all yeast strains examined, including parental strains and strains harboring mutations affecting tRNA function . One of the tRNA Tyr strains harboring and one of the pre-tRNA SerUCG are the same size as the unspliced pre-tRNAs which accumulate in the yeast mutant rna1 . The in vivo tRNA Tyr precursors detected in these studies also appear similar with the RNA species identified when cloned yeast tRNA Tyr is transcribed and processed by Xenopus oocytes and/or Xenopus extracts . We have also studied the precursor and mature tRNA Tyr species from 22 mutants which contain mutations in the SUP4 tyrosine-inserting suppressor locus . The RNA from 2 mutants mapping at the G52 position showing an aberrantly migrating "mature" tRNA Tyr . Although several of those cloned mutant genes showed transcript products of altered size in in vitro transcription studies (1), we did not detect such altered transcripts in vivo. J Biol Chem, 1981 Feb 25, 256(4), 1782 - 5 Inactivation of hydroxymethylglutaryl-CoA reductase from yeast by coenzyme A disulfide; Gilbert HF et al.; The time-dependent inactivation of hydroxymethylglutaryl-CoA reductase from yeast by solutions of hydroxymethylglutaryl-CoA and CoASH is due to the rapid inactivation of the enzyme by oxidized CoA (CoA disulfide) present at trace levels in solutions of hydroxymethylglutaryl-CoA and CoASH . Solutions of hydroxymethylglutaryl-CoA or CoASH incubated for 1.5 h with 10 mM dithiothreitol at pH 7.0, 22 degrees C, do not inactivate the enzyme . Inactivation of hydroxymethylglutaryl-CoA reductase is rapid and complete at concentrations of CoA disulfide comparable to those measured in solutions of hydroxymethylglutaryl-CoA and CoASH . Inactivation of the enzyme by CoA disulfide may be reversed by treating the inactive enzyme with 10 mM dithiothreitol at pH 7.0 Both the inactivation of the enzyme by CoA disulfide and reactivation by dithiothreitol are inhibited by hydroxymethylglutaryl-CoA . Other disulfides such as Ellman's reagent and glutathione disulfide also inactivate the enzyme . A thio-disulfide exchange reaction with a sulfhydryl group on the enzyme forming a mixed disulfide or an intramolecular protein disulfide could account for the enzyme inactivation . The normal function of the sulfhydryl group involved in the inactivation of the enzyme is unknown. Biochim Biophys Acta, 1981 Feb 18, 673(1), 10 - 3 Alpha-glucosidase synthesis in yeast cells depleted of intramitochondrial ATP; Leskova Z et al.; We have studied the dependence on mitochondrial ATP of expression of MAL genes specifying maltose utilization in yeast . It was found that bongkrekic acid does not prevent the maltose induced synthesis of alpha-glucosidase in derepressed cells of the wild-type and corresponding respiratory-deficient mutant of Saacharomyces cerevisiae . The results suggest that expression of nuclear genes specifying alpha-glucosidase and maltose catabolism in yeast is apparently not dependent on the proper function of mitochondrial adenine nucleotide translocase and does not even require the presence of normal levels of ATP in mitochondria. J Biol Chem, 1981 Feb 10, 256(3), 1377 - 84 The amino acid sequence of yeast enolase; Chin CC et al.; Automatic sequencing of yeast enolase and of its chemically and enzymatically produced peptide fragments has established the sequence of 416 of the 436 residues in the enolase subunits . The missing segments have been provided from results from sequencing the DNA of the yeast enolase genes (Holland, M . J., Holland, J . P., Thill, G . P., and Jackson, K . A . (1981) J . Biol . Chem . 256, 1385-1395) . The reported enolase sequence thus represents the results of two completely independent studies, which yielded identical results for 404 of the 436 residues, and which on re-examination are consistent with the reported sequence in all but nine positions . The availability of the entire yeast enolase sequence has permitted a reassessment of structure-function parameters available for the enzyme, and some implications of the sequence information on the secondary, tertiary, and quarternary structure and on the active site components of yeast enolase have been summarized and discussed. J Biol Chem, 1981 Feb 10, 256(3), 1385 - 95 The primary structures of two yeast enolase genes . Homology between the 5' noncoding flanking regions of yeast enolase and glyceraldehyde-3-phosphate dehydrogenase genes; Holland MJ et al.; Segments of yeast genomic DNA containing two enolase structural genes have been isolated by subculture cloning procedures using a cDNA hybridization probe synthesized from purified yeast enolase mRNA . Based on restriction endonuclease and transcriptional maps of these two segments of yeast DNA, each hybrid plasmid contains a region of extensive nucleotide sequence homology which forms hybrids with the cDNA probe . The DNA sequences which flank this homologous region in the two hybrid plasmids are nonhomologous indicating that these sequences are nontandemly repeated in the yeast genome . The complete nucleotide sequence of the coding as well as the flanking noncoding regions of these genes has been determined . The amino acid sequence predicted from one reading frame of both structural genes is extremely similar to that determined for yeast enolase (Chin, C . C . Q., Brewer, J . M., Eckard, E., and Wold, F . (1981) J . Biol . Chem . 256, 1370-1376), confirming that these isolated structural genes encode yeast enolase . The nucleotide sequences of the coding regions of the genes are approximately 95% homologous, and neither gene contains an intervening sequence . Codon utilization in the enolase genes follows the same biased pattern previously described for two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes (Holland, J . P., and Holland, M . J . (1980) J . Biol . Chem . 255, 2596-2605) . DNA blotting analysis confirmed that the isolated segments of yeast DNA are colinear with yeast genomic DNA and that there are two nontandemly repeated enolase genes per haploid yeast genome . The noncoding portions of the two enolase genes adjacent to the initiation and termination codons are approximately 70% homologous and contain sequences thought to be involved in the synthesis and processing messenger RNA . Finally there are regions of extensive homology between the two enolase structural genes and two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes within the 5- noncoding portions of these glycolytic genes. Biochim Biophys Acta, 1981 Feb 5, 672(3), 313 - 6 The effect of spermine on the interaction of AMP deaminase with threonine dehydratase activity in yeast; Yoshino M et al.; Evidence suggesting that AMP deaminase (EC 3.5.4.6) is responsible for the stimulation of threonine dehydratase (EC 4.2.1.16) activity in situ is presented using yeast cells which have been rendered permeable . The addition of polyamine, an activator of AMP deaminase, resulted in the increase in ammonia concentration, which can stimulate the activity of yeast threonine dehydratase . Polyamine may regulate the synthesis of isoleucine and valine, and of the intermediates of citric acid cycle through the activation of AMP deaminase-threonine dehydratase system as a 'cascade system' in yeast. J Clin Microbiol, 1981 Feb, 13(2), 393 - 4 Rapid detection of yeast enzymes by using 4-methylumbelliferyl substrates; Bobey DG et al.; In a preliminary study with a limited number of isolates, the usefulness of 17 4-methylumbelliferyl substrates for identifying yeast isolates was investigated . Substrates to detect acid phosphatase, glucosidase, and pyrophosphate diesterase showed promise. J Inorg Biochem, 1981 Feb, 14(1), 33 - 44 Binding of terbium (III) to yeast enolase; Brewer JM et al.; Several independent criteria indicate 2 mol of terbium (III) bind to yeast enolase in the absence of substrate-fluorescence titrations of enzyme and metal, effects on thermal stability and published ultrafiltration and inhibition experiments . These measurements also suggest the terbium binding sites are the same as those normally occupied by "conformational" magnesium . Terbium binds much more strongly than magnesium, however, and measurements of the kinetics of the absorbance change in the terbium-enzyme on adding excess EDTA suggest the terbium-enzyme dissociation constant is about 1/500 that of the magnesium-enzyme . Measurements of enzyme activity as a function of substrate concentration show that terbium permits no enzymatic activity . However, magnesium competes more effectively with the lanthanide if the substrate analogue 3-aminoenolpyruvate 2-phosphate (AEP) is present . The fluorescence of the lanthanide is not readily observed on exciting the terbium-enzyme at 280 nm, indicating the absence of tyrosines or tryptophans in the coordination sphere of the metal . Excitation of terbium using 488 nm radiation from an argon ion laser shows the fluorescence of the metal is enhanced by binding to the enzyme . EDTA and carbonate have similar effects . This suggests carboxyl groups are involved in binding metal at the conformational sites of yeast enolase . Measurements of lifetimes of enzyme-bound terbium in the presence and absence of D2O indicated three moles of water remained on each of the bound metals, independently of the buffer used . If enzyme-bound terbium is assumed to be nine-coordinate, the metal must bind to six groups from the enzyme . The presence of substrate does not markedly affect the emission spectrum of the bound terbium or the number of water molecules remaining on the metal, but calorimetric measurements show that substrate binds to the terbium enzyme. Arch Microbiol, 1981 Feb, 128(4), 394 - 7 Accumulation of pyrophosphate and other energy-rich phosphorous compounds under various conditions of yeast growth; Ermakova SA et al.; In the cells of hybrid yeast strain Saccharomyces N.C.Y.C . 644 SU3 (Karlsberg collection), a large amount of pyrophosphate (30-300 micro mol per g of dry weight) accumulates whatever the aeration conditions and the contest of glucose in the medium . The content of pyrophosphate is 10-100 times higher than that of ATP . At the early and mid-exponential growth phases two maxima of pyrophosphate accumulation are observable . The periods of maximal pyrophosphate accumulation in yeast coincide with those of the minimal contest of polymeric acid-soluble polyphosphates and intense budding . In the light of the data obtained, the question is discussed as to the relationship between the metabolism of pyrophosphates and acid-soluble polyphosphates in yeats. Cell, 1981 Feb, 23(2), 605 - 14 Transposable elements associated with constitutive expression of yeast alcohol dehydrogenase II; Williamson VM et al.; The yeast structural gene ADR2, coding for the glucose-repressible alcohol dehydrogenase (ADHII), has been isolated by complementation of function in transformed yeast . The chromosomal DNA from nine yeast strains with cis-dominant constitutive mutations (ADR3c) has been investigated by restriction enzyme analysis, using the cloned ADR2 DNA as a hybridization probe . Seven mutants appear to have insertions of approximately 5.6 kg near the 5' end of the ADR2-coding region . Four of these insertions have the same restriction pattern as the yeast transposable element Ty1 . Two differ from Ty1 by the presence of an additional Hind III site, and a seventh insertion differs from Ty1 at a number of restriction sites . All are inserted in the same orientation with respect to the structural gene . A DNA fragment containing the ADR2 gene and adjacent sequences from a constitutive mutant has been cloned and shown by heteroduplex analysis to contain an insertion near the 5' end of the structural gene . The cloned insertion sequence hybridizes to multiple genomic DNA fragments, indicating that it contains a moderately repetitive sequence . Thus it appears that insertion of a transposable element near the 5' terminus of the structural gene can produce constitutive expression of a normally glucose-repressed enzyme . Such insertions seem to be the most common way of generating cis-dominant constitutive mutations of ADHII. Biochim Biophys Acta, 1981 Jan 29, 652(1), 129 - 38 The occurrence of two ribosomal ribonucleases depending on growth phase in yeast . Induction of ribonuclease in glucose-starved cells; Swida U et al.; There are two latent ribonucleases associated with the 40 S subunits of yeast ribosomes which differ in their digestion products, pH optimum, molecular weight, and in their activity during growth phase . The 3'-nucleotide-producing enzyme is active only in the late logarithmic or stationary growth phase, whereas the ribonuclease which produces 5'-nucleotides is present at all growth phases . The enzymes were separated by affinity chromatography and were partially characterized . By changing growth conditions--i.e . decreasing and increasing the glucose concentration in the medium--the activity of the 3'-ribonuclease could be induced or reduced. Biochim Biophys Acta, 1981 Jan 29, 652(1), 234 - 9 Mitochondrially synthesized protein subunits of the yeast mitochondrial adenosine triphosphatase . A reassessment; Orian JM et al.; Evidence is presented that a mitochondrial translation product (Mr, 32,000) previously thought to be a subunit of the membrane sector of the yeast mitochondrial ATPase is a contaminant, consisting of subunit II of the cytochrome oxidase complex and cytochrome b apoprotein . Our data suggest that only two subunits (Mr, 7600 and 20,000) of the mitochondrial ATPase are synthesized in the mitochondria. Biochim Biophys Acta, 1981 Jan 29, 652(1), 82 - 9 Joining of yeast alanine transfer ribonucleic acid half molecules to form a whole molecule by T4 RNA ligase; Wang GH et al.; Covalent joining of the two half molecules of tRNAAla by T4 RNA ligase to form a reconstituted whole molecule was investigated . The two half molecules consisting, respectively, of residues 1-35 and 36-75 were prepared by partial degradation of tRNAAla with RNAase T1 . The 5'-half molecule was treated with alkaline phosphatase to remove the 3'-terminal phosphate group, and the 5'-OH group of the 3'-half molecule was phosphorylated with {gamma-32P}ATP by polynucleotide kinase . The two terminal nucletides to be joined were identified as Guo and Cyd . Prior to the covalent joining reaction, the two modified half molecules in an equimolar mixture were annealed, and the rejoined half molecules, separated by gel electrophoresis, served as the substrate for T4 RNA ligase . Optimum conditions for this ligation, such as RNA ligase concentration, pH, Mg2+ concentration, reaction temperature and time of reaction, were investigated . Under the optimum conditions a yield of about 70% joining of the reconstituted whole molecule was obtained as shown by gel electrophoresis, resistance to hydrolysis by alkaline phosphatase, nearest neighbour analysis and alanine acdeptor activity. Biochim Biophys Acta, 1981 Jan 26, 663(1), 194 - 202 Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes; Aoyama Y et al.; According to Ohba et al . (Ohba, M., Sato, R., Yoshida, Y., Nishino, T . and Katsuki, H . (1978) Biochem . Biophys . Res . Commun . 85, 21-27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of {1,7,15,22,26,30-14C}lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) in the presence of NADPH, NAD+ and molecular oxygen, concomitant with the liberation of 14CO2 derived from C-30 (one of the two methyl groups at the C-4 position) . In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system . In this study it was found that the 14CO2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b5 and by palmitoyl-CoA, a substrate of the cytochrome b5-containing fatty acyl-CoA desaturase system of yeast microsomes . However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) . It is concluded that cytochrome b5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14 alpha-demethylation of lanosterol, by yeast microsomes . It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation . The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation. J Biol Chem, 1981 Jan 25, 256(2), 557 - 9 Binding of citreoviridin to the beta subunit of the yeast F1-ATPase; Gause EM et al.; Citreoviridin, a nonfluorescent inhibitor of bovine and bacterial ATPases, also inhibits the yeast F1 (K1 = 2 microM) . The beta subunit-specific fluorescent ligand, aurovertin, has been used to report the interaction of citreoviridin with the yeast F1-ATPase and the isolated beta subunit . Citreoviridin caused a marked decrease in the fluorescence increment associated with the binding of aurovertin to either intact F1 or the isolated beta subunit . Three lines of evidence indicate that citreoviridin and aurovertin bind to nonidentical sites on the beta subunit: 1) the binding of citreoviridin to the F1 or isolated beta subunit is noncompetitive with respect to aurovertin; 2) the number of aurovertin binding sites (Kd = 0.2 to 0.6 microM) per F1-ATPase molecule remains the same (1.89 +/- 0.6 mol of aurovertin bound per mol of F1) in the presence or absence of citreoviridin; 3) the F1-ATPase obtained from the aurovertin-resistant mutant aur-1 is partly inhibited by citreoviridin. Nature, 1981 Jan 22, 289(5795), 250 - 2 Homology and non-homology at the yeast mating type locus; Sprague GF Jr et al.; Four mutations of the alpha mating type locus of Saccharomyces cerevisiae have been analysed to determine their relationship to the a mating type locus . Mat alpha+ recombinations are produced by mat alpha 2-/MAT but not by mat alpha 1-/MATa diploids . MAT alpha and MATa thus contain regions of homology (coding for at least part of MAT alpha 2) and regions of non-homology (coding for at least part of MAT alpha 1)-the genetic determinant for cell type is larger than the non-homologous sequence seen by DNA-DNA heteroduplexes and genetic analysis . The segment transposed in mating type interconversion includes both types of sequence. Nature, 1981 Jan 22, 289(5795), 244 - 50 A position effect in the control of transcription at yeast mating type loci; Nasmyth KA et al.; The two mating type loci MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters . Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed . It is concluded that sequences to the left of HMRa (and probably HML alpha) must somehow affect transcription initiated at the centre of each locus 700 to 1,400 base pairs away . A possible mechanism for this position effect is discussed. Nature, 1981 Jan 22, 289(5795), 239 - 44 Regulation of transcription in expressed and unexpressed mating type cassettes of yeast; Klar AJ et al.; The genes that control the a, alpha and a/alpha cell types in Saccharomyces are carried on transposable elements known as a and alpha cassettes which reside at three different chromosomal loci . Examination of the transcripts by R-looping and filter hybridization indicates that each cassette is capable of producing two divergent transcripts . Cassettes at the MAT locus are transcribed constitutively . Transcription of cassettes at HML and HMR is prevented by trans-acting negative regulators. Biochem J, 1981 Jan 15, 194(1), 63 - 70 The role of glutathione in amino-acid absorption . Lack of correlation between glutathione turnover and amino-acid absorption by the yeast Candida utilis; Robins RJ et al.; The rate of degradation of glutathione has been determined in the yeast Candida utilis by using a method that minimizes the effect of amino-acid recycling . When yeast are grown in amino-acid-free medium, the half-life of glutathione was found to be 230 min . C . utilis was also found to absorb various L-amino acids rapidly without producing any significant decrease in the half-life of glutathione . While the gamma-glutamyl cycle is thus operating in C . utilis, the rate of degradation of glutathione is found to be 100 times too slow for the cycle to be mediating the transport of these amino acids. Nature, 1981 Jan 15, 289(5794), 144 - 8 Intrachromosomal gene conversion in yeast; Klein HL et al.; We have shown that the yeast Saccharomyces cerevisiae has a mechanism by which information from one gene can be transferred non-reciprocally to a repeated copy of the gene on the same chromosome . This intrachromosomal gene conversion may be important in maintaining sequence homogeneity within families of repeated eukaryotic genes. Nucleic Acids Res, 1981 Jan 10, 9(1), 31 - 45 Efficient and selective initiation by yeast RNA polymerase B in a dinucleotide-primed reaction; Lescure B et al.; Yeast RNA polymerase B catalyzes an efficient abortive initiation on double-stranded DNA templates using the appropriate combination of primer and substrate . The specificity of initiation was investigated using a recombinant plasmid (pJD14 DNA) containing the structural gene for yeast alcohol dehydrogenase I (ADHI) . The combination of the dinucleotide UpA and UTP was 10 fold more efficient with pJD14 DNA than with the vector pBR322 DNA to direct the synthesis of the trinucleotide UpApU . Under these conditions, stable enzyme-DNA complexes were formed and could be retained on nitrocellulose filters . Using the UpA-primed system and a short pulse of RNA synthesis, transcription complexes were located on the yeast part of pJD14 DNA as evidenced by agarose gel electrophoresis . Southern hybridization of the pulsed RNA was restricted to a region, within the yeast DNA fragment, upstream to the initial region of the ADHI gene. Biochim Biophys Acta, 1981 Jan 8, 640(1), 352 - 8 Energy source for lithium efflux in yeast; Rodriguez-Navarro A et al.; The efflux of Li+ in yeast was found to depend on the protonmotive force . The ATP content of the cell regulated the efflux that was also sensitive to the decrease in the cell pH . We propose an electrogenic H+/Li+ antiport as the mechanism for the efflux of Li+. J Cancer Res Clin Oncol, 1981, 102(2), 127 - 39 Polycyclic aromatic hydrocarbons and possible metabolites: convertogenic activity in yeast and tumor initiating activity in mouse skin; Siebert D et al.; The diploid respiratory-deficient strain of yeast D4-RDII was used to assay PAH and urethane as well as some oxygenated derivatives of PAH and the (aliphatic) epoxide hydrolase inhibitor TCPO for convertogenic (mutagenic) activity . As a positive control, the convertogenic ultimate rat liver carcinogen NOAcAAF was used . PAH and urethane were found inactive as convertogens, TCPO was weakly active, whereas oxygenated electrophilic derivatives of PAH, such as K-region oxides, were found strong convertogens . For comparison, some convertogenic key compounds were assayed for their tumor-initiating activity in mouse skin in the standardized system using TPA as a promotor . PAH were stronger initiators than all oxygenated derivatives of PAH tested . TCPO alone exhibited very weak, if any, initiating activity . It was unable to modify initiation to any significant extent, if administered 5 min prior to administration of an initiator . In the absence of correlation between convertogenic and initiating activity the question of the chemical nature of "ultimate initiators" of mouse skin carcinogenesis awaits further investigation. Z Allg Mikrobiol, 1981, 21(9), 643 - 50 {Structure of the cell wall polysaccharide in the food protein yeast Candida spec . H . VI . Isolation and structure of the glucans}; Grimmecke HD et al.; Alkali-soluble glucan was obtained from Candida spec . H by extraction with dilute sodium hydroxide . This minor component is quite heterogeneous in molecular size, has a highly branched structure and contains 19% beta (1,3)-, 24% beta (1,6)- and only 2% beta (1,2)-linkages . Other minor polysaccharide components have been isolated by acetic acid extraction . They have been identified as glycogen and linear beta(1,6)-glucan, respectively . The major component is a 1,2-, 1,3,-, 1,6-glucan branched in 1,2,3-, 1,3,6- and 1,2,6-positions, containing chitin . So we can confirm that different yeasts may differ in glucan structures which vary considerably both in the degree of branching and molecular size. Int J Neurosci, 1981, 13(4), 219 - 27 Ontogenetic alterations of the cerebral cortex in rat caused by a diet containing a lipid fraction extracted from yeast (Candida lipolytica) grown on N-alkanes; Gozzo S et al.; Several ontogenetic aspects of the cerebral cortex were studied in rats whose mothers were fed on a diet, the lipid fraction of which was extracted from yeast (Candida lipolytica) grown on N-alkanes, during part of pregnancy and throughout lactation . Measures of the heights of some cortical regions and specific layers, the maturation of mean cell volumes and of the cell/gray coefficient, especially in layers I, IV and VI, show that the diet affects cerebral cortex ontogeny . These effects on brain ontogeny and on intrinsic and extrinsic neuron connections may explain the electrophysiological and behavioural alterations observed in previous studies. Z Allg Mikrobiol, 1981, 21(3), 211 - 8 {Structure of the cell wall polysaccharide in the food protein yeast Candida spec . H . IV . Structure of the alkali labile oligosaccharide in the mannan-protein-phosphate complex}; Grimmecke HD et al.; Mild alkaline degradation of the proteophosphomannan (PPM) under conditions that effect beta-elimination of the residues, O-glycosidically bound on serine and threonine, release mainly mannose, glucose, mannobioses and mannotrioses with the same structures as those obtained by acetolysis of the polysaccharide component of the PPM . Methylation analysis and paper chromatography demonstrated the structural heterogeneity of the tetra-, penta- and hexasaccharide fractions containing 1,2- and 1,6-linked and 1,2,6-branched manno-oligosaccharides . Methylation analysis and acetolysis, paper chromatography and analysis of the carbohydrate composition of the oligosaccharide fractions DP 7-12 and DP 15-19 demonstrated inner core region like structures containing 1,2- and 1,6-linked mannose, 1,2,6- and 1,3,6-branchpoints and N-acetyl-D-glucosamine. Z Allg Mikrobiol, 1981, 21(3), 201 - 10 {Structure of the cell wall polysaccharide in the food protein yeast Candida spec H . III . Characterization of different phosphate bonds in the mannan-protein-phosphate complex}; Grimmecke HD et al.; The 31P-NMR spectra of the proteophosphomannan (PPM) and also that of mildly hydrolyzed PPM demonstrated phosphomonoester (in both preparations), acid labile and acid stable phosphodiester linkage, and polyphosphate . Decreasing in size by pronase digestion, separation, purification and characterization of the high and low molecular phosphates by 31P-NMR spectroscopy and chemical analysis revealed the mannan protein is phosphorylated in the N-glycosidically linked carbohydrate parts and in the O-glycosidically linked oligosaccharides . Another phosphate serves as a bridge between the serine of the protein and mannose, mannobioses and mannotrioses and between the threonine and a lipophilic acylglycerid unit . The origin of the polyphosphates has been discussed. Z Allg Mikrobiol, 1981, 21(2), 95 - 107 {Cell wall polysaccharide structure in the food protein yeast, Candida spec . H . I . Structure of alkali-stable mannoproteins}; Grimmecke HD et al.; The isolated manno-protein contains about 80% mannose and 10% glucose . Methylation analysis established the highly branched nature of this polysaccharide and the presence of 1,2-, 1,3- and 1,6-linkages, as well as the linkages of the branch points . The research of the acetolysis fragments revealed that the molecule is composed on mannose and mannooligosaccharides with DP2 to DP12 . These oligosaccharides are terminated in the nonreducing end by alpha(1,3)-mannose . Glucose was only found in the monosaccharide fraction corresponding to the nonsubstituted backbone and in the alpha(1,3)-disaccharide fraction (reducing and nonreducing end) of the acetolysis . A heptasaccharide fraction corresponding to the N-glycosidical linkage region between polysaccharide and protein parts of the glycoprotein had been isolated . 1H-NMR spectroscopy and chemical characterization made it probable that the unit with the first side chain, mannopentaose, is linked by di-N,N' -acetylchitobiose or by 4-0-beta-D-glucosyl-N-acetyl-D-glucosamine to the asparagine residue of the protein. Z Allg Mikrobiol, 1981, 21(2), 109 - 16 {Cell wall polysaccharide structure in the food protein yeast, Candida spec . H . II . Characterization of phosphate binding to the mannan-protein-phosphate complex and identification of phosphodiester-bound mono- oligosaccharides}; Grimmecke HD et al.; The proteophosphomannan (PPM) obtained by extraction with citrate buffer, purification by the cetavlon method and gel filtration contains a high proportion of diesterified phosphate groups between the C-6 of a side chain mannose and another glycosidically bound carbohydrate . Mild acid hydrolysis cleaved the phosphate diester linkages to yield mono- and oligosaccharide fractions . Chemical analysis and 1H-NMR studies demonstrated the heterogeneity of the oligosaccharide fractions containing alpha(1,2)-, alpha(1,3)- and alpha(1,6)-linked manno-oligosaccharides . All repeated PPM preparations contained mono-, tri- and heptasaccharides as the phosphate bound main compounds . Galactose, arabinose, fucose, and rhamnose of the monosaccharide fraction and tetra-, penta- and hexasaccharides were not found in all preparations. Acta Microbiol Acad Sci Hung, 1981, 28(4), 339 - 45 Protoplast fusion in the yeast Candida utilis; Delgado JM et al.; Protoplasts obtained by snail enzyme treatment from two stable auxotrophic mutants of the yeast Candida utilis were induced to fuse by the use of polyethylene glycol . The hybrids formed from the auxotrophic parental strains were selected by complementation on stabilized minimal medium . Many of the hybrids were unstable and readily dissociated into their parental strains . Others, in which parental nuclei had fused, gave stable hybrid progeny . cytological and genetic evidence of these processes is presented. Mikrobiologiia, 1981 Jan-Feb, 50(1), 110 - 3 {Yeast capsule ultrastructure}; Gulevskaia SA et al.; The ultrastructure of capsules was studied in 12 yeast species producing extracellular polysaccharides and belonging to ascomycetous, basidiomycetous and asporogenous organisms using the method of ultrathin sections . On the whole, the structure of capsules was similar in these organisms: the capsules are formed by fibrils radially coming out of the outer surface of the cell wall . Unlike those of basidiomycetous yeasts, the capsules of ascomycetous organisms are characterized by a more ordered orientation of fibrils which run strictly parallel to one another . In the capsules of basidiomycetous organisms, fibrils usually interweave, often producing a fibrillar network; in certain species, fibrils stick together forming thick threads on the periphery of the capsule . Moreover, as a rule, these yeasts possess a distinct bright zone (halo) above the outer surface of the cell wall . These results as well as data available about the chemical composition and functions of the capsule indicate that there are radical differences the latter and the cell wall which make it possible to regard the capsule as an individual organelle of the yeast cell rather than as part of the cell wall. Enzyme, 1981, 26(1), 49 - 53 Improved modification of yeast uricase with polyethylene glycol, accompanied with nonimmunoreactivity towards anti-uricase serum and high enzymic activity; Nishimura H et al.; The highly purified uricase from Candida utilis was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2), which was synthesized from monomethoxypolyethylene glycol (MW 5,000) and cyanuric chloride . Modification of approximately 36 out of the total 98 amino groups in the uricase molecule led to the complete loss of the binding ability towards antiuricase serum from rabbit with the retention of high enzymic activity (45% of native uricase) . The modified uricale cleared more slowly from the plasma of mice compared with native uricase. J Immunopharmacol, 1981, 3(2), 171 - 92 Inhibition of yeast phagocytosis and cell spreading by glucocorticoids in cultures of resident murine peritoneal macrophages; Grasso RJ et al.; This study was initiated to determine whether the inhibition of phagocytosis and cell spreading in cortisol-treated cultures of resident murine peritoneal macrophages are glucocorticoid-directed responses . Phagocytosis of heat-killed Saccharomyces cerevisiae and cell spreading were measured in control and steroid-treated macrophage cultures over 6 days . When the cultures were exposed to testosterone, progesterone, or epicortisol, phagocytosis and cell spreading were similar to controls . In contrast, both macrophage functions were inhibited significantly in cultures treated with cortisol, methylprednisolone, dexamethasone, and triamcinolone acetonide . In addition, the rate of phagocytosis was retarded and phagocytic indices (i.e., yeast particle number/cell) were reduced in glucocorticoid-treated cultures . Dose-response studies with dexamethasone demonstrated that the ED50 for the inhibitory effect on phagocytosis was 20 nM . These results indicate that the inhibition of yeast phagocytosis and cell spreading in the steroid-treated cultures are specific glucocorticoid-directed responses. Mol Gen Genet, 1981, 184(3), 347 - 54 The mechanism of interallelic complementation at the INO1 locus in yeast: immunological analysis of mutants; Majumder AL et al.; The ino1 locus of yeast has been demonstrated to be the structural gene for the repressible enzyme, L-myo-inositol-1-phosphate synthase (Donahue and Henry 1981 a) . We have screened a large number of allelic representatives of the ino1 locus for the presence of protein which cross reacts with antibody produced in response to purified wild type inositol-1-phosphate synthase . Approximately 50% of all ino1 representatives screened by immunoprecipitation produce a protein of 62,000 molecular weight, identical in size to the wild type enzyme subunit . These mutants (termed crm+) were tested for expression of the 62,000 MW protein under conditions which are repressing for the wild type enzyme (greater than 25 microM exogenous inositol) . The protein produced by the crm+ mutants, like the active enzyme in wild type yeast, is repressed in the presence of high levels of exogenous inositol . In addition, we have reassessed the interallelic complementation pattern observed among mutants at the ino1 locus . The entire pattern of interallelic complementation is temperature sensitive. Mol Gen Genet, 1981, 184(1), 46 - 51 Elevated recombination and pairing structures during meiotic arrest in yeast of the nuclear division mutant cdc5; Simchen G et al.; A diploid strain of yeast, homozygous for the mutation cdc5-1, undergoes a normal meiosis at 25 degrees C . At the nonpermissive temperature of 34 degrees C, meiosis is arrested at the first meiotic division, after premeiotic DNA replication and recombination commitment have taken place . Haploidisation commitment does not occur at 34 degrees C . Electron microscopy reveals that synaptons (synaptonemal complexes) are formed and the stage of arrest is characterised by a prevalence of "modified synaptons", which consist of paired lateral elements lacking the central elements . Prolonged incubation at this stage of arrest results in unusually high recombination levels, perhaps related to the synaptonal structures observed . Temperature shift-up experiments (transfers of cell from 25 degrees C to 34 degrees C at various times during meiosis) reveal that the CDC5 function is required for both the first and the second divisions of meiosis. Mol Gen Genet, 1981, 183(1), 152 - 7 Radioprotecting action of chemical compounds on gamma-irradiated yeast cells of various genotypes; Petin VG et al.; The radioprotective efficiency of cysteamine and cysteine has been studied on haploid and diploid, Saccharomyces cerevisiae, wild-type and various X-ray repair deficient rad mutants . The correlation between the radioprotecting action of cysteamine and cell repair capacity was demonstrated for diploid yeasts; such a correlation was not expressed for wild-type and rad mutant haploid yeast cells . It was concluded that the radioprotective action may involve cellular recovery processes, which may be mediated by a recombination-like mechanism, for which the diploid state is required . Liquid holding recovery was shown not to participate in radioprotection, judged by the absence of the influence of cysteine on the delay of the first postradiation budding as well as by the additive action of cysteine and liquid holding recovery. Differentiation, 1981, 19(1), 68 - 70 Modulation of a cell surface glycoprotein in yeast: acid phosphatase; Schweingruber ME et al.; Upon inorganic phosphate starvation the cell wall glycoprotein acid phosphatase of yeast Saccharomyces cerevisiae is derepressed . Purified acid phosphatase isolated from early log phase cells differs in reactivity and stability from acid phosphatase from late log phase cells indicating that the two enzymes are structurally different . This demonstrates that the yeast cell has not only the capacity to regulate the amount of acid phosphatase but also the ability to vary (modulate) the structure of the secreted enzyme . Modulation of acid phosphatase may be a mechanism which is involved in morphogenetic and behavioral differentiation of the yeast cell. Carcinogenesis, 1981, 2(11), 1201 - 5 The genetic activity of dinitropyrenes in yeast: unusual dose response curves for induced mitotic gene conversion; Wilcox P et al.; 1,6-Dinitropyrene (1,6DNP) and 1,8-dinitropyrene (1,8DNP) were tested for their ability to induce mitotic gene conversion at the trp 5 and his 4 loci in the yeast Saccharomyces cerevisiae JD1 . Both compounds were shown to be potent inducers of gene conversion in yeast, with 1,6DNP being somewhat more active than 1,8DNP . Unusual dose-response curves were obtained in that toxicity and genetic activity decreased at the higher concentrations examined . This reduction in genetic activity may reflect a decrease in the ability of yeast cells to convert the dinitropyrenes to their mutagenic forms when the concentration of the compounds exceeds a certain level. CRC Crit Rev Biochem, 1981, 11(3), 209 - 54 Yeast enolase: mechanism of activation by metal ions; Brewer JM; Yeast enolase as prepared by current procedures is inherently chemically homogeneous, though deamidation and partial denaturation can produce electrophoretically distinct forms . A true isozyme of the enzyme exists but does not survive the purification procedure . The chemical sequence for both has been established . The enzyme behaves in solution like a compact, nearly spherical molecule of moderate hydration . Strong intramolecular forces maintain the structure of the individual subunits . The enzyme as isolated is dimeric . If dissociated in the presence of magnesium ions and substrate, then the subunits are active, but if the dissociation occurs in the absence of metal ions, they are inactive until they have reassociated and undergone a first order "annealing" process . Magnesium (II) enhances association . The interaction between the subunits is hydrophobic in character . The enzyme can bind up to 2 mol of most metal ions in "conformational" sites which then allows up to 2 mol of substrate or some substrate analogue to bind . This is not sufficient for catalysis, but conformational metal ions do more than just allow substrate binding . A change in the environment of the metal ions occurs on substrate or substrate analogue binding . There is an absolute correlation between the occurrence of a structural change undergone by the 3-amino analogue of phosphoenolpyruvate and whether the metal ions produce any level of enzymatic activity . For catalysis, two more moles of metal ions, called "catalytic", must bind . There is evidence that the enzymatic reaction involves a carbanion mechanism . It is likely that two more moles of metal ion can bind which inhibit the reaction . The requirement for 2 mol of metal ion per subunit which contribute in different ways to catalysis is exhibited by a number of other enzymes. Neoplasma, 1981, 28(3), 281 - 9 The inhibitory effect of vinylfurans on the glycolysis in tumor and yeast cells; Drobnica L et al.; Most of the eighteen vinylfurane derivatives studied fully inhibit the glycolysis of both Ehrlich ascites carcinoma (EAC) cells and respiratory deficient yeast Saccharomyces cerevisiae at concentrations lower than 0.5 mmol/l . The inhibition of glycolysis is a consequence of some thiol enzymes inactivation . This concerns namely hexokinase (EC 2.7.1.1), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and especially 6-phosphofructokinase (EC 2.7.1.11) . Interference of vinylfurans with energy metabolism resulted in the depression of biosynthetic processes followed (14C-precursors incorporation into proteins and nucleic acids) and finally in the loss of EAC cell transplantability. Mol Gen Genet, 1981, 181(4), 420 - 3 Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA . IV . Characterization of the omega neutral allele; Bos JL; The omega locus controls the polarity of recombination and transmission of genetic markers in the 21S ribosomal RNA region in yeast mtDNA . Polarity is observed in crosses between omega+ and omega- strains . These two strains differ by the presence of an intervening sequence in the 21S ribosomal RNA gene of omega+ strains . Mutations of the omega- allele, omega neutral (omegan), can eliminate the polarity effect . We have made DNA:RNA hybrids containing ribosomal RNA from an omegan strain and mtDNA from Saccharomyces carlsbergensis (identical to omega- in the nucleotide sequence of the omega region) . These hybrids contain no mismatch at the omega region detectable by digestion with S1 nuclease . We conclude that omegan differs from omega- only in a point mutation or analogous small alteration and that the omegan mutation can result either in a Cr phenotype (omeganCr) or in the phenotypic suppression of pre-existing Cr mutations (omegenCs) . All results can be explained by a model which postulates interaction in the ribosome between the Cr and omegan regions of the ribosomal RNA and interference of the omegan mutation with splicing of the precursor ribosomal RNA in omega+ strains . The mechanism of omega-directed polarity is discussed. Folia Microbiol (Praha), 1981, 26(2), 107 - 11 A simple procedure to obtain yeast hexokinase free of glucosephosphate isomerase and mannosephosphate isomerase; Cabrera V et al.; A hexokinase preparation was obtained from a Saccharomyces cerevisiae mutant strain deficient in glucosephosphate isomerase (GPI) and mannosephosphate isomerase (MPI) by precipitation with ammonium sulfate . The supernatant fraction corresponding to 40-60 % saturation showed the lowest content in GPI and MPI activity . The fraction was used without further purification in the determination of glucose, either free or in a mixture with fructose and mannose . The results were similar to those obtained with pure commercial hexokinase. Folia Microbiol (Praha), 1981, 26(2), 103 - 6 Use of permeabilized yeast cells as a system of enzyme immobilization . Its use for the preparation of mannose 6-phosphate; Pascual C et al.; It was shown that hexokinase contained inside permeabilized yeast cells can be used successfully in the phosphorylation of mannose for the production of mannose 6-phosphate . We propose that enzymes entrapped within the semipermeable cell membrane be considered as an extension of the enzyme immobilization concept, since it shares many of its advantages and several of its properties with some unique characteristics. Genetika, 1981, 17(5), 814 - 21 {Combined action of UV light and alpha particles on yeast cells of different genotypes}; Komarov VP et al.; Combined action of ultraviolet (UV) light and alpha-particles on yeast cells of different genotypes has been studied . Under combined action, after small doses of UV-light the oscillated changes of cell survival were registered for wild-type cells independent of the ploidy and the sequence of application of radiations . Additive effect of high doses of UV-light and ionizing radiation was expressed for strains incapable of the recovery of damages induced by ionizing radiation . For yeast cells possessing such a capability, the synergistic interaction of damages inflicted by high doses of UV-light and alpha-particles was noted . Possible reasons of the observed cell responses are discussed. Clin Exp Immunol, 1981 Jan, 43(1), 208 - 14 Polymorphonuclear neutrophil iodination response as an estimate of defective yeast opsonization; Roberton DM et al.; Polymorphonuclear neutrophil iodide uptake can be used as a measure of yeast opsonization and optimal assay conditions are described for the identification of sera defective in this function . The use of standard normal and defective sera permits correction of inter-assay variations resulting from the use of different cell donors . The iodination assay correlated well (r = 0.74, P less than 0.001) with a direct assay of yeast opsonization when both were used to measure this function in a panel of 72 sera . Differences in results between the two assay systems for some sera may be explained by a requirement for two different surface-associated opsonic molecules in the initiation of neutrophil metabolic activity. Proc Natl Acad Sci U S A, 1981 Jan, 78(1), 494 - 8 Gene conversion at the var1 locus on yeast mitochondrial DNA; Strausberg RL et al.; Alleles of the var1 locus on yeast mtDNA determine the apparent size of the mitochondrial translation product, var1 polypeptide . We have analyzed most of the different var1 alleles in our collection, which number at least 15, and have developed procedures and a genetic rationale for determining their origin and predicting their behavior in crosses . The var1 alleles are characterized by two genetically defined segments, designated a and b, which can move from one var1 allele to another by asymmetric gene conversion . We show that the a segment behaves as an entity in recombination; it is either present in or absent from different var1 alleles . The b segment usually, but not always, recombines as an entity; in some cases, only portions of the b segment recombine by gene conversion . Thus, the total number of electrophoretically resolvable var1 species we observe is explained by the assortment of a, b, and partial b segments . Each segment recombines at a characteristic frequency; however, one example is presented which shows that the recipient can modulate the frequency of gene conversion . Finally, we show that, like the 21S rDNA region (omega), there is polarity of gene conversion within var1. Proc Natl Acad Sci U S A, 1981 Jan, 78(1), 415 - 9 Yeast tRNA precursor mutated at a splice junction is correctly processed in vivo; Colby D et al.; Yeast mutants with decreased expression of a tRNATyr gene were obtained by selection for functional inactivation of the tyrosine-inserting ochre suppressor SUP4 and subsequent screening for production of the tRNA gene product in vivo . One mutant with reduced suppressor activity was characterized by a decreased quantity of the suppressor-specific tRNA; a precursor to this tRNA, matured at both 5' and 3' termini but still containing a 14-nucleotide intervening sequence, was present in an amount greater than 7-fold that in the parent . By RNA sequence analysis of the accumulated precursor, we have identified the mutation as an A leads to G transition at the 5' splice junction . Similar analysis of the mature tRNA produced in this mutant demonstrated that the intervening sequence was accurately excised . We conclude that the specific sequence of nucleotides at this splice junction affects the efficiency but not the fidelity of processing. Proc Natl Acad Sci U S A, 1981 Jan, 78(1), 238 - 42 Cloning of yeast gene for trichodermin resistance and ribosomal protein L3; Fried HM et al.; Yeast cells sensitive to the eukaryotic protein synthesis inhibitor trichodermin have been transformed with autonomously replicating recombinant plasmids carrying DNA fragments of the genome of a trichodermin-resistant yeast strain . After selection for trichodermin-resistant cells, several transformants yielded a plasmid containing a 13.5-kilobase (kb) DNA fragment that encodes the trichodermin resistance gene, tcm1, and the gene for ribosomal protein L3, the largest of the yeast ribosomal proteins . Cells carrying this plasmid are resistant to trichodermin and to the related drug verrucarin A as well as to the unrelated drug anisomycin . This pattern of resistance is similar to that exhibited by strains carrying a chromosomal copy of tcm1 . Moreover, polyribosomes prepared from transformed cells are resistant to trichodermin when tested in an in vitro protein synthesis assay . Subcloning of the 13.5-kb DNA fragment revealed that the gene for tcm1 and the gene for protein L3 are contained within a 3.2-kb segment . These results suggest that the gene for trichodermin resistance in yeast specifies ribosomal protein L3. Mol Gen Genet, 1981, 181(3), 346 - 51 Isolation of yeast mutants sensitive to the bifunctional alkylating agent nitrogen mustard; Ruhland A et al.; Mutants of Saccharomyces cerevisiae with enhanced sensitivity to the DNA cross-linking agent nitrogen mustard (HN2) have been isolated and partially characterized with respect to their phenotypic and genetic properties . The screening technique, based on HN2-sensitivity as sole criterion, yields approxiamtely 1 sensitive isolate in 200 clones when applied to an intensively mutagenized population of a resistant parent strain . Mutants characterized so far are all due to recessive nuclear genes and represent at least seven complementation groups . They exhibit different degrees as well as different patterns of sensitivity towards monofunctional and bifunctional alkylating agents, and ultraviolet light. Chromosoma, 1981, 82(3), 333 - 40 Nuclear morphology of yeast under thymidylate starvation; Moens PB et al.; During early meiotic development the yeast Saccharomyces cerevisiae has a characteristic nuclear dense body (NDB) . It is shown that the NDB can also be induced in vegetatively growing cells through the inhibition of thymidylate synthetase which causes depletion of the dTMP pool and arrests DNA synthesis . The observations on NDBs and recombination levels suggest that thymidylate-stressed cells may activate parts of the meiotic pathway and, conversely, cells on sporulation medium may sense, among other things, reduced thymidylate levels and respond to the several stimuli by entering the meiotic pathway. Mol Gen Genet, 1981, 181(1), 147 - 9 Correspondent reaction of mitotic recombination in yeast and sister chromatid exchange in human lymphocytes; Fahrig R; In the lower eukaryote Saccharomyces cerevisiae, 4,5,6-trichloro-2-(dichlorophenoxy)phenol and acridine orange cause different specific genetic alterations, either gene mutations or recombinations . These specific effects were used to characterize the mechanism of sister chromatid exchange (SCE) formation in human lymphocytes . Assuming that genetically active substances have comparable effects in lower and higher eukaryotes, the observations provide indirect evidence for a connection between induced mitotic recombination in yeast and SCEs in human lymphocytes and suggest that SCEs may be the consequence of a repair process. Biochimie, 1981 Jan, 63(1), 67 - 9 {Isolation of yeast protoplasts using various preparations of the hepato-pancreatic juice of Helix pomatia}; Diatewa M et al.; Conversion of large amounts of Saccharomyces cerevisiae cells to protoplasts is studied using various preparations extracted from Helix pomatia hepato-pancreatic juice . The most favourable yield in two hours incubations (88 per cent) is obtained with 20 ml cytohelicase, a chitinase and glucanase enriched extract, per 400 g of yeast cells, harvested at the end of the logarithmic growth phase and preincubated in presence of 2-mercaptoethanol. Z Naturforsch {C}, 1981 Jan-Feb, 36(1-2), 142 - 8 Initiation of protein synthesis in yeast: binding of Met-tRNAi; Kreutzfeldt C; Conditions for the binding of Met-tRNAi to 40 s ribosomal subunits and to proteins isolated out of the yeast ribosomal KCl wash were investigated . Sucrose density gradient experiments revealed that binding of Met-tRNAi to 40 s ribosomal subunits was catalyzed in a AUG and GTP dependent reaction . Binding of Met-tRNAi to proteins of the ribosomal KCl wash as assayed by the Millipore filter technique was found to be independent of AUG, GTP and 40 s ribosomal subunits . Additions of GTP yielded only slight stimulation, whereas Mg2+ caused dissociation of complexes . It was concluded that these reactions were most likely catalyzed by initiation factor eIF-2 although stimulation by GTP did not occur. Biokhimiia, 1981 Jan, 46(1), 140 - 7 {Cause of the activity loss of the alternative pathway of electron transport in cyanide-resistant mitochondria of the yeast Candida lipolytica}; Akimenko VK et al.; The cause of the activity loss of alternative pathway of electron transport in mitochondria of the yeast Candida lipolytica has been investigated . Incubation of cyanide-resistant mitochondria at 25 degrees was shown to cause the loss by mitochondria of their ability to oxidize substrates in the presence of 1 mM cyanide . This suggests that in the course of incubation the alternative pathway loses its activity . Repeated washing of mitochondria with a solution containing 2,5 mM EDTA inhibits, while Ca2+, Mn2+, Cu2+ and Zn2+ (but not Sr2+) enhance the process of the activity loss of the alternative pathway . The loss of the cyanide-resistant respiration is also observed during incubation of mitochondria in the presence of phospholipases A, C and D or lysolecithin . In all cases studied the reactivation of the cyanide-resistant respiration of mitochondria is attained by addition of azolectin . The loss of cyanide-resistant respiration is accompanied by the activity reduction of the main respiratory chain, which is restored by addition of cytochrome c and Mg2+ . These data indicate that the activity loss of the alternative pathway is not related to inactivation of any components in the alternative pathway itself or in the main respiratory chain . The most probable cause of the activity loss in the destruction of reducing equivalents in the alternative pathway of a donor as a result of a break of the structural entity of the internal membrane of mitochondria due to the detersive action of the phospholipid lysoforms produced either by endogenic or exogenic phospholipases. Acta Histochem Suppl, 1981, 23, 211 - 7 Polyethylene glycol induced membrane fusion in yeast protoplasts; Svoboda A; Freeze-etching and ultrathin section techniques were used to demonstrate ultrastructural change in plasma membranes during fusion of Saccharomyces cerevisiae protoplasts . Incubation of protoplasts in 30% (w/v) polyethylene glycol fed to strong reduction in the number of particles in areas of close membrane contact and disappearance of invaginations . On sections, neighbouring plasma membranes merged to pentalaminar or trilaminar structures . After incubation in regeneration medium, these membrane changes progressed to form rounded bulging areas with crater-like openings which are thought to be channels through which coalescence of partner's cytoplasms may occur. Acta Histochem Suppl, 1981, 23, 151 - 5 Plasma membrane particles in yeast protoplasts; Necas O et al.; Investigations of plasma membrane particles in Saccharomyces cerevisiae protoplasts are shortly reviewed . Particle arrangement into a hexagonal patterns is considered to reflect some minute changes in the biomembrane organization rather than functional differentiation . Plasma membrane invaginations are proved to be rigid structures capable of migrating in the fluid domain of the membrane . Density of the particles per unit area is related to the degree of membrane stretching which suggest that upon membrane extension new protein molecules are shifted towards the hydrophobic layer of the membrane. Acta Biochim Pol, 1981, 28(1), 51 - 9 On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo; Kudlicki W et al.; Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with {32P}orthophosphate . Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors . Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B . Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec . Biol . Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins . The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases . The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit. Nature, 1981 Jan 1, 289(5793), 33 - 7 Isolation of a gene from Drosophila by complementation in yeast; Henikoff S et al.; Transformation of mutant yeast cells by cloned genomic DNA from a higher eukaryote has made it possible to isolate a Drosophila DNA sequence that complements a yeast adenine-8-mutation . A 0.8-kilobase poly(A)-containing RNA is transcribed from the cloned Drosophila segment in transformed yeast cells and can account for functional expression of the gene. J Histochem Cytochem, 1981 Jan, 29(1), 45 - 8 Fluorescence of yeast vitally stained with ethidium bromide and propidium iodide; Corliss DA et al.; Both ethidium bromide and propidium iodide stain growing yeast . As visualized in the fluorescence microscope, ethidium stains the nucleus and cytoplasm in wild type yeast and in those grown in 10% dextrose, with brightly fluorescent cytoplasmic granules being present in both . Under the latter conditions, the mitochondria are repressed but not absent . In rho 0 cells, in which the mitochondrial DNA is absent, ethidium appears to bind to the cell wall or membrane preferentially with no cytoplasmic granules being visible . In all cell types, propidium appears to bind the cell wall or membrane with no cytoplasmic granules being visible in any cell . The staining patterns thus suggest greater differences in the binding of these two types to mitochondrial DNA in situ than is suggested by their in vitro behavior . These differences in binding could explain their different mutagenic capacities.. Folia Microbiol (Praha), 1981, 26(1), 65 - 7 Use of ruthenium red for visualization of the regenerating cell wall in yeast protoplasts; Havelkova M; Ruthenium red was used to stain the surface of fresh and regenerating protoplasts in Saccharomyces cerevisiae . The presence of a dense layer, 10--40 nm wide, was demonstrated on the surface of most of the fresh protoplasts . This layer could be removed neither by repeated washings of protoplasts nor ;by their centrifugation . Ruthenium red proved to be a very useful dye for regenerating walls since it stained both the amorphous and the fibrillar components, but to a different degree, thus permitting their easy differentiation. J Supramol Struct Cell Biochem, 1981, 15(2), 119 - 28 Effect of exogenous fatty acids on growth, membrane fluidity, and phospholipid fatty acid composition in yeast; Esfahani M et al.; The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-delta 11-eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity . There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 15:0, and 16:0 . We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism. Nature, 1980 Dec 25, 288(5792), 669 - 74 Crystal structure of yeast tRNAAsp; Moras D et al.; Two independent, three-dimensional structures of yeast tRNAAsp, mainly differing by the conformation of the D loop, have been obtained from a multiple isomorphous replacement (MIR) X-ray analysis at 3.5-A resolution . The folding of the ribose-phosphate backbone is similar to that found for tRNAPhe; major differences concern the relative positioning of the acceptor and anticodon stems, and the conformation of the loops in the two molecules . Crystal packing involves self-complementary GUC anticodon interactions. J Biol Chem, 1980 Dec 25, 255(24), 12087 - 93 Purification and properties of proteinase A from yeast; Meussdoerffer F et al.; Proteinase A (EC 3.4.23.6) was purified from commercial bakers' yeast in five steps, including hydrophobic chromatography and affinity chromatography . After the last step the enzyme appeared homogeneous on polyacrylamide gel electrophoresis and in the analytical ultracentrifuge . A molecular weight of 41,500 was determined for proteinase A . The amino acid composition includes 43% polar residues and 12% aromatic amino acids . Proteinase A is a glycoprotein containing 7.5% mannose and 1% of glucosamine and galactosamine . The temperature and the pH stability of the enzyme have been determined . At pH 6, the proteinase exhibits a remarkable stability even in 6 M urea . Proteinase A splits hemoglobin with an optimum at pH 3.0 and casein and azocasein with an optimum at pH 6.0 . The enzyme is inhibited by pepstatin, diazoacetyl-DL-norleucine methyl ester and by 1,2-epoxy-3-(4-nitro-phenoxy) propane. J Biol Chem, 1980 Dec 25, 255(24), 11927 - 41 Assembly of the mitochondrial membrane system . Structure and nucleotide sequence of the gene coding for subunit 1 of yeast cytochrme oxidase; Bonitz SG et al.; the oxi3 locus of yeast mitochondrial DNA has been sequenced in Saccharomyces cerevisiae D273-10B . The sequence was obtained from the mitochondrial genomes of a series of cytoplasmic "petite" mutants selected for the retention of genetic markers in the oxi3 locus . The oxi3 locus has been ascertained to code for Subunit 1 of cytochrome oxidase . The Subunit 1 gene is 9,979 nucleotides long, consisting of seven to eight exons that account for only 16% of the gene sequence . The coding sequences have been identified on the basis of protein sequence homology with Subunit 1 of human cytochrome oxidase . The yeast Subunit 1 is 510 amino acid residues long and has a molecular weight of 56,000 . In addition to the exon sequences, the Subunit I gene contains six to seven introns . The first four introns have long reading frames that are continuous with the exon coding sequences . These reading frames are potentially capable of coding for basic proteins with molecular weights ranging from 30,000 to 80,000 . The first two introns of the gene have a sequence homology of 50%, while the reading frame of the fourth intron is 70% homologous with an intron of the apocytochrome b gene . At least five stable transcripts have been found by Northern blot hybridizations with single-stranded DNA probes containing either exon or intron sequences . A 1.9-kolobase transcript hybridizes only with probes from the exon regions of the gene . This RNA species has been tentatively identified as the fully processed messenger of Subunit 1 . Other transcripts are detected with intron probes . Three transcripts with sizes of 2.5, 2.4, and 0.85 kilobases appear to be stable excision products from the first, second, and fifth introns. J Biol Chem, 1980 Dec 25, 255(24), 11922 - 6 Assembly of the mitochondrial membrane system . Physical map of the Oxi3 locus of yeast mitochondrial DNA; Bonitz SG et al.; The oxi3 locus of yeast mitochondrial DNA is currently thought to code for Subunit 1 of cytochrome oxidase (Tzagoloff, A., Macino, G., and Sebald, W . (1979) Annu . Rev . Biochem . 48, 419-441) . The respiratory competent strain of Saccharomyces cerevisiae D273-10B/A48 was used to obtain cytoplasmic "petite" clones enriched for genetic markers in the oci3 locus . The most complex clone studied (DS6) was ascertained to have a mitochondrial genome with a tandemly repeated segment of mtDNA 16.5 kilobases in length . The oxi3 locus was dissected by mutagenesis of DS6 with ethidium bromide and selection of new clones having less complex genotypes . Six derivative clones with genome sizes ranging from 2.3 to 6.1 kilobases have been extensively analyzed . Most of the restriction sites present in the segments of mtDNA retained by the clones have been mapped, thereby providing a detailed restriction map of the oxi3 gene . Based on the physical locations of the most distal oxi3 mutations, the gene spans approximately 10,000 nucleotides and occupies the region of wild type mtDNA from 44 to 58 map units. Biochim Biophys Acta, 1980 Dec 15, 633(3), 376 - 85 Evidence for a highly specific protein kinase phosphorylating two strongly acidic proteins of yeast 60 S ribosomal subunit; Kudlicki W et al.; Two distinct, cyclic AMP-independent protein kinase (ATP : protein photransferase, EC 2.7.1.37) from yeast have been isolated and highly purified . The first of the enzymes, protein kinase 1 A, phosphorylates casein and phosvitin, and its cellular protein substrate is unknown . The second enzyme, protein kinase 1 B, phosphorylates two strongly acidic proteins, L44 and L45, of the 60 S ribosomal subunit. C R Seances Acad Sci D, 1980 Dec 15, 291(13), 1037 - 40 {Importance of the parameters of temperature and natural light on stability of 2 types of L(+) lactate:cytochrome oxidoreductases (cytochromes b2) extracted from Hansenula anomala yeast}; Prats M; In this communication, we present the effect of two parameters, temperature and daylight-at high ionic strength-on the stability of H-flavocytochrome b2 solutions prepared using {H-flavocytochrome b2 (S) or not {H-flavocytochrome b2 (n) n-butanol during extraction . These two enzyme preparations presented the same behaviour towards these two parameters: in the dark, temperature was critical; in daylight, an opposite effect was observed for this parameter: the stability of samples was smaller at 0 degrees C . However, the evolution of life of the classical kinetic parameters (Km and Vm) of the enzymatic reaction is presented for three samples of H-flavocytochrome b2 (n) or (s) exposed to daylight at 20 +/- 0,1 degrees C. Biochim Biophys Acta, 1980 Dec 11, 610(2), 221 - 8 Rapid purification of yeast mitochondrial DNA in high yield; Hudspeth ME et al.; A procedure is presented for the rapid isolation of mitochondrial DNA (mtDNA) in high yield from Saccharomyces cerevisiae . Yeast cells, which may be grown to late stationary phase, are broken by a combination of enzymatic and mechanical means; mtDNA is then isolated from a crude mitochondrial lysate by a single cycle of bisbenzimide-CsCl buoyant density centrifugation . mtDNA so isolated is at least 99.5% pure, and has a mean duplex molecular weight of 24.5 . 10(6) . In addition to mtDNA and bulk nuclear DNA, several other yeast nucleic acid species, identified as ribosomal DNA and a mixture of duplex RNAs, form discrete bands in these gradients. C R Seances Acad Sci D, 1980 Dec 8, 291(12), 941 - 4 {Parameters influencing inactivation-dissociation/reactivation-association phenomena induced by variations of ionic strength of L (+)lactate: cytochrome c oxidoreductase (cytochrome b2) extract of the yeast Hansenula anomala}; Prats M; H-flavocytochrome b2, a tetramer enzyme, is inactivated, at low ionic strength and can be reactivated, increasing the ionic strength of the medium . The inactivation-reactivation process was structurally manifested by a dissociation-association phenomenon between subunits . It was clearly shown that the inactivation-dissociation process appeared independent of enzyme concentration whereas the reactivation-association phenomenon was enzyme concentration dependent . However, proteins protect H-flavocytochrome b2 from inactivation-dissociation, only when electrostatic interactions are possible between the two proteins: Horse heart cytochrome c was a good protector whereas serum albumin had no protector effect. Biochim Biophys Acta, 1980 Dec 5, 620(3), 429 - 39 Effect of unsaturated fatty acids on sterol biosynthesis in yeast; Boll M et al.; Lipid-depleted yeast, grown anaerobically, contains only very low amounts of sterols . The hydroxymethylglutaryl-CoA reductase activity, the regulatory enzyme of sterol synthesis in yeast, is also low . Aeration of such cells in a buffer containing a carbon source induces hydroxymethylglutaryl-CoA reductase activity and increases sterol synthesis . The velocity of the increase depends on the carbon source present during the aeration period . Glucose and sugars that are easily converted to glucose were found to be most effective . A supplement of unsaturated fatty acids during anaerobic growth causes a several-fold greater velocity of the enzyme induction and of sterol biosynthesis . Linolenic acid (30 microM) accelerated sterol biosynthesis about 7-fold . Activities of galactokinase and galactose-1-phosphate uridyltransferase, which are involved in the conversion of galactose to glucose, increased several-fold in the supplemented cells within 6 h of aeration, concomitantly with stimulation of sterol synthesis from galactose . It is suggested that the stimulation of enzyme induction and sterol biosynthesis in fatty acid supplemented cells is due to a completion of the protein-synthesizing apparatus during cell growth . A markedly enhanced capacity of these cells to incorporate leucine into acid-precipitable protein supports this assumption. Can J Microbiol, 1980 Dec, 26(12), 1428 - 37 Lipid composition and the transition from yeast-like to chlamydospore cells of Pullularia pullulans; Regulez P et al.; The lipid composition of the fungus Pullularia pullulans has been investigated at different stages of the morphogenetic transition from yeast-like, through the so-called "large cells," to chlamydospores . The first 3 days of culture correspond to the period of exponential growth . There is a rapid consumption of glucose and ammonium, until the latter becomes exhausted, and a concomitant decrease of pH down to values of about 2 . A decrease in fatty acid unsaturation takes place at this stage, together with an increase in the proportion of long-chain aldehydes . The period between days 3 and 6 is dominated by the large cells . They accumulate large amounts of triacylglycerols; phospholipids and free sterols are also synthesized in this period, suggesting de novo synthesis of membranes . Chlamydospores can be seen from the 6th day on . Simultaneously, a decrease in free sterols and phospholipids takes place, while saturated triacyglycerol production goes on . The lipid composition of chlamydospores suggests that these are resistance forms, induced by the hostile environmental conditions of the medium after the end of the exponential growth. J Cell Sci, 1980 Dec, 46, 289 - 97 Observations on the mitochondrial reticulum in the yeast Candida utilis as revealed by freeze-fracture electron microscopy; Keyhani E; Freeze-fracture of Candida utilis yeast cells grown to early logarithmic phase (5 h) and stationary phase (24 h) revealed a branched mitochondrial reticulum both in longitudinal and transverse cross-fracture studies . In contrast, the longitudinal and transverse sections of permanganate-fixed cells showed that the small ovoid or spherical mitochondria were located at the periphery of the cell, without formation of a reticulum . Data indicate that the separate mitochondrial profiles seen in thin sections of permanganate-fixed yeast cells are not separate round or ovoid mitochondria, but rather are cross-sections of the mitochondrial reticulum. Biochim Biophys Acta, 1980 Dec 1, 633(2), 176 - 80 Conversion of 5'-methylthioadenosine into S-adenosylmethionine by yeast cells; Shapiro SK et al.; 5'-Methylthio{U-14C}adenosine was used as a culture supplement for Candida utilis . The resulting S-adenosylmethionine was hydrolyzed into its structural components . Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine . Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound . The (U-14C)-labeled adenine of 5'-methylthio{U-14C}adenosine did not contribute to the labeling of the amino acid components of the sulfonium compound. Cell, 1980 Dec, 22(3), 799 - 805 Histone H2B genes of yeast encode two different proteins; Wallis JW et al.; The two genetically unlinked histone H2B genes isolated from Saccharomyces cerevisiae have been sequenced . The genes encode H2B proteins that are 130 amino acids in length and that differ by 4 amino acids . The changes betwen them are Ala leads to Ser, Lys leads to Ala, Thr leads to Val and Ala leads to Val at amino acid positions 2, 3, 27 and 35, respectively . A comparison of yeast H2B histones with those of higher eucaryotes demonstrates a high degree of homology clustered mainly at the carboxyl terminus . There is extensive base substitution between the two H2B genes in nucleotides that do not affect the amino acid sequence . DNA prelude sequences show 64% divergence . The coding regions differ at 49 of the 390 bases (12.6% divergence) . 41 of these changes are in silent positions . By using the number of amino acid differences in the proteins we estimate that the two H2B genes are the result of an ancient duplication event. J Inorg Biochem, 1980 Dec, 13(4), 353 - 66 EPR of Cu+2 binding to apo-yeast enolase; Dickinson LC et al.; We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion . An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA) . the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK . The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands . In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound . The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites . In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding . Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2 . A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data. Biochim Biophys Acta, 1980 Dec 1, 633(2), 211 - 27 Identification of porphyrin present in apo-cytochrome C oxidase of copper-deficient yeast cells; Keyhani E et al.; Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells . Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells . The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a . When solubilized mitochondria from {3H}leucine and delta-amino{14C}levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained . Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed {3H}leucine associated with six bands and delta-amino{14C}levulinic acid resolved in a single band . HCl fractionation of copper-deficient mitochondria labeled with delta amino{14C}levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a . Thin-layer chromatography of the radioactivity found in 20% HCl showed an RF value identical to that of purified porphyrin a . When delta-amino{3H}levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells was converted into heme a, and this conversion was prevented by cycloheximidine . These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur . This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficiency. Biochemistry, 1980 Nov 11, 19(23), 5168 - 75 Yeast 3-phosphoglycerate kinase: sulfate and substrate binding, their effect on the conformational state of the enzyme; Roustan C et al.; Anions and particularly sulfate are known to interact with 3-phosphoglycerate kinase and to induce an increase of its catalytic efficiency . The present work affords information on the location of the anionic site and on the conformational change produced by the sulfate binding . We have established that sulfate is able, first, to modify the environment of some critical amino acids (cysteine and arginines) located in the N-terminal half of the protein, second, to induce perturbation of aromatic residues as judged by spectrophotometry, and, third, to slightly decrease the magnitude of the Cotton effect at 233 nm . All these modifications are produced by sulfate concentrations required for the activation of the enzyme . The most striking result consists in a large change in the hydrodynamic properties of the protein upon sulfate interaction as determined by analytical ultracentrifugation studies . Thus, sulfate modifies the shape of the molecular, causing it to become more compact . Furthermore, a study of the binary and ternary complexes between yeast 3-phosphoglycerate kinase and its substrates suggests that such a change of the shape of the molecular only occurs in sulfate-enzyme with or without substrates and in ATP (with or without Mg2+)-3-phosphoglycerate-enzyme complexes. Nucleic Acids Res, 1980 Nov 11, 8(21), 5007 - 16 Characterization of tRNA genes in tRNA region II of yeast mitochondrial DNA; Newman D et al.; We have isolated individual mitochondrial tRNAs from a petite mutant OI-P2-1 known to contain a limited subset of mitochondrial tRNA genes and have mapped these genes on the wild type genome of the yeast strains MH41-7B and D273-10B . To obtain DNA for fine structure mapping and DNA sequence analysis of these genes, we screened a yeast mitochondrial DNA-pBR322 recombinant bank with the isolated tRNAs . We report here the fine structure mapping of recombinant clones containing the tryptophan, formyl methionine and proline tRNA genes as well as the DNA sequence of the proline tRNA gene . The combination of restriction mapping and DNA sequence analysis has enabled us to locate these genes precisely on the wild type genome and to determine their direction of transcription. Nucleic Acids Res, 1980 Nov 11, 8(21), 4919 - 26 The structure of the yeast ribosomal RNA genes . 2 . The nucleotide sequence of the initiation site for ribosomal RNA transcription; Bayev AA et al.; The 5'-terminal coding sequence for the 37 S precursor to rRNA of Saccharomyces cerevisiae is identified by reverse transcriptase extension and protection mapping with nuclease S1 . The sequence of a 419 bp rDNA fragment containing the transcription initiation site and its adjacent region is determined. Nucleic Acids Res, 1980 Nov 11, 8(21), 4841 - 50 Sequence of 863 nucleotides encompassing the PstI site of the yeast 2 micron plasmid; Elleman TC et al.; The sequence of part of the larger unique region of the yeast 2 micron plasmid cloned in pMB9 has been determined . The sequence extends from the single EcoRI site in this region to the AvaI site and includes the single PstI site and HpaI site . A notable feature of this sequence is the presence of tandem repeats of 124 residues beginning at the HpaI site and extending beyond the AvaI site . The sequence was determined independently by both the Maxam-Gilbert procedure applied to isolated restriction fragments, and by the chain-termination procedure applied to restriction fragments cloned in the single-stranded phage M13mp2 and purified by plaque selection. J Biol Chem, 1980 Nov 10, 255(21), 10188 - 93 Sulfhydryl groups of yeast phosphofructokinase-specific localization on beta subunits of fructose 6-phosphate binding sites as demonstrated by a differential chemical labeling study; Tijane MN et al.; Yeast phosphofructokinase contains 83 +/- 2 cysteinyl residues/enzyme oligomer . On the basis of their reactivity toward 5,5-dithiobis(2-nitrobenzoic acid), the accessible cysteinyl residues of the native enzyme may be classified into three groups . For titrations performed with N-ethylmaleimide, subdivisional classes of reactivity are evidenced . In each case, the 6 to 8 most reactive cysteines are not protected by fructose 6-phosphate from chemical labeling and do not seem involved in subsequent enzyme inactivation . Differential labeling studies as well as direct protection experiments in the presence of fructose 6-phosphate, indicate that 12 -SH groups/enzyme oligomer (i.e . three -SH groups per binding site) are protected by the allosteric substrate from the chemical modification . Specific labeling by the differential method of the cysteinyl residues protected by fructose 6-phosphate and further separation of the two types of subunits constituting yeast phosphofructokinase, show that the substrate binding sites are localized exclusively on subunits of beta type . Thus, alpha subunits are not implicated directly in the catalytic mechanism of yeast phosphofructokinase reaction. Nature, 1980 Nov 6, 288(5786), 60 - 3 Leaky +1 and -1 frameshift mutations at the same site in a yeast mitochondrial gene; Fox TD et al.; Two mutations in a mitochondrial structural gene, which cause leaky premature polypeptide chain termination and leaky growth, are +1 and -1 frameshifts in the same run of five T residues . The partial restoration of reading frame is probably due to ribosomal frameshifting at this site, and may be promoted by the unique structure of the yeast mitochondrial t RNAPhe. J Bacteriol, 1980 Nov, 144(2), 772 - 80 Significance of ribosomal ribonucleic acid synthesis for control of the G1 period in the cell cycle of the heterobasidiomycetous yeast Rhodosporidium toruloides; Ya