FEBS Lett, 1984 Apr 9, 169(1), 73 - 8 Nucleotide substitutions in a yeast mitochondria cis-acting mutant located in the last intron of the apocytochrome b gene; Bonjardim CA et al.; The region of mitochondrial DNA corresponding to the intron mutant M6-200 in Saccharomyces cerevisiae D273-10B has been isolated, and the nucleotide sequence of a 519 bp RsaI fragment has been determined . Three nucleotide substitutions were found at nucleotides +2650 (G----T), +2668 (G----A) and +2798 (A----G), all within the genetically defined location in the gene . Particular significance can be attributed to the first two changes (+2650 and +2668), that can be genetically isolated from the third substitution and, in addition, alter conserved sequence features detected in a study {(1982) Biochimie 64, 867-881} of fungal mitochondrial introns. Eur J Biochem, 1984 Apr 2, 140(1), 143 - 6 Catalytical mechanism of the phenylalanyl-tRNA synthetase from yeast . Reactivity of ATP in the absence of phenylalanine; Thiebe R; Phenylalanyl-tRNA synthetase catalyses an AMP-ATP exchange under conditions where no aminoacylation of tRNA occurs . A plausible explanation for this reaction had not been given so far . The results of the present investigation provide evidence for the following interpretation . tRNAPhe induces a polarisation of the ATP in complex with the enzyme; this stimulates (a) the formation of phenylalanyl-adenylate in the presence of phenylalanine, (b) the hydrolysis of ATP in the absence of phenylalanine and AMP and (c) the transfer of diphosphoryl onto AMP in the presence of AMP, especially when phenylalanine is absent. Eur J Biochem, 1984 Apr 2, 140(1), 157 - 61 Synthesis and properties of (4-13C)NAD+ . Observation of its binding to yeast alcohol dehydrogenase by 13C-NMR spectroscopy; Oberfrank M et al.; Starting from (13C)formic acid, acetone and cyanoacetamide samples of (4-13C)nicotinic acid and (4-13C)-nicotinamide were synthesised in an overall and additive yield of 11% . 1H-NMR and mass spectroscopy showed 90% enrichment of 13C in the expected position . NADase-catalysed exchange between thionicotinamide-adenine dinucleotide and (4-13C)nicotinamide furnished (4-13C)NAD+ which was purified, characterized and quantified by 1H-NMR and 13C-NMR spectroscopy and by enzymic assay . The 13C-NMR signal of (4-13C)beta-NAD+ (146.09 ppm) was broadened and shifted (147.83 ppm) upon binding to yeast alcohol dehydrogenase. Mol Cell Biol, 1984 Apr, 4(4), 771 - 8 Transformation of protoplasted yeast cells is directly associated with cell fusion; Harashima S et al.; The frequency of cell fusion during transformation of yeast protoplasts with various yeast plasmids with a chromosome replicon (YRp or YCp) or 2 mu DNA (YEp) was estimated by two methods . In one method, a mixture of protoplasts of two haploid strains with identical mating type and complementary auxotrophic nuclear markers with or without cytoplasmic markers was transformed . When the number of various phenotypic classes of transformants for the nuclear markers was analyzed by equations derived from binominal distribution theory, the frequency of nuclear fusion among the transformants was 42 to 100% in transformations with the YRp or YCp plasmids and 28 to 39% with the YEp plasmids . In another method, a haploid bearing the sir mutation, which allows a diploid (or polyploid) homozygous for the MAT (mating type) locus to sporulate by the expression of the silent mating-type loci HML and HMR, was transformed with the plasmids . Sporulation ability was found in 43 to 95% of the transformants with the YRp or YCp plasmids, and 26 to 31% of the YEp transformants . When cytoplasmic mixing was included with the nuclear fusion, 96 to 100% of the transformants were found to be cell fusants . Based upon these observations, we concluded that transformation of yeast protoplasts is directly associated with cell fusion. J Bacteriol, 1984 Apr, 158(1), 337 - 9 Activation of trehalase by membrane-depolarizing agents in yeast vegetative cells and ascospores; Thevelein JM; The membrane-depolarizing agents 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazone, and nystatin are known to cause a rapid increase in the cyclic AMP level in fungal cells . Addition of these proton ionophores to yeast stationary-phase cells or ascospores causes an immediate 10-fold increase in trehalase activity . This observation is in agreement with a role for cyclic AMP-induced phosphorylation in the activation process of trehalase . It also provides an explanation for previous results on the induction of trehalose breakdown by 2,4-dinitrophenol in resting yeast cells. EMBO J, 1984 Apr, 3(4), 829 - 34 Processing of yeast mitochondrial messenger RNAs at a conserved dodecamer sequence; Osinga KA et al.; The yeast mitochondrial genes coding for cytochrome c oxidase subunit I ( COX1 ) and the ATPase subunits 8 and 6 are organized in one transcription unit . Precise mapping of RNA termini with S1 nuclease and primer extension analysis shows that the 3' end of the COX1 mRNA and the 5' end of the ATPase precursor RNA are juxtaposed within a conserved dodecamer sequence (5'- AAUAAUAUUCUU -3') . Sequence comparison reveals that this motif is present downstream of nearly all protein-encoding genes, including extragenic unassigned reading frames ( URFs ) and two URFs located within introns . Also the 3' terminus of an RNA species derived from the URF -containing intron of the large rRNA gene maps within such a dodecamer sequence . It is likely, therefore, that this motif serves as a processing point in the generation of mature mRNA . From a comparison of the various transcription units, we infer that RNAs that originate from an endonucleolytic cleavage at this sequence have stable 3' termini, while further processing of the 5' ends occurs . The efficiency of the initial cleavage varies between the different positions at which the motif is present. Radiat Res, 1984 Apr, 98(1), 74 - 81 Comparisons of the effects of vacuum-uv and far-uv synchrotron radiation on dry yeast cells of different uv sensitivities; Hieda K et al.; Far-uv-sensitive (rad l/rad l) and wild-type cells of diploid Saccharomyces cerevisiae were irradiated in vacuum at 155, 170, 220, and 250 nm using synchrotron radiation (SR) . Inactivation, gene conversion at leu l, and membrane damage as judged by methylene blue penetration were measured . Radiations of all these wavelengths killed dry yeast cells . In the vacuum uv, radiation at 155 and 170 nm induced membrane damage but not gene conversion, whereas far-uv radiation at 220 and 250 nm induced gene conversion but not membrane damage . The far-uv-sensitive strain showed no enhanced sensitivity to vacuum-uv radiation . These results indicate that damage to the cell membrane is considerably more important than to nuclear DNA for yeast cell inactivation by vacuum-uv radiation. Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 905 - 12 Sequence of tRNA Ile IAU from brewer's yeast; Pixa G et al.; The nucleotide sequence of tRNAIle from brewer's yeast Saccharomyces cerevisiae was determined . Its primary structure is pG-G-U-C-U-C-U-U-m1G-m2G- C-C-C-A-G-D-D-G-G-D-D-A-A-G-G-C-A-C-C-G-U-G-C-U-I-A-U-t6 A-A-C-G-C-G-G-G-GA-D-m5 C-A-G-C-G-G-T-psi-C-G-m1 A-U-C-C-C-G-C-U-A-G-A-G-A-C-C-A-C-C-A . Its anticodon is I-A-U . It should therefore recognize the three isoleucine codons and is for this reason probably the only isoacceptor tRNA for isoleucine in brewer's yeast . It presents a large homology with its counterpart from Torulopsis utilis (87%). Nucleic Acids Res, 1984 Mar 26, 12(6), 2955 - 68 Structure and function of the nontranscribed spacer regions of yeast rDNA; Skryabin KG et al.; The sequences of the nontranscribed spacers (NTS) of cloned ribosomal DNA (rDNA) units from both Saccharomyces cerevisiae and Saccharomyces carlsbergensis were determined . The NTS sequences of both species were found to be 93% homologous . The major disparities comprise different frequencies of reiteration of short tracts of six to sixteen basepairs . Most of these reiterations are found within the 1100 basepairs long NTS between the 3'-ends of 26S and 5S rRNA (NTS1) . The NTS between the starts of 5S rRNA and 37S pre-rRNA (NTS2) comprises about 1250 basepairs . The first 800 basepairs of NTS NTS2 (adjacent to the 5S rRNA gene) are virtually identical in both strains whereas a variable region is present at about 250 basepairs upstream of the RNA polymerase A transcription start . In contrast to the situation in Drosophila and Xenopus no reiterations of the putative RNA polymerase A promoter are present within the yeast NTS . The strands of the yeast NTS reveal a remarkable bias of G and C-residues . Yeast rDNA was previously shown to contain a sequence capable of autonomous replication (ARS) (Szostak, J.W . and Wu, R (1979), Plasmid 2, 536-554) . This ARS, which may correspond to a chromosomal origin of replication, was located on a fragment of 570 basepairs within NTS2. Nucleic Acids Res, 1984 Mar 26, 12(6), 2705 - 15 Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence; Haumont E et al.; We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs . For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U) . This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34 . Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme . Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all . This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes. J Mol Biol, 1984 Mar 25, 174(1), 163 - 73 Structure of the ribotrinucleoside diphosphate codon UpUpC bound to tRNAPhe from yeast . A time-dependent transferred nuclear Overhauser enhancement study; Clore GM et al.; The structure of the ribotrinucleoside diphosphate UpUpC, the codon for phenylalanine, bound to yeast tRNAPhe in solution is elucidated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons . The glycosidic bond and ribose conformations are low anti and 3'-endo, respectively, typical of an A-RNA type structure . The main chain torsion angles are all within the range of those expected for A-RNA but small differences from those in conventional A-RNA 11 result in a special structure with a larger rotation per residue (40 to 45 degrees compared to 32.7 degrees in R-RNA 11) and almost perfect stacking of the bases . These two structural features, which are similar to those found in the anticodon triplet of the monoclinic crystal form of tRNAPhe, can account for the known greater stability of the codon-anticodon complex relative to an equivalent double helical RNA trimer with a conventional A-RNA structure. J Biol Chem, 1984 Mar 25, 259(6), 3714 - 9 Yeast LEU1 . Repression of mRNA levels by leucine and relationship of 5'-noncoding region to that of LEU2; Hsu YP et al.; Yeast LEU1 encodes the second enzyme in leucine biosynthesis . A 3.5-kilobase pair (kb) yeast genomic DNA fragment which complements a leu1 auxotroph was isolated by yeast transformation . After recloning into an integrating vector, a subfragment (of the 3.5-kb fragment) directs a URA3 marker to integrate at the LEU1 locus . About 1.9 kb was sequenced from the 5'-end of the 3.5-kb insert, and a long open reading frame and potential ATG start codon were located . S1 nuclease mapping showed a major start for LEU1 transcripts at 79 nucleotides upstream of the ATG codon . Northern blots with a LEU1-specific probe showed the size of the LEU1 transcript (about 2.9 kb) is consistent with the size of the enzyme and steady state levels of the transcript are sharply reduced in cells grown in the presence of an elevated leucine concentration . The latter observation correlates with the repression by leucine of LEU1 gene product levels . Other work has shown that the level of the LEU2 gene product is also repressed by leucine . Sequence comparisons between LEU1 and LEU2 show that the LEU2 5'-sequences which are cognate to leucine are not found in LEU1; and three blocks of nucleotide sequence homology between LEU1 and LEU2 occur in the 330 nucleotides upstream of the respective start codons. Biochem Biophys Res Commun, 1984 Mar 15, 119(2), 447 - 51 Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance; Tijssen JP et al.; Non-penetrating cations, like UO2+(2) and Eu3+, are bound to the outside of yeast cells in a reversible fashion . Binding of these ions was attended with a decrease of the 31P NMR polyphosphate signal . Subsequent addition of EDTA to the suspension restored the original spectrum . These experiments confirm the localization of a polyphosphate fraction outside the plasma membrane of yeast. FEBS Lett, 1984 Mar 12, 168(1), 61 - 4 Inhibition of ribonuclease activity during RNA synthesis in isolated yeast nuclei by cadmium; Schulz-Harder B et al.; We have developed an efficient transcription system in isolated yeast nuclei . If MnCl2 is substituted by CdCl2, degradation of newly synthesized RNA is markedly reduced . This effect is due to the inhibition of nuclear ribonuclease activity, since microsomal ribonuclease activity is less affected by the cation . The extent to which the addition of CdCl2 to the in vitro transcription assay inhibits ribonuclease activity is demonstrated by the measurements of the size of newly synthesized RNA . Efficient RNA synthesis in this system is not affected up to a concentration of 0.1 M CdCl2. Nucleic Acids Res, 1984 Mar 12, 12(5), 2407 - 19 In vitro generation of specific deletions in DNA cloned in M13 vectors using synthetic oligodeoxyribonucleotides: mutants in the 5'-flanking region of the yeast alcohol dehydrogenase II gene; Chan VL et al.; Deletion mutants are particularly useful in defining the boundaries of noncoding genetic functions . Such mutants can be precisely generated using synthetic oligodeoxyribonucleotides as mutagens . In this paper we describe the application of this method to recombinant DNA cloned in a phage M13-derived vector . The mutagenic oligodeoxyribonucleotides, 20 and 21 nucleotides in length, were used to delete a tract of 20 dA-dT base-pairs and an adjacent 22 base-pair perfect dyad from the ADR3 locus, the 5'-flanking regulatory region of the ADR2 gene, of Saccharomyces cerevisiae with high efficiency. J Biol Chem, 1984 Mar 10, 259(5), 3124 - 6 Solution X-ray scattering studies of the yeast phosphofructokinase allosteric transition . Characterization of an ATP-induced conformation distinct in quaternary structure from the R and T states of the enzyme; Laurent M et al.; The allosteric transition of yeast phosphofructokinase has been studied by solution x-ray scattering . The scattering curves corresponding to the native enzyme (T conformation) were found to be similar to the curves recorded in the presence of saturating concentrations of fructose 6-phosphate (R conformation) or AMP (R or R' conformation) . However, the curves obtained in the presence of ATP are clearly different: the radius of gyration increases and the secondary minima and maxima are systematically shifted to lower angles, suggesting a swelling of the enzyme in the presence of ATP . These results give the first direct evidence for the existence of an ATP-induced T' conformation, distinct in quaternary structure from the R and T states of the enzyme oligomer, in agreement with our previous modeling of yeast phosphofructokinase regulation . X-ray scattering data are discussed in relation to the distinct molecular mechanisms of the ATP and fructose 6-phosphate allosteric effects involving, respectively, sequential and concerted conformational changes of the enzyme oligomer. J Biol Chem, 1984 Mar 10, 259(5), 2886 - 95 Investigations of the metal ion-binding sites of yeast inorganic pyrophosphatase; Knight WB et al.; Yeast inorganic pyrophosphatase was found to bind two Mn2+ per subunit in the absence of phosphate and three Mn2+ per subunit in the presence of phosphate . Kinetic studies of the pyrophosphatase-catalyzed hydrolysis of Cr(NH3)4PP and Cr(H2O)4PP were carried out with Mn2+ and with Mg2+ as activators . The results from these studies suggest that three divalent cations per pyrophosphatase active site are required for catalysis . NMR and EPR studies were conducted to evaluate the relative location of the metal ion binding sites on the enzyme . The two Mn2+ ions bound to the free enzyme are in close enough proximity to magnetically interact . Analysis of the NMR and EPR data in terms of a dipolar relaxation mechanism between Mn2+ ions provides an estimate of the distance between them of 10-14 A . When the diamagnetic substrate analog {Co(NH3)4PNP}- or intermediate analog {Co(NH3)4 (P)2}- are bound to pyrophosphatase, two Mn2+ ions still bind to the enzyme and their magnetic interaction increases . In the presence of these Co3+ complexes, the Mn2+--Mn2+ separation decreases to 7-9 A . Several NMR and EPR experiments were conducted at low Mn2+ to pyrophosphatase ratios (approximately 0.3), where only one Mn2+ ion binds per subunit, in the presence of Cr3+ or Co3+ complexes of PNP or PP . Analysis of the Mn2+--Cr3+ dipolar relaxation evident in proton NMR and EPR data provided for the calculation of Mn2+--Cr3+ distances . When the substrate analog CrPNP was present, the Mn2+--Cr3+ distance was congruent to 7 A whereas, when Cr(P)2 was bound to pyrophosphatase, the Mn2+--Cr3+ distance was congruent to 5 A . These results strongly support a model for the catalytic site of pyrophosphatase that involves three metal ion cofactors. Cell, 1984 Mar, 36(3), 741 - 51 Sequence of the preprotoxin dsRNA gene of type I killer yeast: multiple processing events produce a two-component toxin; Bostian KA et al.; The preprotoxin gene of the 1.9 kb M1 dsRNA genome from type I killer yeast has been sequenced employing a partial-length cDNA derived from an in vivo transcript . A single open reading frame, commencing with AUG at M1 dsRNA bases 14-16, terminates with UAG at 963-965 and codes for a 316 amino acid protein, believed to be identical to the 34 kd preprotoxin species, M1-P1, synthesized by in vitro translation of denatured M1 dsRNA . N-terminal sequencing of M1-P1 confirms this prediction . Secreted toxin is shown to consist of two dissimilar, disulfide-bonded subunits, alpha and beta, of apparent size 9.5 and 9.0 kd, respectively, whose N-terminal sequences are also found in the predicted preprotoxin sequence . Its proposed domains consist of delta, a 44 amino acid N-terminal segment, followed by alpha and beta, which are separated by gamma, a large central glycosylated segment . Processing sites, domain functions, and the potential role of gamma in immunity are discussed. Biophys Chem, 1984 Mar, 19(2), 171 - 81 A new theoretical index of biochemical reactivity combining steric and electrostatic factors . An application to yeast tRNAPhe; Lavery R et al.; A new theoretical index of the chemical reactivity of sites within macromolecules is developed, which combines both steric and electrostatic factors . It is applied to the study of yeast tRNAPhe and the results obtained are compared with known experimental reactivities . A comparison indicates the superiority of the new index over the sole use of the surface accessibility. Biochem J, 1984 Mar 1, 218(2), 405 - 13 Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda; Rytka J et al.; The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins . Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation . Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type . The excreted porphyrins comprised mainly dehydroisocoproporphyrin . Analysis of uroporphyrinogen decarboxylase activity in the cell-free extract revealed a 70-80% decrease of activity in the mutant and showed that the relative rates of the different decarboxylation steps were modified with the mutant enzyme . A 2-3-fold increase in 5-aminolaevulinate synthase activity was measured in the mutant . The biochemical characteristics of the Sm1 mutant are very similar to those described for porphyria cutanea tarda. Cell, 1984 Mar, 36(3), 645 - 53 Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast; Langford CJ et al.; Our previous deletion experiments have shown that a short region of yeast nuclear gene introns containing the conserved sequence 5'-TACTAACA-3' is essential for splicing . In this report we show that the chemically synthesized decanucleotide 5'-TGTACTAACA-3', when introduced into a hybrid gene forming unspliceable RNA molecules, results in the generation of spliceable transcripts . Single A----C transversions in the fourth or eighth position of this sequence eliminated its intron-generating capacity . The C----T transition in the fifth position, generated by sodium bisulphite mutagenesis, did not affect the efficiency and accuracy of splicing . These results clearly demonstrate the biological significance of this conserved intron sequence and shed further light on its possible functioning. Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1475 - 9 Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA; Pluta AF et al.; The termini of macronuclear DNA molecules from the protozoan Oxytricha fallax share a common sequence and structure, both of which differ markedly from those deduced for yeast telomeres . Despite these differences, terminal restriction fragments from O . fallax macronuclear DNA can support telomere formation in yeasts . Two linear plasmids (LYX-1 and LYX-2) constructed by ligating BamHI-digested total Oxytricha macronuclear DNA to a yeast vector were analyzed . One end of LYX-1 and both ends of LYX-2 are derived from the Oxytricha DNA that encodes rRNA (rDNA) whereas the other end of LYX-1 is from an Oxytricha fragment other than rDNA . After propagation in yeast, both ends of LYX-1 and LYX-2 retain the C4A4 repeat characteristic of the O . fallax terminal sequence . In addition, both ends of both plasmids acquire 300-1000 base pairs of DNA containing the sequence (C-A)n, a sequence found near the termini of yeast chromosomes . Thus, at least two different Oxytricha termini display distinctive properties in yeast cells in that linear plasmids containing them are not degraded nor are they integrated into chromosomal DNA . These Oxytricha termini may act directly as telomeres in yeast; alternatively, the Oxytricha DNA may serve as a signal that results in the elaboration of a yeast telomere on the ciliate DNA. Eur J Biochem, 1984 Mar 1, 139(2), 201 - 8 The duck alpha A globin but not the yeast actin gene is transcribed by a HeLa cell extract; Horcher R et al.; We have investigated the transcription in a HeLa whole-cell extract of two evolutionary widely separated structural genes coding for duck alpha A globin and yeast actin . Transcription of isolated DNA fragments of the duck alpha A globin gene increases linearly up to relatively high concentrations of DNA . Size analyses and S1 mapping of the transcripts synthesized in vitro on either linear DNA fragments or supercoiled templates reveal that the alpha A globin RNA is initiated at the in vivo cap site and remains unspliced . The same assay conditions were used to transcribe the yeast actin gene . In contrast to the duck gene, size analyses and S1 mapping of the RNA products synthesized on both linear DNA fragments and the supercoiled template containing the actin gene show that the transcripts found in vitro do not stem from the in vivo cap site . The promoter of the yeast actin gene is not recognized in this system in vitro. J Hosp Infect, 1984 Mar, 5(1), 83 - 91 Oral ketoconazole and amphotericin B for the prevention of yeast colonization in patients with acute leukaemia; Donnelly JP et al.; Forty-eight neutropenic patients with acute leukaemia were randomly allocated to receive, as antifungal prophylaxis, either ketoconazole, 400 mg once daily (K), or amphotericin B tablets and lozenges (A), or both ketoconazole and amphotericin B together (K + A) . Antifungal prophylaxis was considered to have failed if (1) there was evidence of increasing colonization of the oropharynx or faeces with Candida spp . or other yeasts, or (2) if systemic antifungal therapy was begun empirically . Prophylaxis failed in nine of 17 patients given K, in four of 19 given A, and in four of 12 given K + A . The differences between the three regimens were not statistically significant, neither was there any significant difference in the mean duration of neutropenia before prophylaxis failed . The absorption of ketoconazole was impaired when patients were neutropenic . We conclude that ketoconazole was neither more nor less effective than amphotericin B in the prevention of yeast colonization in neutropenic patients. Biochemistry, 1984 Feb 28, 23(5), 797 - 801 Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate; Gonzalez MA et al.; In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled {gamma-17O,18O}ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio{17O,18O}phosphate proceeds with inversion of configuration . Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb {Webb, M . R . (1982) Methods Enzymol . 87, 301-316} . We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O . These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate. Nucleic Acids Res, 1984 Feb 24, 12(4), 1889 - 900 Initiation of transcription in yeast mitochondria: analysis of origins of replication and of genes coding for a messenger RNA and a transfer RNA; Osinga KA et al.; The initiation of transcription of the yeast mitochondrial genes coding for subunit I of cytochrome c oxidase (COX1) and for tRNA1Thr has been examined . COX1 messenger RNA synthesis is initiated in a conserved nonanucleotide sequence (ATATAAGTA) which we have previously found immediately upstream of ribosomal RNA genes at positions at which RNA synthesis starts . The 5'-end of the precursor of tRNA1Thr is located in a variant nonanucleotide motif (TTATAAGTA), which may be characteristic for tRNA genes . Using a partially purified fraction of mtRNA polymerase, we demonstrate that RNA synthesis is precisely initiated in vitro in nonanucleotide sequences preceding both ribosomal RNA-, tRNA- and messenger RNA-encoding genes and origins of replication. J Mol Biol, 1984 Feb 15, 173(1), 1 - 13 Location of DNAase I sensitive cleavage sites in the yeast 2 micron plasmid DNA chromosome; Fagrelius TJ et al.; We have studied the uniformity with which the yeast 2 micron plasmid DNA within its nucleoprotein complex is protected from digestion by DNAase I . To probe for relatively unprotected regions, plasmid nucleoprotein complexes were digested with DNAase I to yield a preparation in which approximately half of the circular DNA molecules had been converted to full-length linear molecules . The sites of the double-strand breaks were then mapped in relation to restriction endonuclease sites using end-label probes . The most prominent sensitive sites were found at positions very close to the beginning and end of a 122 base-pair sequence with dyad symmetry located within the 599 base-pair inverted repetition of the plasmid . The sequence is known to be necessary for plasmid site-specific recombination . Other sensitive sites were mapped to the 5'-side of known coding regions . A unique plasmid sequence located to one side of the replication origin was also sensitive to DNAase I digestion yet did not yield discrete cleavage sites . Cleavage of plasmid DNA stripped of proteins did not result in the appearance of distinct fragments as found after cleavage of the same DNA within the nucleoprotein complex . We conclude from these results that, when complexed with proteins, specific plasmid DNA sequences involved in transcription, replication and recombination are more accessible to nuclease digestion. FEBS Lett, 1984 Feb 13, 167(1), 165 - 9 Nucleotide sequence of a yeast tRNAArg3A gene and its transcription in a homologous in vitro system; Villanueva J et al.; Twelve bacterial clones containing complementary sequences to yeast tRNAArg3 were isolated from a gene library . The size of the yeast BamHI inserts ranges from 5.4 to 10 MDa . There are at least 6 copies of this gene in different loci of the yeast genome . Insert from clone pYAT-3 was mapped, and the presence of a tRNAArg3A gene was confirmed by DNA sequence . The coding region is colinear with the transcriptional product . Unlike other reported tRNAArg3A genes, this one is not linked to a tRNAAsp gene . In vitro transcription using a yeast extract produces a transcript of 76 +/- 1 bases. J Biol Chem, 1984 Feb 10, 259(3), 1661 - 6 Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation . II . Lanosterol metabolism by purified P-450(14)DM and by intact microsomes; Aoyama Y et al.; A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen . This conversion did not occur anaerobically or when either P-450(14)DM, the reductase, or NADPH was omitted from the system . In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns . Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol . Upon aerobic incubation of microsomes from semianaerobically grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol . This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-450(14)DM . It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase. J Biol Chem, 1984 Feb 10, 259(3), 1655 - 60 Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation . I . Purification and spectral properties; Yoshida Y et al.; A form of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation (tentatively called "P-450(14)DM") was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoretic homogeneity . An apparent monomeric Mr = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Both optical and EPR spectra of oxidized P-450(14)DM are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm . As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-450(14)DM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state . Oxidized P-450(14)DM was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350- and 414-nm troughs possessed larger and relatively smaller {theta} values, respectively, than those reported for other low spin ferric cytochromes P-450 . Lanosterol was the only compound which caused a Type I spectral change in oxidized P-450(14)DM . The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-450(14)DM adduct was in a spin state equilibrium. Cell, 1984 Feb, 36(2), 309 - 18 Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway; Julius D et al.; Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway . In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro . At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates . In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed . The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus . Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5). J Biochem (Tokyo), 1984 Feb, 95(2), 589 - 92 Nucleotide sequence of an essential region for autonomous replication of cloned yeast mitochondrial DNA; Mabuchi T et al.; A 341 bp sequence from yeast mtDNA was cloned, which consisted of an upstream 98 bp AT stretch and a downstream 206 bp AT stretch separated by a single 37 bp GC cluster . Cleavage of this GC cluster did not cause loss of the autonomously replicating function of this sequence . The recloned first 98 bp AT stretch was incapable of replication, while the recloned 206 bp AT stretch could replicate . We were able to confine an essential sequence for autonomous replication within a 186 bp AT stretch . Sequencing data revealed a sequence of ATATAAAT and stem and loop structures within the AT stretch. Biochem J, 1984 Feb 1, 217(3), 641 - 7 The regulatory properties of yeast pyruvate kinase; Morris CN et al.; The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic . The findings were represented empirically by the exponential model for a regulatory enzyme . The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively {Ainsworth (1977) J . Theor . Biol . 68, 391-413} . The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account . The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme {Ainsworth, Kinderlerer & Gregory (1983) Biochem . J . 209, 401-411}, and it is suggested that they have a common origin in charge interactions within the active site. Genetics, 1984 Feb, 106(2), 207 - 26 Healing of broken linear dicentric chromosomes in yeast; Haber JE et al.; In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome . Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores . The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle) . The Ura+ Mal+ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura+ Mal+, others Ura+ Mal- and others Ura- Mal+ . The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.--Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic breakage have demonstrated that these stable derivatives arose from mitotic breakage of the dicentric chromosome, followed by one of several different healing events . The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome . A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere . In two instances we recovered chromosome III partially duplicated with a novel right end . We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres. Radiat Res, 1984 Feb, 97(2), 329 - 40 Interpretation of the dose and LET dependence of RBE values for lethal lesions in yeast cells; Frankenberg D; Survival data on yeast cells proficient or deficient in the repair of DNA double-strand breaks (dsb) and data on the induction of dsb are used to interpret the dose dependence of the RBE value for lethal lesions after irradiation at high dose rate followed by 72-hr liquid holding providing optimum conditions for repair of potentially lethal lesions (RBEDP, DP = delayed plating) . The radiations applied are conventional (150 kV), soft (50 kV), and ultrasoft (4 kV) X rays, 30-MeV electrons (or 60Co gamma rays), and 3.5-MeV alpha particles . Analysis shows that the dose dependence of the RBEDP value can be explained by the combination of two dose-independent RBE values, one for the single-particle traversal effect (RBEspt) and the other for the accumulation of dsb (RBEdsb) due to the traversal of more than one particle through the cell nucleus . Furthermore, it is shown that the LET dependence of RBEspt values describing the linear component of the lethal lesions must be considered separately for "electron" and "particle" radiations. Biochimie, 1984 Feb, 66(2), 127 - 34 Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (L-lactate cytochrome c reductase) upon binding of pyruvate, the reaction product; Tegoni M et al.; Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b2 moiety were carried out at room temperature, in the presence of added pyruvate or in its absence . Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate, the reaction product in the oxidation of L-lactate . The proportion of flavin semiquinone nearly reaches then 100 per cent. Infect Immun, 1984 Feb, 43(2), 467 - 71 Influence of yeast mannan on release of myeloperoxidase by human neutrophils: determination of structural features of mannan required for formation of myeloperoxidase-mannan-neutrophil complexes; Wright CD et al.; Structural features of mannan which participate in the formation of myeloperoxidase-mannan-neutrophil complexes have been studied by using a battery of structurally modified mannans . Mannan was isolated from Saccharomyces cerevisiae X2180 wild type and modified by strong alkaline degradation, selective (mild) alkaline degradation, and selective acetolysis . Mannose oligosaccharides bound to the peptide portion of mannan appeared to be required for binding of mannan to the neutrophil . The interaction of mannan with myeloperoxidase appeared to occur through phosphate groups of the mannan outer chain . The myeloperoxidase-mannan interaction was determined to be ionic in nature . The mannan-neutrophil interaction may involve cell membrane receptors for mannose. FEBS Lett, 1984 Jan 30, 166(2), 321 - 5 Vacuoles are not the sole compartments of proteolytic enzymes in yeast; Emter O et al.; Localization in vacuoles, the lysosome-like organelle of yeast, was checked for several newly detected proteolytic enzymes . While aminopeptidase Co and carboxypeptidase S were found in vacuoles, proteinase D and proteinase E as well as a variety of other proteolytic activities detectable with the aid of chromogenic peptide substrates do not reside in this cell compartment. J Biol Chem, 1984 Jan 25, 259(2), 1004 - 10 Yeast cells recover from mating pheromone alpha factor-induced division arrest by desensitization in the absence of alpha factor destruction; Moore SA; Saccharomyces cerevisiae MATa cells arrest cell division in response to the mating pheromone, alpha factor . After some interval of time the cells resume division . It is demonstrated here that cells recover from division arrest by becoming insensitive to the alpha factor under conditions of low cell density (10(2) cells/ml) where no alpha factor destruction occurs . The time of desensitization occurs later at higher alpha factor concentrations . Using the technique of perfusion photomicroscopy developed in this study, it was found that 95% of the desensitized cells remain insensitive to the alpha factor for greater than or equal to 3 generations at the alpha factor concentration where recovery occurred . Upon recovery, cells have generation times which are similar or identical to the calculated time from the cdc28 "start" step of cell division to cell separation . Therefore, the abnormally large recovered cells behave as if they have no growth time requirement for cell division for several generations . Desensitization was found to occur asymmetrically for parent and daughter cells . While parents (cells which have budded) are insensitive to alpha factor, their daughters (cells which have never budded and which were formed from desensitized parents during continuous perfusion with alpha factor) show 54% with a delayed generation time compared to control daughter cells. FEBS Lett, 1984 Jan 23, 166(1), 131 - 5 Reaction of thionitrobenzoate-modified yeast cytochrome c with monomeric and dimeric forms of beef heart cytochrome c oxidase; Darley-Usmar VM et al.; Thionitrobenzoate-modified yeast cytochrome c was shown to react with both monomeric and dimeric forms of beef heart cytochrome c oxidase through subunit III . This cytochrome c derivative was found to inhibit electron transfer in the dimer but not in the monomer . These results are interpreted to show that the high affinity binding site for cytochrome c is a cleft at the interface between monomers in the cytochrome c oxidase dimer. Nature, 1984 Jan 12-18, 307(5947), 178 - 80 Human hepatitis B vaccine from recombinant yeast; McAleer WJ et al.; The worldwide importance of human hepatitis B virus infection and the toll it takes in chronic liver disease, cirrhosis and hepatocarcinoma, make it imperative that a vaccine be developed for worldwide application . Human hepatitis B vaccines are presently prepared using hepatitis B surface antigen (HBsAg) that is purified from the plasma of human carriers of hepatitis B virus infection . The preparation of hepatitis B vaccine from a human source is restricted by the available supply of infected human plasma and by the need to apply stringent processes that purify the antigen and render it free of infectious hepatitis B virus and other possible living agents that might be present in the plasma . Joint efforts between our laboratories and those of Drs W . Rutter and B . Hall led to the preparation of vectors carrying the DNA sequence for HBsAg and antigen expression in the yeast Saccharomyces cerevisiae . Here we describe the development of hepatitis B vaccine of yeast cell origin . HBsAg of subtype adw was produced in recombinant yeast cell culture, and the purified antigen in alum formulation stimulated production of antibody in mice, grivet monkeys and chimpanzees . Vaccinated chimpanzees were totally protected when challenged intravenously with either homologous or heterologous subtype adr and ayw virus of human serum source . This is the first example of a vaccine produced from recombinant cells which is effective against a human viral infection. J Biol Chem, 1984 Jan 10, 259(1), 412 - 7 Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway; Runge KW et al.; Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a {3H}mannose suicide enrichment . Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro . When UDP-{3H}Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose . When dolicholphospho{3H}glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides . These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide . We also present evidence that both mutants transfer Man9GlcNAc2 to protein. FEBS Lett, 1984 Jan 9, 165(2), 251 - 3 Investigation of the role of the substrate metal ion in the yeast inorganic pyrophosphatase reaction; Ting SJ et al.; The substrate activities of a series of tripositive metal ion-pyrophosphate complexes with yeast inorganic pyrophosphatase were examined . While the Michaelis constants for these complexes were shown to be between one and two orders of magnitude greater than that of the natural substrate, {Mg(H2O)4PPi}2-, the turnover numbers were in general comparable to that of {Mg(H2O)4PPi}2- . These data suggest that the nature of the metal ion cofactor effects substrate binding but in most cases not catalysis . Thus, the role of the metal ion in catalysis is probably restricted to that of an electron sink. Biochemistry, 1984 Jan 3, 23(1), 69 - 73 Stimulation of yeast RNA polymerase II transcription by critical values of supercoiling; Pedone F et al.; RNA chains of discrete length were obtained in vitro by yeast RNA polymerase II directed transcription of a supercoiled plasmid . On the basis of the amount and the molecular weight of the RNA chains synthesized in the absence of reinitiation events, the number of actively transcribing RNA polymerase molecules has been calculated . A stimulation of transcriptional activity was found to be related to the torsional strength of negative supercoiling of the template . The DNA unwinding angle measured in the complexes formed with the enzyme in the presence of three ribonucleoside triphosphates equals 485 +/- 30 degrees, marking a melting effect of 14 base pairs per bound enzyme molecule. Eur J Biochem, 1984 Jan 2, 138(1), 77 - 81 Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys; Vacher J et al.; In this paper we describe the construction of a yeast tRNACys UGA suppressor . After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil . The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities . The level of aminoacylation was similar to that of the parent molecule . Readthrough of the UGA termination codon in beta-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions . We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes . This may be related to the low suppressor activity; however, it is also possible that other features of tRNACys structure may be unadapted to efficient UCA anticodon function. Eur J Biochem, 1984 Jan 2, 138(1), 169 - 77 The biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast . DNA sequence analysis of the nuclear gene coding for the 14-kDa subunit; De Haan M et al.; The nuclear gene coding for the imported 14-kDa subunit of the ubiquinol-cytochrome c reductase of yeast mitochondria has been sequenced in an attempt to define regulatory and protein topogenic elements . The gene has a length of 381 base pairs and is potentially capable of encoding a polypeptide of 14561 Da . It is transcribed into a single low-abundance RNA of 680 nucleotides whose 5' and 3' termini map, respectively, 30-35 nucleotides upstream and 180-190 nucleotides downstream of the initiator and termination codons . Consistent with the estimated low level of the mRNA, codon usage in the gene is not strongly biased and other features, characteristic of highly expressed genes in yeast, are absent . The 14-kDa protein is predicted to be a predominantly hydrophilic protein, with only a single, short hydrophobic stretch located between positions 19-38 . Comparison with other imported mitochondrial proteins so far sequenced has failed to reveal unifying features that might serve as targeting elements . Steady-state levels of the 14-kDa and 11-kDa subunits are reduced in mit- mutants which synthesize truncated forms of apocytochrome b and in these, newly synthesized subunits exhibit a specifically increased turnover rate . We suggest that association of these two subunits with the complex may be mediated or enhanced by interaction with other subunits, in particular cytochrome b. Eur J Cell Biol, 1984 Jan, 33(1), 19 - 23 Monitoring yeast spindles in the fluorescence microscope; Baumstark-Khan C et al.; Formation of the complete spindles during the budding process of Saccharomyces uvarum was investigated by fluorescence microscopy of protoplasted cells . Protoplasts were treated with anti-tubulin antibodies and DAPI, a fluorescent dye staining DNA . Thus, both chromatin and spindles could be visualized . Duplication as well as formation of separated spindle pole bodies during the different stages of budding are documented, demonstrating the occurrence and behaviour of microtubules during yeast cell cycle. Antonie Van Leeuwenhoek, 1984, 50(4), 369 - 78 Trichosporon adeninovorans sp . nov., a yeast species utilizing adenine, xanthine, uric acid, putrescine and primary n-alkylamines as the sole source of carbon, nitrogen and energy; Middelhoven WJ et al.; A new yeast species, Trichosporon adeninovorans, was isolated from soil by the enrichment culture method . Apart from adenine, the strain utilized uric acid, guanine, xanthine, hypoxanthine, 6,8-dihydroxypurine, putrescine, propylamine, butylamine, pentylamine, hexylamine and octylamine as sole source of carbon, nitrogen and energy . The structure of the cell wall of Tr . adeninovorans was ascomycetous . On the subcellular level growth on adenine or uric acid was accompanied with the development of microbodies in the cell . These cell organelles probably were the site of urate oxidase, an enzyme that, after growth on purine substrates, together with allantoinase was present at high activities . Low activities of adenine amidohydrolase and xanthine dehydrogenase were also demonstrated. Antonie Van Leeuwenhoek, 1984, 50(3), 249 - 60 The induction of mycelial development in Aureobasidium pullulans (IMI 45533) by yeast extract; Cooper LA et al.; Germ tube and subsequent mycelial development from yeast-like and swollen cells of Aureobasidium pullulans (IMI 45533) was induced by yeast extract in defined liquid medium . This morphogenetic transition was dependent on inoculum size; pH effects were not involved and once mycelial development was induced in the cells it continued even in the absence of yeast extract . The progeny of mycelium and future generations were unaffected by yeast extract . Cessation of germination was not due to any obvious medium changes but appeared to be partly due to the production of a germination inhibitor, which could also be produced by control cells grown in the absence of yeast extract. Antonie Van Leeuwenhoek, 1984, 50(3), 219 - 25 Candida lignophila sp . nov., a new basidiomycetous yeast anamorph from rotting wood of Drimys winteri; Dill I et al.; Two strains of an undescribed Candida species were isolated from samples of a rotting trunk of Drimys winteri collected on the isle of Chiloe in South Chile . A description of the new species Candida lignophila is given and its relationship to other species is discussed with particular emphasis on its typical basidiomycetous properties as well as on its ecological habitat. J Immunopharmacol, 1984, 6(4), 305 - 21 The immunomodulatory effect of yeast glucan on delayed hypersensitivity; Perez HA et al.; The effect of yeast beta-1, 3-glucan as an immunopotentiator of delayed type-hypersensitivity reactions (DHR) was studied . Delayed-type-hypersensitivity reactions in mice sensitized intraperitoneally (IP) with sheep red blood cells (SRBC) and pretreated three days previously with glucan given IP were significantly increased . However, mice sensitized IP with SRBC three days after the subcutaneous (SC) administration of glucan showed depressed DHR . Glucan given at the same site but not at distance strongly potentiated the DHR induced by SC sensitization with SRBC . Subcutaneous injection of glucan and SRBC given together also resulted in a sustained DHR which persisted twelve days after sensitization when DHR of control mice had waned. Antonie Van Leeuwenhoek, 1984, 50(4), 379 - 81 The yeast Candida sequanensis sp . nov; Saez H et al.; A strain of an undescribed Candida species was isolated from animal fodder . A description of the new species is given and its distinction from the most closely resembling species of the genus is discussed. Antonie Van Leeuwenhoek, 1984, 50(4), 305 - 20 Nematospora sinecauda sp . nov., a yeast pathogen of mustard seeds; Holley RA et al.; An undescribed yeast species was recovered from oriental (Brassica juncea) and yellow (B . hirta) mustard seeds . The new species most closely resembled Nematospora coryli but its asci were rarely cylindrical . The asci and ascospores of N . sinecauda were smaller and the spores did not possess a whip-like appendage . During germination a sprout cell formed first on the smooth anterior surface of the spore above the median ridge . The posterior region of the spore was decorated with interrupted concentric ridges . A description of the new species is given. Mol Gen Genet, 1984, 197(3), 519 - 21 Mutagenesis in yeast-misreplication or misrepair? Kilbey BJ. Evidence from the phenotype of mutants which partially block mutagenesis and from experiments made to time induced mutagenesis relative to cell division in yeast is used to question whether mutagenesis in yeast should be regarded as an error-prone repair phenomenon. Sabouraudia, 1984, 22(6), 487 - 91 Immunodiffusion studies on the antigens of Histoplasma capsulatum: comparison of mycelial histoplasmin with the yeast phase reagent Histolyn-CYL; Owens RD et al.; Immunodiffusion assays were performed to determine if H and M antigens associated with mycelial histoplasmin were present in the yeast phase reagent, Histolyn-CYL (H-CYL) . Neither H nor M antigens could be detected in H-CYL . However, when H-CYL was reacted against human histoplasmal sera a positive reaction was evidenced in all instances in which a response was elicited with a reference mycelial immunodiffusion reagent. Enzyme, 1984, 31(4), 197 - 208 Studies on NADPH-cytochrome c reductase . II . Steady-state kinetic properties of the crystalline enzyme from ale yeast; Tryon E et al.; From a study of the steady-state kinetics (at pH 7.6, 30 degrees C) of the reduction of cytochrome c, a 'ping-pong' mechanism may be postulated for the crystalline NADPH-cytochrome c reductase from ale yeast, Saccharomyces cerevisiae {1}, a result derivable from a three-substrate ordered system with a rapid equilibrium random sequence in substrates, NADPH and FAD, followed by reactions of the third substrate, Cyt C3+ . On this basis, estimates for the kinetic parameters were made together with the inhibitor dissociation constants for NADP+ (competitive with respect to NADPH as variable substrate, but noncompetitive with respect to cytochrome c3+ as the variable substrate) . A noncompetitive type of inhibition was also found for cytochrome c2+ with NADPH as variable substrate, in confirmation of the proposed mechanism . With 2,6-dichloroindophenol as the acceptor, in place of cytochrome c3+, a value for KNADPH could be estimated which agreed with that estimated above, with cytochrome c3+ as the acceptor, again, in confirmation of the postulated mechanism . The reactions with molecular O2 catalyzed by the enzyme with NADPH as the reductant have been studied polarographically, and its Km for O2 estimated to be about 0.15 mmol/l at pH 7.6, 25 degrees C . The product of the reaction appears to be H2O2, which acts as a noncompetitive inhibitor for NADPH (Ki = 0.5 mmol/l), and tentatively an enzyme ternary complex containing oxygen and FADoh (semiquinone of FAD) may be assumed to be the kinetically important intermediate, which may be postulated to be in quasi-equilibrium with an enzyme ternary complex containing Oo2 (superoxide) and FAD. Sabouraudia, 1984, 22(1), 1 - 5 Effects of divalent cations and functionally related substances on the yeast to mycelium transition in Sporothrix schenckii; Alsina A et al.; In a minimal basal medium with glucose at pH 4.0 and 25 degrees C, a lowering of the magnesium and zinc concentrations or increase in the calcium concentration of the medium favoured the yeast-mycelium transition in Sporothrix schenckii . Addition of zinc (1 and 10 mM) inhibited mycelial development and induced reversion to a yeast-like morphology . EDTA and EGTA also delayed germ tube formation, possibly by their calcium-chelating effects or by altering intracellular concentrations of this or other ions . Ionophore X537A also caused a delay in germ tube formation, possibly by interfering with magnesium metabolism in these cells. J Cell Sci Suppl, 1984, 1, 43 - 58 Structural and functional analysis of a yeast centromere (CEN3); Carbon J et al.; Structure-function analysis of a yeast (Saccharomyces cerevisiae) centromere (CEN3) has been carried out by altering the nucleotide sequence of the DNA within and surrounding the centromere of yeast chromosome III, and observing the behaviour of the resulting altered chromosomes during mitotic and meiotic cell divisions . A centromere substitution vector (pJC3-13) was constructed, which contains in the proper orientation: the DNA sequences that normally flank the chromosome III centromere, a wild-type URA3 gene for selection, and a unique BamHI restriction site for insertion of various DNA sequences to be assayed for centromere activity . Cleavage of the plasmid DNA with EcoRI generates a linear DNA fragment whose ends are homologous with the regions flanking the centromere . Transformation of the appropriate homozygous ura3 diploid yeast strain with this linear DNA results in URA3+ transformants in which the CEN3 region on one copy of chromosome III has been replaced by the URA3 gene and the DNA sequence previously inserted into the vector . These studies identify a 289 base-pair (bp) DNA fragment from the CEN3 region that retains full centromere function when used to replace the normal CEN3 sequence . Centromeres function equally well in either orientation, and the chromosome XI centromere (CEN11) can be used to replace CEN3, with no observable effect on mitotic or meiotic chromosome segregation . Various DNA restriction fragments occurring within the CEN3 region were used alone or in combinations to replace the normal CEN3 sequence . Yeast centromeres contain a high A + T region about 82-89 bp in length (element II) flanked by a highly conserved 11 bp sequence (III) and a less-conserved 14 bp sequence (I) . The experiments demonstrate that both regions II and III are necessary for normal centromere function, although centromeres containing III plus truncated or rearranged portions of the high A + T region II retain partial activity . Chromosomes of the latter type often give abnormal segregation patterns through meiosis, including separation and random segregation of sister chromatids during the first meiotic division. Antonie Van Leeuwenhoek, 1984, 50(5-6), 799 - 805 The adventures of the yeast genus Endomycopsis Dekker; von Arx JA et al.; The yeast genera Endomyces, Endomycopsella, Guilliermondella and Saccharomycopsis are delimited by the size, structure and pigmentation of the ascospores; they include mycelial yeasts formerly classified in the invalid genus Endomycopsis . The ultrastructure of the cell wall and the septa of yeasts is briefly discussed. Mol Gen Genet, 1984, 197(3), 491 - 6 A hybrid DNA sequence containing the replication origin of the multicopy yeast plasmid 2 micron circle and an additional repeated sequence can convert maltose-negative into maltose-positive strains; Rodicio R et al.; Yeast DNA pools were prepared by ligating partial Sau3A genomic digests from strains carrying various MAL genes into the BamHI site of the yeast-Escherichia coli shuttle vector YRp7 . They were used to transform recipient yeast strains that could not utilize maltose since they lacked a classical MAL gene . Transformants were obtained that could use maltose and also formed normal levels of maltase . They were unstable . They would lose the selective marker TRP1 of YRp7 alone, together with the ability to utilize maltose or only the ability to utilize maltose . The insertion of one of the plasmids was used as a hybridization probe for the others and found to share homologous sequences with all . They were then shown to contain the replication origin of the yeast 2 micron circle plasmid and additional sequences . These additional sequences were used to probe genomic digests of total yeast DNA . They hybridized at various degrees of efficiency with several bands, indicating that they were part of a family of repeated sequences . Apparently, it was the combination of the replication origin of the 2 micron circles with the additional sequences that promoted maltose utilization. Mol Gen Genet, 1984, 197(3), 351 - 7 Conformational alterations in the proximal portion of the yeast invertase signal peptide do not block secretion; Brown PA et al.; Various amino acid insertions have been introduced into the proximal portion of the signal sequence of secreted yeast invertase . The altered invertase genes have been reintroduced into yeast and monitored for their ability to direct synthesis of secreted invertase in vivo . The insertions should alter the signal polypeptide local secondary structure as predicted by the Chou and Fasman rules (1978) . Secretion of these altered invertase polypeptides is not blocked by the amino acid insertions. Mol Gen Genet, 1984, 197(2), 345 - 6 A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance; Boeke JD et al.; Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection . Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3- mutants grow normally . This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells. Microbiol Immunol, 1984, 28(9), 997 - 1007 Lethal effect of neutral mannan fraction of bakers' yeast in mice; Nagase T et al.; A simple polysaccharide, the neutral mannan from Saccharomyces cerevisiae wild type strain (WNM) was found to kill ddY strain mice by intravenous administration, showing a LD50 value of 12.2 mg/kg . On the other hand, the acidic mannan fraction from the same yeast containing phosphate (WAM025), and chemically phosphorylated WNM (WNM-P) were practically non-toxic . Concerning the relationship between chemical structure and lethal effect of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger lethality than those with less branched structures . Against C3H/HeJ strain mice with no responsiveness to lipopolysaccharide, the LD50 value of WNM was as high as 75 mg/kg . Pretreatment with 500 mg/kg of D-mannose, N-acetyl-D-glucosamine, D-galactose, and L-fucose prevented mice from the lethal effects of WNM . However, WNM (LD100) did not show any lethal effect in mice for 2 to 12 hr after treatment with dexamethasone, an anti-inflammatory steroid. Radiat Environ Biophys, 1984, 23(4), 287 - 94 Release of P and K from yeast cells irradiated by vacuum UV below 170 nm; Matsumoto S et al.; Yeast cells were irradiated with monochromatic synchrotron radiation (SR) under wet conditions in the wavelength region from 160 to 185 nm at INS-SOR, Tokyo . By the particle-induced X-ray emission (PIXE) method applied to whole cells several elements were found to be released from the irradiated cells at the wavelengths shorter than 170 nm . The most drastic release occurred with phosphorus, followed potassium . Sulphur and calcium were not released over the whole wavelength region studied . It was also revealed that the release of these elements paralleled the cell inactivation . The cause of these element releases upon vaccuum-UV irradiation was inferred in relation to the dissociation of H2O molecules located in the vicinity of the cell surface region. Mol Gen Genet, 1984, 196(2), 266 - 74 Organization and processing of the mitochondrial oxi3/oli2 multigenic transcript in yeast; Simon M et al.; In the present article, we confirm our previous proposal (Faye and Simon 1983a, b) that the oxi3 and oli2 genes belong to the same transcription unit . Furthermore, we have shown that a primary polycistronic transcript covers oxi3, aap1, oli2 and extends beyond URF2 . Transcriptional analysis of this region revealed several cleavage points . The examination of the DNA sequence at and surrounding these cleavage points disclosed that some of them take place at or near specific sequences found also in other known multigenic transcripts . Two of the major cleavages involve the stem-loop structure of GC rich clusters . We discuss the possibility that some of these cleavage sites serve as post-transcriptional processing signals and may be necessary for the maturation of the precursor RNA. Mol Gen Genet, 1984, 195(1-2), 260 - 6 Replicon size of yeast ribosomal DNA; Walmsley RM et al.; The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9K bp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 microns long, on chromosome XII . Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined . We have used immobilised rRNA as a probe to examine the size of radioactively labelled rDNA replicons resolved on alkaline sucrose gradients . The replicons were found to be uniformly sized, about 5 repeat units in length, and groups of 4 adjacent replicons may be activated simultaneously . These observations suggest that replicon initiation events are not determined solely by the recognition of specific DNA sequences that function as origins of replication. Prep Biochem, 1984, 14(2), 163 - 71 An evaluation of methods used to prepare yeast mitochondria for transcriptional studies; Rickwood D et al.; This report describes experiments which compare conditions necessary to isolate mitochondria uncontaminated with nuclear chromatin using a KDL grinding mill. Microbiol Immunol, 1984, 28(6), 651 - 7 Pyrogenicity of yeast mannans in rabbits; Nagase T et al.; A few yeast mannans free from protein and phosphorus showed pyrogenic activity in rabbits although the extent of this activity was considerably lower than that of the bacterial lipopolysaccharides (LPS) . The pyrogenic activity was not abolished by treatment with sodium deoxycholate . This result showed that the mannans themselves participated in the pyrogenicity, excluding any possibility of LPS contamination in the mannans . Concerning the relationship between chemical structure and pyrogenicity of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger pyrogenicity than that of a less branched one. Biochem Int, 1984 Jan, 8(1), 105 - 12 Yeast mitochondrial inner membrane 30 kd hydrophobic protein: properties and interaction with phospholipids; Alkoutayni M et al.; A 30 kd hydrophobic protein is extracted from yeast mitochondrial inner membrane . It is present in wild yeast strains but absent in mitochondrial DNA lacking mutants . The isoelectric point of the protein and its solubility in various organic solvents are determined . The fluorescence of a tryptophan residue near the surface of the 30 kd protein dissolved in butanol-1, can be quenched by phospholipids containing unsaturated fatty acids . Results are in accordance with the 30 kd protein being an integral protein of the yeast mitochondrial inner membrane. Mol Gen Genet, 1984, 195(3), 487 - 90 UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast; Lawrence CW et al.; The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis . The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles . The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes . If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis. Mol Gen Genet, 1984, 194(3), 489 - 93 Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha; Roggenkamp R et al.; The biosynthesis of methanol oxidase, a peroxisomal enzyme in the methanol-utilizing yeast Hansenula polymorpha, was studied in vitro . Translation of Hansenula mRNA in a rabbit reticulocyte lysate yields methanol oxidase protein in high amounts . The apparent molecular mass of the protein was found to be identical to the subunit of the functional multimeric enzyme, which indicates the absence of an N-terminal extension typical of most transported proteins . The regulation of methanol oxidase by glucose repression and depression as well as by induction of methanol was shown to be controlled at the level of transcription . Two mutants of Hansenula polymorpha, unable to grow on methanol as a carbon and energy source were shown to be affected in methanol oxidase synthesis. Mol Gen Genet, 1984, 194(1-2), 7 - 14 Genetic mode of action of cocarcinogens and tumor promoters in yeast and mice; Fahrig R; In experiments with yeast, cocarcinogens were found to be comutagenic and antirecombinogenic , tumor promoters to be corecombinogenic and antimutagenic . Substances that were cocarcinogens as well as tumor promoters had an intermediary effect . These results were confirmed in the mammalian spot test: By in vivo treatment of mice with the cocarcinogen catechol and the tumor promoter limonene carcinogen-induced recombination due to mitotic crossing over and gene mutations was reduced and enhanced, respectively . Our results support the hypothesis that mutagenesis is the mechanism by which chemicals induce malignancy, and that cocarcinogens modify the process by enhancement of mutagenicity whereas tumor promoters effect carcinogenesis by increase of the spontaneous frequency of recombination . In addition, induced mitotic recombination in mammals in vivo has been demonstrated for the first time. Gene, 1984 Jan, 27(1), 23 - 33 Cloning and expression of a yeast copper metallothionein gene; Butt TR et al.; The induction of a copper-binding metallothionein (Cu-MT) was studied in yeast, Saccharomyces cerevisiae, and a relationship between copper resistance and intracellular levels of Cu-MT in these eukaryotes was established . Poly(A)-containing RNA from a copper-resistant (Cur) yeast strain, which synthesized abundant quantities of Cu-MT and in which Cu-MT gene transcription was enhanced 50-fold upon exposure to CuSO4, was used to screen yeast genomic DNA clones . Restriction analysis revealed common XbaI and KpnI sites in five genomic clones isolated . The transcription of these clones was regulated by copper . Transformation of a copper-sensitive (Cus) yeast strain by one of these clones confers copper resistance in yeast . The results suggest that the expression of the Cu-MT gene is, in part, responsible for mediating copper resistance in yeast. Mikrobiologiia, 1984 Jan-Feb, 53(1), 5 - 9 {Intracellular pool of free amino acids in dehydrated yeast organisms}; Novichkova AT et al.; The dehydration of Saccharomyces cerevisiae was found to result in a noticeable decrease of the free amino acids content in the cells and in a considerable increase of cytoplasmic membrane permeability for these compounds . When the dehydrated organisms were reactivated, the normal permeability of the cytoplasmic membrane gradually restored and the pool of free amino acids increased in the cells. J Cell Biol, 1984 Jan, 98(1), 44 - 53 Genes required for completion of import of proteins into the endoplasmic reticulum in yeast; Ferro-Novick S et al.; Yeast secretory mutants sec53 and sec59 define a posttranslational stage in the penetration of glycoprotein precursors into the endoplasmic reticulum (ER) . In the previous report we showed that at the restrictive temperature (37 degrees C) these mutants accumulate enzymatically inactive and incompletely glycosylated forms of the secretory enzyme invertase and the vacuolar enzyme carboxypeptidase Y . Cell fractionation experiments reveal that these precursor forms remain firmly bound to the ER membrane . However, upon return to the permissive temperature (24 degrees C), the invertase precursors are glycosylated, become partially active, and are secreted . Thermoreversible conversion does not require protein synthesis, but does require energy . In contrast to the effect of these mutations, inhibition of oligosaccharide synthesis with tunicamycin at 37 degrees C causes irreversible accumulation of unglycosylated invertase . The effect of the drug is exaggerated by high temperature since unglycosylated invertase synthesized in the presence of tunicamycin at 25 degrees C is secreted . A portion of the invertase polypeptide accumulated at 37 degrees C is preserved when membranes from sec53 and sec59 are treated with trypsin . In the presence of Triton X-100 or saponin, the invertase is degraded completely . The protected fragment appears to represent a portion of the invertase polypeptide that is embedded in or firmly associated with the ER membrane . This association may develop early during the synthesis of invertase, so that in the absence of translocation, some of the completed polypeptide chain remains exposed on the cytoplasmic surface of the ER. J Cell Biol, 1984 Jan, 98(1), 35 - 43 Yeast secretory mutants that block the formation of active cell surface enzymes; Ferro-Novick S et al.; Yeast cells secrete a variety of glycosylated proteins . At least two of these proteins, invertase and acid phosphatase, fail to be secreted in a new class of mutants that are temperature-sensitive for growth . Unlike the yeast secretory mutants previously described (class A sec mutants; Novick, P., C . Field, and R . Schekman, 1980, Cell., 21:205-420), class B sec mutants (sec 53, sec 59) fail to produce active secretory enzymes at the restrictive temperature (37 degrees C) . sec 53 and sec 59 appear to be defective in reactions associated with the endoplasmic reticulum . Although protein synthesis continues at a nearly normal rate for 2 h at 37 degrees C, incorporation of {3H}mannose into glycoprotein is reduced . Immunoreactive polypeptide forms of invertase accumulate within the cell which have mobilities on SDS PAGE consistent with incomplete glycosylation: sec 53 produces little or no glycosylated invertase, and sec 59 accumulates forms containing 0-3 of the 9-10 N-linked oligosaccharide chains that are normally added to the protein . In addition to secreted enzymes, maturation of the vacuolar glycoprotein carboxypeptidase Y, incorporation of the plasma membrane sulfate permease activity, and secretion of the major cell wall proteins are blocked at 37 degrees C. J Biochem (Tokyo), 1984 Jan, 95(1), 109 - 15 A kinetic study on the binding of monomeric and polymeric derivatives of NAD+ to yeast alcohol dehydrogenase; Yamazaki Y et al.; The binding to yeast alcohol dehydrogenase of NAD+ and its five derivatives (N6-{2-{N-{2-{N-(2-methacrylamidoethyl)carbamoyl}ethyl} carbamoyl}ethyl}-NAD (I), N6-{N-{2-{N-(2-methacrylamidoethyl) carbamoyl}ethyl}carbamoylmethyl}-NAD (II), copolymer of I with acrylamide (PA-I), copolymer of II with acrylamide (PA-II), and copolymer of I with N,N-dimethylacrylamide (PDMA-I} were studied statically and kinetically by the stopped-flow method by using the quenching of the enzyme fluorescence in the presence of pyrazole . Apparent dissociation constants and apparent rate constants were determined therefrom . It was concluded that (1) the N6-CH2CH2CO group (of I) is effective in making the derivative bind more strongly as well as faster than NAD+, while the N6-CH2CO group (of II) is not; and (2) the binding of the polymer derivatives of NAD+ to the enzyme is not essentially weaker and slower than that of native NAD+, but is even faster in some cases . The coenzymic activities of the above compounds were also determined with yeast alcohol dehydrogenase, pig heart malate dehydrogenase, and rabbit muscle lactate dehydrogenase. Int J Biochem, 1984, 16(2), 177 - 82 The stimulation of yeast mitochondrial protein synthesis by low molecular weight cytoplasmic factors: requirement for intact mitochondria and lack of effect by folate derivatives; Finzi E et al.; Protein synthesis by GTP-supplemented yeast mitochondria is stimulated by a fraction of molecular weight less than 2,000 isolated from yeast high-speed supernatant (S-150) . The low molecular weight fraction works independently of the respiratory chain as the stimulation effect is not cyanide-sensitive . Stimulation of mitochondrial protein synthesis by cytoplasmic factors is dependent upon the method of mitochondrial isolation . The low molecular weight stimulatory factor(s) are not reduced folate derivatives which supply formyl groups required for initiation of mitochondrial protein synthesis. EMBO J, 1984 Jan, 3(1), 107 - 11 Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA; Skipper N et al.; Complementary DNA (cDNA) copies of the M1-1, toxin-coding region of the yeast M1 double-stranded RNA (dsRNA) have been cloned and sequenced . These sequences, in combination with the known terminal sequence of M1-1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation . Potential glycosylation sites in the preprotoxin peptide are identified . Based on its methionine content the extracellular yeast toxin appears to be contained within the C-terminal region of the precursor. Br J Cancer Suppl, 1984, 6, 157 - 61 Recovery from transcription inhibition in irradiated yeast cells; Weber KJ et al.; Repair process operating on radiation damaged DNA may be investigated by studying its functional integrity, e.g . its ability to serve as template for transcription . We measured the synthesis of ribosomal RNA and of the inducible enzyme arginase in yeast cells and followed their recovery after X-ray, alpha-particle and heavy ion exposure . Transcription inhibiting lesions formed upon X-irradiation in yeast cells are repaired during post-exposure incubation ("liquid-holding") . The inactivation curves are strictly exponential immediately after irradiation . After the "liquid holding" treatment the inactivation curves are still exponential but with a progressive decrease of the slopes as a function of incubation time . This indicates that a constant fraction of the lesions is repaired per time interval . But even after long incubation times (24 h) there is still a sizeable unrepaired fraction . Comparing different yeast strains and different irradiation temperatures recovery can also be demonstrated in cells exposed to alpha-particles . In addition, recovery is detected by the arginase assay after irradiation of yeast with very heavy charged particles, e.g . titanium ions. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 337 - 41 Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast; Karin M et al.; Copper resistance in yeast is controlled by the CUP1 locus . The level of resistance is proportional to the copy number of this locus, which can be found in up to 15 tandemly iterated copies . To elucidate the molecular mechanisms controlling the amplification and expression of the CUP1, locus, we determined its full nucleotide sequence . We have also identified and mapped two transcription units within the basic amplification unit of CUP1 in laboratory yeast strains . One of those transcription units is inducible by copper and encodes a low molecular weight copper binding protein--copper chelatin . The increased production of chelatin, due to both gene amplification and induction of transcription, leads to increased resistance of yeast cells to copper ions. J Inorg Biochem, 1984 Jan, 20(1), 39 - 52 Activation of yeast enolase by Cd(II); Spencer SG et al.; Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+ . The activity is weakly stimulated, then inhibited by increasing ionic strength . The activity increases, then falls with increasing Cd2+ concentration . The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme . The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH . This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's . The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme . We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc . The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex . The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7 . There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor . The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen . The apparent reaction rate constant increases linearly with Cd2+ concentration . Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly . The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants. J Cell Biochem, 1984, 24(3), 229 - 42 Mitochondrial membrane biogenesis: characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase; McEwen JE et al.; A nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene (COX5) by complementation . This mutant, E4-238 {24}, and its revertant, JM110, produce variant forms of subunit V . In comparison to the wild-type polypeptide (Mr = 12,500), the polypeptides from E4-238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively . These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis-acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type . Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4-238, with DNA from a yeast nuclear DNA library in the vector YEp13 . One plasmid, YEp13-511, with a DNA insert of 4.8 kilobases, was characterized in detail . It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V . The availability of mutants altered in the structural gene for subunit V (COX5) and of the COX5 gene on a plasmid, together with the demonstration that plasmid-encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase. Gene, 1984 Jan, 27(1), 13 - 21 Cloning of the vaccinia virus telomere in a yeast plasmid vector; DeLange AM et al.; The vaccinia virus DNA telomere, which contains a covalently closed hairpin structure, has been cloned in a yeast plasmid vector . Restriction mapping indicates that the cloned vaccinia telomere is maintained in yeast not in its native hairpin configuration but as an inverted repeat structure, within a circular plasmid, with the sequences of the viral hairpin now at the axis of symmetry of an imperfect palindrome . As such, the cloned telomere resembles the telomeric replicative intermediate observed during vaccinia virus DNA replication . Small deletions and duplications in the viral inverted repeats of different clones suggest a model in which the observed circular plasmids were generated in yeast by the replication of hybrid linear DNA molecules consisting of the linearized yeast vector flanked by two hairpin-containing vaccinia termini. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 367 - 70 Regulated expression of a human interferon gene in yeast: control by phosphate concentration or temperature; Kramer RA et al.; The promoter/regulator region from the yeast repressible acid phosphatase gene was used to construct a vector for the regulated expression of cloned genes in yeast . The gene for human leukocyte interferon was inserted into this vector . Yeast cells transformed with the resulting plasmid produced significant amounts of interferon only when grown in medium lacking inorganic phosphate . Mutants in two acid phosphatase regulatory genes (coding for a defective repressor and a temperature-sensitive positive regulator) were used to develop a yeast strain that grew well at a high temperature (35 degrees C) but produced interferon only at a low temperature (23 degrees C), independent of phosphate concentration. Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 8 - 12 Steps in processing of the mitochondrial cytochrome oxidase subunit I pre-mRNA affected by a nuclear mutation in yeast; Simon M et al.; In Saccharomyces cerevisiae, the mitochondrial gene encoding the subunit I of cytochrome c oxidase (oxi-3 gene) is interrupted by intervening sequences . In this report, a nuclear mutation {referred to as mss51 in Faye, G . & Simon, M . (1983) Cell 32, 77-87} that specifically affects the processing of oxi-3 pre-mRNA was further characterized . DNA probes covering each oxi-3 exon-intron boundary were individually hybridized to wild-type and mutant mitochondrial RNA . By a technique relying on the S1 nuclease resistance or sensitivity of the RNA X DNA hybrids thereof, we have shown which site needs the MSS51 gene product to be cleaved . The mutation in the MSS51 gene gave rise to a complex pattern of splicing: the third intron was excised efficiently but the first two introns remained bracketed by their flanking exons . Further, the fourth and fifth introns were only partially split from their common exon and remained fused to their upstream and downstream flanking exon, respectively . Several plausible roles for the MSS51 gene product are discussed. Nature, 1984 Jan 26-Feb 1, 307(5949), 386 - 8 Preferential integration of yeast transposable element Ty into a promoter region; Eibel H et al.; Mobile genetic elements have been identified in several eukaryotic organisms and some classes have been found to share common structural features with the proviral forms of animal retroviruses . The representatives of this class of mobile elements in the yeast Saccharomyces cerevisiae are called Ty elements, which could be a useful model system for studying the transposition of retrovirus-like elements . Here we have attempted to answer two questions often raised in discussions of the biological importance of transposition: what is the frequency of spontaneous Ty transposition, and are there certain chromosomal regions into which Ty elements preferentially integrate? We chose the LYS2 gene to investigate these questions because it allows direct selection of both mutants and revertants . We have found that 2% of spontaneous lys2 mutants are caused by Ty transposition with a preferential integration into the transcription initiation region. Mol Gen Genet, 1984, 197(3), 515 - 6 Multiple genes control particulate phosphofructokinase of yeast; Parmar L et al.; Mutants of Saccharomyces cerevisiae lacking the particulate phosphofructokinase define at least four unlinked genes, PFK2, PFK3, PFK4 and PFK5 . A structural role of PFK2 is indicated . Mutations in the other three have pleiotropic effects. Biomed Biochim Acta, 1984, 43(10), 1083 - 9 Influence of inorganic phosphate on the kinetic properties of yeast phosphofructokinase; Przybylski F et al.; Yeast phosphofructokinase is effectively activated by inorganic phosphate . In the absence of other allosteric stimulators, inorganic phosphate increases the maximum activity of the enzyme only . In the presence of the activators AMP and fructose 2,6-bisphosphate inorganic phosphate causes changes in the maximum activity and the enzyme affinity to fructose 6-phosphate . Inorganic phosphate augments the sensitivity of phosphofructokinase to the activators AMP and fructose 2,6-bisphosphate and increases the respective maximum activities . The extent of activation of the enzyme by inorganic phosphate prevails at low levels of fructose 6-phosphate and high ATP concentrations. Biomed Biochim Acta, 1984, 43(4), 535 - 40 Cooperation of fructose-2,6-bisphosphate and AMP in the activation of yeast phosphofructokinase; Nissler K et al.; Yeast phosphofructokinase is effectively activated by AMP and fructose-2,6-bisphosphate . Both effectors influence the sensitivity of the enzyme with respect to fructose-6-phosphate and increase the respective maximum activities . The dependence of phosphofructokinase activity on the concentration of fructose-2,6-bisphosphate was measured at different AMP concentrations and vice versa . By AMP the half activation constant for fructose-2,6-bisphosphate is decreased by one order of magnitude . The affinity to AMP is significantly increased by fructose-2,6-bisphosphate . AMP increases the maximum activity of the enzyme with respect to fructose-2,6-bisphosphate only slightly, while the maximum activity with respect to AMP is drastically increased by fructose-2,6-bisphosphate . The interaction of the two activators is most pronounced at low levels of fructose-6-phosphate and at high concentrations of ATP. Biomed Biochim Acta, 1984, 43(4), 413 - 8 Interaction of Cibacron blue F3G-A with yeast phosphofructokinase; Frenzel J et al.; The binding of Cibacron blue F3G-A to yeast phosphofructokinase was investigated by means of ultracentrifugation . Four moles of Cibacron blue are tightly bound per subunit of phosphofructokinase (dissociation constant = 0.26 microM) . This stoichiometry does not correspond to the stoichiometry of ATP binding to yeast phosphofructokinase (two moles of ATP per subunit) . Moreover, 32 moles of the dye are bound per subunit of phosphofructokinase to a second class of binding sites with low affinity (dissociation constant = = 53 microM) . The action of Cibacron blue on yeast phosphofructokinase cannot be explained completely in terms of its function as ATP analogue. Biochimie, 1984 Jan, 66(1), 49 - 58 Studies on the structure of yeast phosphofructokinase; Chaffotte AF et al.; In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase . This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations . The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods . However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer . On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase . Obviously, some methods of molecular weight determination have led to erroneous results . In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution. Acta Microbiol Pol, 1984, 33(2), 119 - 30 Effect of some quaternary benzylammonium salts on physiology of yeast; Kolodynski J et al.; In order to determine the biological activity of eight compounds belonging to a group of quaternary ammonium salts, their influence on the active methionine transport, the integrity of cell membranes, respiration, and viability of Saccharomyces cerevisiae and some other yeast species has been investigated . The earliest effect observed during ammonium salts action on yeast cells is an immediate methionine transport abolishment followed by its fast leakage, which indicates increasing cell membrane degradation . Gradual decline of other biological functions such as respiration and viability is thus a result of disintegration and lack of tightness of the cell membranes . The studied compounds are characterized by a rather unspecific spectrum of action on yeast resulting in irreversible damage of cell walls and cell membranes, which in consequence leads to cell death. J Biochem (Tokyo), 1984 Jan, 95(1), 131 - 6 Purification and properties of factors in yeast mitochondria stabilizing the F1F0-ATPase-inhibitor complex; Hashimoto T et al.; A previously found yeast-mitochondrial protein fraction stabilizing the inactivated complex between mitochondrial ATPase and intrinsic ATPase inhibitor (Hashimoto, T., et al . (1983) J . Biochem . 94, 715-720) was separated into two proteins by high performance liquid chromatography on a cation exchanger . The molecular weights of the factors were estimated to be 9,000 and 15,000 daltons by sodium dodecyl sulfate (SDS)-gel electrophoresis . Both factors were required to stabilize a complex of inhibitor and proton-translocating ATPase (F1F0-ATPase) either in its purified form or in mitochondrial membranes . On the other hand both factors together could not stabilize a complex of the inhibitor and F1-ATPase, suggesting that both factors act together with the F0-portion . The factors also facilitated very efficiently the binding of ATPase inhibitor to F1F0-ATPase in the presence of ATP and Mg2+ . Both the 15,000 and 9,000 dalton stabilizing factors were hardly distinguishable from delta- and epsilon-subunit, respectively, on an SDS-gel electrophoregram, but immuno-diffusion assay showed that neither factor was present in the purified F1-ATPase containing the delta- and epsilon-subunit. Teratog Carcinog Mutagen, 1984, 4(4), 365 - 75 Comparative genetic activity of cis- and trans-1,2-dichloroethylene in yeast; Bronzetti G et al.; The cis and trans isomers of 1,2-dichloroethylene were tested for mutagenic effects in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system, an S9 mouse liver fraction, and by an in vivo intrasanguineous host mediated assay . The effects of the same agents on aminopyrine N-demethylase activity and cytochrome P-450 level in liver were studied in nonpretreated and in phenobarbital + beta-naphtoflavone-pretreated mice . In the suspension test, both isomers exhibited dose dependent toxicity, and survival was lower with metabolic activation than without . In this test also, both isomers exhibited genetic activity as measured by increases in recombinants at the ade 2 locus in experiments with metabolic activation . In the host-mediated assay, only the cis isomer showed evidence of mutagenic activity with significant increases in convertants at the trp locus and revertants at the ilv locus . Such mutagenic activity was found both after acute and chronic doses and in liver, kidney, and lung tissue . The two isomers exhibited different effects with respect to aminopyrine N-demethylase activity and cytochrome P-450 level . In general, the trans isomer appeared to emphasize induction of enzyme activity or level while the cis isomer more frequently tended to inhibit activity or destroy the enzyme. Mol Gen Genet, 1984, 197(3), 420 - 4 A yeast with linear molecules of mitochondrial DNA; Kovac L et al.; Mitochondrial DNA from the yeast strain SR23, tentatively allocated to the species Candida rhagii, consists of linear molecules 30 kb long . This has been demonstrated by restriction analysis and selective radioactive labelling of terminal restriction fragments . Preliminary sequence analysis indicated that the two ends of the molecule are formed by inverted repeats . The arrangement of several genes in the mitochondrial genome of C . rhagii SR23 was established by specific hybridisation with probes prepared from mitochondrial DNA of Saccharomyces cerevisiae . The arrangement is unique, with genes coding for the two ribosomal RNAs placed widely apart . Intron(s) may be present in the gene coding for cytochrome b. Nucleic Acids Symp Ser, 1984, (15), 177 - 80 Terminal structures of F2, a linear DNA plasmid from yeast: two different terminal structures in one linear DNA molecule; Kikuchi Y et al.; The terminal structure of F2, a linear double-stranded DNA plasmid of 3.9 kb from yeast, was analyzed . Results obtained by electrophoretic analyses of terminal restriction fragments of F2 showed that one end of F2 has terminal protein at the 5'-terminus, but another end has hairpin structure . This is a novel type of autonomously replicating DNA molecule. Mol Gen Genet, 1984, 197(2), 219 - 24 Nuclear genes coding for four subunits of the yeast ubiquinol-cytochrome c reductase complex are present in single copies in the haploid genome and at least two of these are located on different chromosomes; Van Loon AP et al.; Genes coding for the 40 kilodaltons (kDa), 17-kDa, 14-kDa and 11-kDa subunits of the ubiquinol-cytochrome c reductase in yeast are present in single copies in the haploid genome . We have mapped each gene to a unique genomic environment and demonstrate that integration of cloned segments into nuclear DNA by homologous crossing-over with the endogenous gene results in the replacement of the corresponding chromosomal restriction fragment by fragments of predicted sizes . Chromosomal mapping, carried out by the procedure of Falco and Botstein 1983, indicates that the gene for the 17-kDa subunit lies on chromosome VI and that for the 11-kDa subunit on chromosome XII. J Biol Chem, 1983 Dec 25, 258(24), 15037 - 45 The catalytic mechanism of yeast thiosulfate reductase; Chauncey TR et al.; Thiosulfate reductase catalyzes the desulfuration of thiosulfonates while oxidizing GSH to GSSG . Kinetic studies of the enzyme-catalyzed reaction between GSH and benzenethiosulfonate have been carried out, and direct evidence for the occurrence of glutathione persulfide as an immediate product of the reaction has been obtained . The formal mechanism of this enzymic reaction has been shown to be rapid equilibrium-ordered with GSH as the leading substrate. J Mol Biol, 1983 Dec 15, 171(3), 345 - 52 Homologous proteins encoded by yeast mitochondrial introns and by a group of RNA viruses from plants; Zimmern D; Hensgens et al . (1983a) have demonstrated the existence of distant homology (averaging 19.6%) between the central sections of seven proteins encoded by introns (and one product of an apparently independent gene) in yeast mitochondrial DNA . The homologous regions are typically segments of about 115 amino acids within open reading frames of about 10(3) bases . Genetic studies indicate that at least two of these proteins are required for the splicing of mitochondrial transcripts . This paper reports that two distantly related proteins of Mr 30,000 that are encoded by different strains of tobacco mosaic virus both contain central sections whose amino acid sequences are 15% to 23% identical in a single alignment to those of one group of four intron-encoded proteins, and possess certain groups of conserved residues also characteristic of the mitochondrial proteins . Genetic studies implicate these proteins in the spreading of viral lesions . While this level of identity cannot establish conclusively that the proteins are related, it suggests the possibility of a functional and/or evolutionary connection that would, if borne out, have important implications. J Biol Chem, 1983 Dec 10, 258(23), 14271 - 5 Dicyclohexylcarbodiimide blocks proton ejection and affects antimycin binding but not electron transport in complex III from yeast mitochondria; Clejan L et al.; Treatment of complex III with dicyclohexyldicarbodiimide (DCCD) either before or after incorporation into liposomes resulted in a loss of electrogenic proton movements; however, only minimal decreases in cytochrome c reductase activity were noted in the liposomes containing DCCD-treated complex III . Thus, DCCD appears to act by "uncoupling" proton translocation from electron transport . A decreased sensitivity of the ubiquinol:cytochrome c reductase activity to antimycin was also noted in the DCCD-treated complex III . This loss of sensitivity to antimycin was reflected in a decreased binding of antimycin to the complex after DCCD treatment from 9.5 nmol/mg of protein in the control to 3.8 nmol/mg of protein in the DCCD-treated complex . DCCD also affected the red shift observed after antimycin addition to dithionite-reduced complex III resulting in a broad peak with no sharp maximum . Similarly, DCCD treatment of yeast mitochondria resulted in a complete loss in the red shift after antimycin addition to mitochondria previously reduced with succinate . No loss in enzymatic activity was observed in the DCCD-treated mitochondria . These results suggest that DCCD concomitant with the inhibition of proton ejection in the cytochrome b-c1 region of the respiratory chain causes modifications in the properties of cytochrome b which alter the binding of antimycin without significantly affecting the electron transfer activity of this cytochrome. J Biol Chem, 1983 Dec 10, 258(23), 14065 - 8 oli1 Transcripts in wild type and in a cytoplasmic "petite" mutant of yeast; Thalenfeld BE et al.; Subunit 9 of ATPase is known to be encoded in the oli1 gene of yeast mitochondrial DNA . The oli1 transcripts of wild type and of a cytoplasmic "petite" mutant have been analyzed by hybridization of mitochondrial RNA to various DNA fragments from the internal and flanking regions of the gene and by S1 nuclease mapping of the 5