Biochim Biophys Acta, 1983 May 4, 757(1), 77 - 84 Synthesis of retinylphosphate mannose in yeast and its possible involvement in lipid-linked oligosaccharide formation; Lehle L et al.; A membrane fraction from Saccharomyces cerevisiae as well as a mannosyltransferase purified therefrom was shown to catalyze the transfer of mannose from GDPmannose to retinyl phosphate . The product formed has chromatographic and chemical properties characteristic for retinylphosphate mannose . The enzyme requires divalent cations . Mg2+ is more effective than Mn2+ with an optimum concentration around 25 mM . Amphomycin at a concentration of 0.1 mg/ml inhibits the reaction to 50% . Glycosyl transfer was specific for mannose residues from GDPmannose and did not occur with dolichylphosphate mannose nor with UDP galactose; UDPglucose is a poor donor . Formation of retinylphosphate mannose is inhibited by dolichyl phosphate . This observation as well as similarities between retinylphosphate mannose and dolichylphosphate mannose synthesis in respect to ion requirement, inhibition by amphomycin are suggestive that both reactions are catalyzed by one and the same enzyme . In experiments studying the glycosyl donor specificity in the assembly of lipid-linked oligosaccharide intermediates involved in N-glycosylation of proteins, it could be demonstrated that retinylphosphate mannose can replace dolichylphosphate mannose in the final steps of mannosylation. FEBS Lett, 1983 May 2, 155(1), 50 - 4 Interactions of antibodies against soluble phosphofructokinase with the soluble and particulate enzymes from yeast; Huse K et al.; Antibodies obtained from rabbits against soluble yeast phosphofructokinase also react with the particulate yeast phosphofructokinase . Their effects on the activity of the soluble enzyme recognized as inactivation or slight activation depend on the specific immune response of an individual animal yielding antisera with different proportions of inactivating and activating antibodies . The availability of particulate phosphofructokinase to complex inactivating antibodies specifically allows a separation of activating and inactivating antibodies from each other by a simple extraction procedure. Sci Sin {B}, 1983 May, 26(5), 504 - 12 Assay of biological activity of synthetic yeast alanine transfer RNA (tRNAAlay); Shen QX et al.; The biological activity of the synthetic tRNAAlay was studied with an extremely sensitive method . tRNAAlay accepted alanine in the presence of rat liver aminoacyl-tRNAAlay-synthetase (this was called the accepting activity) . The aminoacylated tRNAAlay was conveniently precipitated by ethanol with good recovery . The efficiency of transferring alanine from the aminoacylated tRNAAlay into the protein was determined in in vitro rabbit reticulocyte lysate cell-free protein-synthesizing system (this was called the incorporation activity) . Both accepting and incorporation activities could be determined in one assay with only 5-7 pmoles of tRNAAlay either in ligation mixture or in purified form . Our results show that the accepting activities of the synthetic products were 51.6-65.6% and 91.3-106.0% of that of natural and reconstituted natural tRNAAlay respectively . The efficiency of the incorporation of alanine in the aminoacylated tRNAAlay into the protein was 61.6-63.1%, corresponding to 90.6-91.7% and 97.2-115.8% of that of the natural and the reconstituted natural tRNAAlay respectively. Sci Sin {B}, 1983 May, 26(5), 495 - 503 Synthesis of the 5'-half molecule of yeast alanine tRNA; Wang DB et al.; In this paper, we report the synthesis of the 5'-half molecule of yeast alanine tRNA (tRNAAlay) by ligating three oligonucleotide fragments corresponding to the nucleotide sequences 1-13, 14-22 and 23-35 respectively under the catalysis of T4 RNA ligase (Fig . 1) . Because of the high purity of the oligonucleotide fragments and the excellent quality of T4RNA ligase and polynucleotide kinase we prepared, the isolation steps were simplified and the overall yields were much higher . The ligating yield of the docosamer (IV) was 75%, that of the pentatriacontamer (V), 90%, and the isolated yield of the final product was 21% calculated on the basis of the tridecamer (III) used in the first reaction . Under the action of T4 RNA ligase the synthetic 5'-half molecule was joined with the natural 3'-half molecule forming a semi-synthetic tRNAAlay, which possessed the biological activities of both accepting (3H)-alanine and incorporating it into proteins . The correctness of the structure of the synthetic 5'-half molecule was verified by both chemical analysis and biological activity assay. Sci Sin {B}, 1983 May, 26(5), 482 - 94 Synthesis of the 3'-half molecule of yeast alanine tRNA; Wang DB et al.; This paper deals with the synthesis of the 3'-half molecule of yeast alanine transfer RNA (tRNAAlay) by ligation with T4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45(I), 46-57(II) and 58-76(III) in succession extending from the 3'-end to the 5'-end . First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5'-phosphorylation of the nonadecamer (III), ligation with the dodecamer (II) and the 5'-phosphorylation of the ligation product formed; with one isolation step and obtained the 5'-phosphorylated 31mer(46-76) (IV) in an overall yield of 70% . Then the 31mer(IV) as a donor was ligated with 3 times of decamer (I) to form the 41mer(36-76) (V), the 3'-half molecule of tRNAAlay . The yield was 67% . After 5'-phosphorylation, (V) was ligated with the natural 5'-half molecule to form the semi-synthetic tRNAAlay, which was biologically active, i.e . accepting and transferring (3H)-alanine into proteins. Sci Sin {B}, 1983 May, 26(5), 464 - 81 Total synthesis of yeast alanine transfer ribonucleic acid; Wang DB et al.; By a combination of chemical and enzymatic methods, small oligonucleotides with lengths varying from 2 to 8 nucleotides were synthesized from mononucleotides . The small oligonucleotides were then ligated with T4 RNA ligase into six large oligonucleotides (9 to 19 nucleotides long) which were further ligated to form two half molecules with 35 and 41 nucleotides respectively . Finally, the two synthetic half molecules were annealed and ligated to obtain the whole molecule of yeast alanine tRNA (tRNAAlay) . Prior to this, two semi-syntheses were performed, i.e . ligation of the synthetic 5'-half molecule with the natural 3'-half molecule and that of the natural 5'-half molecule with the synthetic 3'-half molecule . Both the semi-synthetic tRNAAlay and the synthetic tRNAAlay occupy the same position as the natural tRNAAlay after electrophoresis on a 20% polyacrylamide gel . They have the same chemical composition (containing 9 modified nucleotides of 7 different species) and structure as the natural tRNAAlay and are biologically active, i.e . accepting and transferring alanine into proteins in a cell-free protein synthesizing system, the accepting activity of the synthetic product is 52-66% of that of the natural tRNAAlay and 91-106% of that of the reconstituted product of the two natural half molecules . The incorporation activity of alanine into proteins of the synthetic 3H-alanine tRNAAlay is 63%, corresponding to 91% of that of the natural tRNAAlay and 115% of that of the reconstituted product of the two natural half molecules . To the best of our knowledge, this is the first time that a natural RNA with biological activity is synthesized. Mutat Res, 1983 May, 120(2-3), 111 - 9 Combined mutagenic action of chemicals and radiation in diploid yeast; Anjaria KB et al.; The interaction between the mutagenic action of chemicals and radiation was studied by using a diploid yeast strain Saccharomyces cerevisiae BZ 34 with mitotic gene conversion as the end-point . The cells were treated with EMS, MMS or 4-NQO alone or in combination with gamma-radiation . The 2 alkylating agents EMS and MMS produced an additive mutagenic response, whereas 4-NQO exhibited an antagonistic effect in the combined treatment with gamma-radiation. Cell, 1983 May, 33(1), 211 - 9 Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo; Neff NF et al.; A DNA fragment from yeast (Saccharomyces cerevisiae) was identified by its homology to a chicken beta-tubulin cDNA and cloned . The fragment was shown to be unique in the yeast genome and to contain the gene for yeast beta-tubulin, since it can complement a benomyl-resistant conditional-lethal mutation . A smaller subfragment, when used to direct integration of a plasmid to the benomyl resistance locus in a diploid cell, disrupted one of the beta-tubulin genes and concomitantly created a recessive lethal mutation, indicating that the single beta-tubulin gene of yeast has an essential function . Determination of the nucleotide sequence reveals extensive amino acid sequence homology (more than 70%) between yeast and chicken brain beta-tubulins. Cell, 1983 May, 33(1), 195 - 202 Processing of yeast mitochondrial RNA: involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion; Weiss-Brummer B et al.; Revertants have been obtained from six mutants of the box9 cluster, which are supposed to be defective in RNA splicing as a result of alterations in a splice signal sequence . This sequence is in the 5' part of intron 4 of the cob gene, 330 to 340 bp downstream from the 5' splice site . Sequencing reveals that reversion to splicing competence is achieved by restoration of the wild-type box9 sequence; by creation of novel box9 sequences; and by introduction of a second site or suppressor mutation (sup-) compensating for the effect of the primary box9- mutation . The sup- mutation alters a sequence in intron 4,293 bp upstream from the box9- primary mutation . The box9 sequence and this upstream sequence can base pair to form an intramolecular hybrid in intron RNA in which box9- and sup- are compensatory base pair exchanges (G----A and C----U, respectively) . Thus intramolecular hybrid structures of intron RNA are essential for RNA splicing. Proc Natl Acad Sci U S A, 1983 May, 80(10), 3035 - 9 Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus; Jensen R et al.; The ultimate product of yeast mating-type interconversion is a stable a/alpha diploid cell . A haploid cell carrying the HO gene gives rise to a diploid cell in a two-step process: first, the cell switches mating type as a result of genetic rearrangement (cassette substitution) catalyzed by HO; then, cells of opposite type mate to form a/alpha diploids . Mating-type interconversion does not occur in a/alpha diploids despite the presence of the HO gene . We have identified a plasmid carrying the HO gene by screening a yeast clone bank (constructed in vector YEp13) for plasmids that allow mating-type switching by ho cells . The yeast segment responsible for mating-type interconversion integrates by homology at the ho locus, thus confirming that it carries HO . Using the HO gene as a probe, we find that strains with an active mating-type interconversion system produce HO RNA, whereas a/alpha HO/HO cells do not and that this inhibition requires products of both the MATa1 and MATa2 genes . Thus, mating-type interconversion does not occur in a/alpha HO/HO cells because the HO gene product is not synthesized . These results demonstrate the following: (i) The mating-type locus, proposed on genetic grounds to be a regulatory locus, controls expression of an unlinked gene (HO) at the level of RNA production . (ii) The HO gene is under negative feedback control: its expression is inhibited after successful completion of diploidization (formation of a/alpha diploids). Cancer Lett, 1983 May, 19(1), 33 - 9 Augmentation of antitumor activities in hamster macrophages by yeast cell walls treated with lymphokines in vitro; Mashiba H et al.; The effect of the treatment of yeast cell walls (YCW) with PPD-induced lymphokines (LK) was studied . The rate of macrophage spreading increased 1 h and 24 h after incubation with YCW treated with 16-fold or 64-fold diluted LK . Phagocytosis was slightly enhanced after 1 h incubation . Cytostatic activity was obtained when incubated with YCW treated with 16-fold or 64-fold diluted LK, but not with LK of higher or lower dilution . Tumor-inhibitory effect was observed when lymphoma cells were mixed with LK-treated YCW and inoculated s.c. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2704 - 8 Identification of AAS genes and their regulatory role in general control of amino acid biosynthesis in yeast; Penn MD et al.; In yeast, most amino acid biosynthetic pathways are coregulated: starvation for a single amino acid results in derepression of enzyme activities for many different biosynthetic pathways . This phenomenon is referred to as "general control of amino acid biosynthesis." In this paper we describe the isolation and characterization of 43 amino acid analog-sensitive (aas-) mutants that are perturbed in this general regulatory system . These 43 mutations define four unlinked complementation groups, AAS101, AAS102, AAS103, and AAS104, two of which identify previously unreported genes involved in general control . These aas mutants are unable to derepress a number of amino acid biosynthetic genes, resulting in increased sensitivity to amino acid analogs, reduced growth rates, and reduced enzyme activity levels under amino acid starvation conditions . Thus, the AAS+ gene products function as positive regulatory elements for this system . We show that the AAS genes mediate these effects by regulating the mRNA levels of genes under their control. J Bacteriol, 1983 May, 154(2), 1002 - 4 Cloning of genes that complement yeast hexokinase and glucokinase mutants; Walsh RB et al.; Genes complementing the glucose-negative fructose-negative Saccharomyces cerevisiae triple mutant strain (hxkl hxk2 glk1), which lacks hexokinase PI, hexokinase PII, and glucokinase, were obtained from a pool of yeast DNA in the multicopy plasmid YEp13. Vopr Med Khim, 1983 May-Jun, 29(3), 11 - 4 {Cloning of the SV40 virus genome simultaneously with yeast vector YEp 13}; Granovskii NN et al.; Recombinant plasmides were constructed, which contained all the genome of virus SV 40 and were able to replicate in bacterial and yeast cells . The plasmides were dissimilar in the orientation of the virus SV 40 genome towards the vector YEp 13 . Reversed correlation was found between molecular weight and the transformating activity of plasmides for yeast cells. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2432 - 6 RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA; Elder RT et al.; The RNA homologous to the yeast transposable element Ty1 is one of the more abundant poly(A)+ RNAs in many strains of the yeast Saccharomyces cerevisiae . The 5' and 3' ends of Ty1 RNA have been determined from analysis of cDNA . The 5' end is 245 bases into the left delta sequence measured from the left side of the Ty1 element . The delta sequence is a direct repeat of about 340 base pairs present at each end of the Ty1 element . The Ty1 transcription includes 93-97 bases of the left delta sequence and continues through the entire internal portion of the element and through about 295 bases of the right delta sequence before reaching the 3' end located 38-46 bases from the right side of the right delta sequence . Because the delta sequences present at each end of a single Ty1 element have identical or very similar DNA sequences, these end points for Ty1 RNA raise several questions about the expression of Ty1 elements . First, what are the initiation and termination signals, because the Ty1 transcript must read through a DNA sequence that is identical to the 3' end at about 50 bases from the 5' end? Second, why is the direction of transcription of the Ty1 element opposite to that of genes that are overexpressed after the insertion of a Ty1 element? Third, because the Ty1 RNA itself has direct repeats of about 45 bases, a structure analogous to retrovirus RNAs, is the Ty1 RNA an intermediate in the transposition of Ty1? J Biol Chem, 1983 Apr 25, 258(8), 5291 - 9 Homologous nucleotide sequences at the 5' termini of messenger RNAs synthesized from the yeast enolase and glyceraldehyde-3-phosphate dehydrogenase gene families . The primary structure of a third yeast glyceraldehyde-3-phosphate dehydrogenase gene; Holland JP et al.; Genomic DNA containing a third yeast glyceraldehyde-3-phosphate dehydrogenase structural gene has been isolated on a bacterial plasmid designated pgap11 . The complete nucleotide sequence of this structural gene was determined . The gene contains no intervening sequences, codon usage is highly biased, and the nucleotide sequence of the coding portion of this gene is 90% homologous to the other two glyceraldehyde-3-phosphate dehydrogenase genes (Holland, J . P., and Holland, M . J . (1980) J . Biol . Chem . 255, 2596-2605) . Based on the extent of nucleotide sequence divergence among the three glyceraldehyde-3-phosphate dehydrogenase genes, it is likely that they arose as a consequence of two duplication events and the gene contained on the hybrid plasmid designated pgap11 is a product of the first duplication event . All three structural genes share extensive nucleotide sequence homology in the 5'-noncoding regions adjacent to the three respective translational initiation codons . The gene contained on pgap11 is not homologous to the others downstream from the respective translational termination codon, however . The 5' termini of messenger RNAs synthesized from the three glyceraldehyde-3-phosphate dehydrogenase and two yeast enolase genes have been mapped to sites ranging from 36 to 82 nucleotides upstream from the respective translational initiation codons . In each case the 5' terminus of the mRNA maps to a region of strong nucleotide sequence homology which is shared by all five structural genes . These latter data confirm that all five structural genes are expressed during vegetative cell growth and further support the hypothesis that a portion of the 5'-noncoding flanking region of the yeast glyceraldehyde-3-phosphate dehydrogenase and enolase genes evolved from a common precursor sequence. J Biol Chem, 1983 Apr 25, 258(8), 4930 - 6 Association equilibria and reacting enzyme gel filtration of yeast hexokinase; Furman TC et al.; The association-dissociation of yeast hexokinase has been re-examined and the size of the reacting form of the enzyme has been investigated under a variety of conditions . Both Sephacryl S-200 and high performance liquid chromatography on Bio-Sil have been employed with continuous effluent monitoring under reacting and nonreacting conditions . The reacting enzyme was monomeric under all conditions, suggesting that the dimer is not an important catalytic species in normal assays . The reacting enzyme remained as a monomer under the following conditions: 0.1-15 micrograms/ml loading concentration, from 30 to 100 mM ionic strength, with 2 mM citrate, with 50% D2O, and at 160 atm hydrostatic pressure . The dissociation constant for the nonreacting hexokinase was 22 (+/- 2) micrograms/ml (uncorrected for 5-fold dilution) in 100 mM triethanolamine, pH 6.5, and 25 degrees C . Glucose or MgATP had dissociative effects under all conditions studied, but MgATP was much less effective and only slightly more effective than an equivalent ionic strength . NaCl, between 30 and 80 mM, promoted dissociation, with a concomitant conformational change suggested by nonlinearity of log-log plots . The extent of dissociation with MgCl2 was slightly greater than an equivalent NaCl ionic strength and the shape of the association curves suggested the formation of an elongated dimer in the presence of MgCl2 . The conclusion is made that hexokinase is monomeric under most assay conditions and that the dissociation is predominantly the result of the glucose interaction . High performance liquid chromatography has been shown to be a useful method of assessing the association state of enzymes under both reacting and nonreacting conditions. J Biol Chem, 1983 Apr 25, 258(8), 4944 - 9 Import of proteins into mitochondria . Isolated yeast mitochondria and a solubilized matrix protease correctly process cytochrome c oxidase subunit V precursor at the NH2 terminus; Cerletti N et al.; The cytoplasmically made subunit V was isolated from enzymically active yeast cytochrome c oxidase and its NH2-terminal amino acid sequence was determined to be (formula; see text) In order to exclude that this NH2 terminus had been generated by proteolysis during the lengthy isolation of the subunit, subunit V was directly immunoprecipitated from yeast cells that had been pulse-labeled with {35S}methionine; radiochemical sequencing revealed methionine at position 12, in agreement with the sequence given above . When the precursor to subunit V was synthesized in vitro in the presence of either {35S}methionine, {3H}leucine, or {3H}histidine and then incubated either with isolated yeast mitochondria or the partially purified matrix protease (Bohni, P . C., Daum, G., and Schatz, G . (1983) J . Biol . Chem . 258, 4937-4943), it was converted to a polypeptide co-migrating with mature subunit V on dodecyl sulfate-polyacrylamide gels . Radiochemical sequence analysis of the processed in vitro product showed that it contained histidine, leucine, and methionine in positions 4, 6, and 12, respectively, exactly as the authentic mature protein . In contrast, the unprocessed precursor contained methionine only at position 9, but not at position 12; thus, the precursor has a NH2 terminus different from the mature polypeptide . Similarly, if the in vitro synthesized cytochrome b2 precursor is incubated with isolated mitochondria, it is converted to a polypeptide which co-migrates with mature cytochrome b2 and, like the latter, contains leucine and methionine in positions 4 and 6, respectively . These data show that isolated yeast mitochondria convert the precursors to polypeptides which have the NH2 terminus of the authentic mature polypeptides . In the case of cytochrome c oxidase subunit V, correct NH2-terminal processing was also demonstrated with the purified matrix protease. J Biol Chem, 1983 Apr 25, 258(8), 4668 - 71 In vitro initiation and termination of ribosomal RNA transcription in isolated yeast nuclei; Lohr D et al.; Using the Hg-agarose affinity chromatography/gamma-sulfhydryl nucleotide technique of Reeve, et al . (Reeve, A., Smith, M., Pigiet, V., and Huang, R . (1977) Biochemistry 10, 4464-4469) and high resolution electrotransfer of DNA electrophoretograms to diazobenzyloxymethyl paper, we have analyzed the transcription from the 5 S and 35 S ribosomal RNA genes in isolated yeast nuclei . In vitro initiation from these complex gene loci exhibits the same fidelities as in vivo with respect to initiating nucleotide, polymerase activity, and initiating and terminating region of DNA template . The efficiency of the experimental approach is enhanced by the use of RNase-deficient yeast strains . Thus, this system can be used to study structural aspects affecting transcription at these loci. J Biol Chem, 1983 Apr 25, 258(8), 4687 - 9 Transport of the precursor to neurospora ATPase subunit 9 into yeast mitochondria . Implications on the diversity of the transport mechanism; Schmidt B et al.; Isolated yeast mitochondria were able to take up Neurospora ATPase subunit 9 in vitro although the homologous yeast protein is synthesized within the mitochondria and inserted into the membrane from the matrix side (Tzagoloff, A., and Meagher, P . (1972) J . Biol . Chem . 247, 594-603) . The transfer of the protein was dependent on an energized mitochondrial inner membrane . It was accompanied by proteolytic processing of the precursor to the mature protein with the correct NH2 terminus as determined by Edman degradation of the transferred protein . The possibility is discussed that there are common features in the uptake machinery neither specific for one species nor specific for individual precursor proteins in the same species. Nature, 1983 Apr 21, 302(5910), 681 - 7 The yeast tRNATyr gene intron is essential for correct modification of its tRNA product; Johnson PF et al.; Precise deletion of the intervening sequence of a yeast tRNATyr ochre suppressor gene (SUP6) significantly reduced its suppressor activity relative to that of the unaltered gene . This is probably the result of the absence of the pseudouridine modification, normally present at the middle anticodon position of mature suppressor tRNA, in tRNA synthesized in vivo from the deleted gene. Exp Cell Res, 1983 Apr 15, 145(1), 209 - 17 The timing of the S phase and other nuclear events in yeast meiosis; Williamson DH et al.; The length of the premeiotic S phase in individual cells of a homothallic strain of Saccharomyces cerevisiae was determined by pulse-labelling synchronously sporulating cultures with {3H}adenine and following changes in the frequency of cells in S by DNA-specific whole cell autoradiography . The average S phase was found to be at least 2-3 times as long as in mitotic diploids, and it was concluded that activation of replication origins in meiosis is considerably staggered . The timing of S in relation to other meiotic milestones was established by counting different nuclear morphologies visualised by DAPI-fluorescent staining . This allowed tentative identification of pachytene nuclei as well as first and second nuclear divisions. Biochim Biophys Acta, 1983 Apr 13, 751(2), 201 - 9 2-hydroxyethylhydrazine as a potent inhibitor of phospholipid methylation in yeast; Nikawa J et al.; The effect of 2-hydroxyethylhydrazine on the phosphatidylethanolamine methylation pathway in yeast was studied . 2-Hydroxyethylhydrazine inhibited the growth of cells . The concentration required for 50% inhibition was 66 microM . The growth rate decreased by 2-hydroxyethylhydrazine was restored by the addition of a low concentration of choline . Incorporation of radioactivity from L-{3-14C}serine, L-{methyl-14C}methionine and S-adenosyl-L-{methyl-14C}methionine into phosphatidylcholine was markedly reduced by 2-hydroxyethylhydrazine . The restoration of growth by choline was not due to the reversal of the inhibition, but to the formation of phosphatidylcholine via the CDPcholine pathway . Thus, the site of action of 2-hydroxyethylhydrazine in vivo was the phosphatidylethanolamine methylation pathway . Experiments with methylation mutants indicated that all three steps of methylation were sensitive to 2-hydroxyethylhydrazine . 2-Hydroxyethylhydrazine was shown to inhibit the methyltransferase after it had become chemically or metabolically transformed in cells . 2-Hydroxyethylhydrazine-resistant mutants were obtained and were found to have a defect in choline transport activity . Genetic data indicated that the uptake of 2-hydroxyethylhydrazine into cells is mediated by the choline transport system. J Biol Chem, 1983 Apr 10, 258(7), 4356 - 63 Control of the transfer of oxidizing equivalents between heme iron and free radical site in yeast cytochrome c peroxidase; Ho PS et al.; A procedure has been developed for obtaining yeast cytochrome c peroxidase with the heme iron in the Fe(IV) state, without the concomitant formation of the protein free radical that occurs in the ES compound resulting from the oxidation of ferric peroxidase with hydrogen peroxide . In this procedure, ferrous peroxidase, prepared either by photochemical reduction or by trapping the dithionite-reduced enzyme with carbon monoxide, is oxidized with a stoichiometric amount of hydrogen peroxide . The resulting Fe(IV) enzyme oxidizes ferrocyanide monophasically, with a rate constant of 4 x 10(3) M-1 S-1 . The optical spectrum of the free radical was obtained as the difference between the spectra of the ES and Fe(IV) compounds . EPR spectra of ES compound prepared with {16O}-and {17O}hydrogen peroxide are identical, demonstrating that no fragment of the oxidant is associated with the free radical . The heme in the Fe(IV) enzyme is stable and does not oxidize the free radical site either intra- or intermolecularly . On the other hand, previous results from the presteady state kinetics of reduction and reductive titrations of the ES compound with ferrocyanide imply that the heme and free radical sites exchange oxidizing equivalents, in particular that the radical site once reduced can be reoxidized, either intra- or intermolecularly, by the ferryl heme . To resolve these contradictions, we propose a catalytic mechanism for cytochrome c peroxidase in which the radical site can exist in two conformations having very different reduction potentials and in which a significant flow of oxidizing equivalents between heme and free radical sites occurs only (i) during the hydrogen peroxide oxidation of the resting Fe(III) enzyme to form compound ES and (ii) within the initial transient intermediate formed upon the one-electron reduction of this oxidized product. Br Poult Sci, 1983 Apr, 24(2), 159 - 68 Calcium and phosphorus utilisation in chickens fed on diets high in N-alkane-grown yeast; Oguntona T et al.; Interactions between calcium (Ca) and phosphorus (P) were studied in young chickens fed on diets high in n-alkane-grown yeast and in chicks fed on control soya-fishmeal diets for 14 d . Additions of inorganic Ca to diets containing 300 g yeast/kg caused increases in body-weight gain, gain:food ratio and bone mineralisation up to a total dietary concentration of 13.9 g Ca/kg . At all additions of Ca, bone mineralisation was inferior in yeast-fed chicks compared with control chicks . Supplementation of high Ca diets (16.8 g Ca/kg) with inorganic P led to further improvements in body-weight gain, food intake and food utilisation of chicks fed on high-yeast diets . Bone mineralisation also improved but was always inferior in the yeast-fed chicks compared with control chicks . It was concluded that Ca and P supplementation was necessary in high-yeast diets due to low dietary Ca concentrations and low availability. Mutat Res, 1983 Apr, 109(1), 31 - 40 Yeast mutants affecting the spontaneous mutation frequency on both the mitochondrial and nuclear genomes; Johnston LH et al.; We have previously described two complementation groups of mutants affecting the spontaneous mutation frequency on the mitochondrial genome (Johnston, 1979) . In a further search for such mutants the majority isolated fell into one of these groups, 3 into group I and 14 into group II . There are now a total of 12 alleles in the first group and 19 in the second complementation group, suggesting that these are the major classes of mutants affecting spontaneous mutation frequency in the mitochondrion . However, this search also identified a third complementation group, consisting of 2 mutants, which, like the existing groups, is recessive and is coded on the nuclear genome . In contrast to complementation groups I and II, these new mutants have no effect on spontaneous petite mutagenesis and they also increase the spontaneous mutation frequency on the nuclear genome . This nuclear mutation activity may be novel, as it complemented the existing nuclear mutators mut1-mut10 . None of the three complementation groups has any detectable phenotype, other than the mutator activity. Biochem J, 1983 Apr 1, 211(1), 199 - 218 The complete amino acid sequence of yeast phosphoglycerate kinase; Perkins RE et al.; The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined . The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene . The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes . Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis . Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure . The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371) . Both these regions contribute to the nucleoside phosphate-binding site . A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes . The yeast has strikingly fewer methionine, cysteine and tryptophan residues. Pediatr Res, 1983 Apr, 17(4), 296 - 300 Yeast opsonisation and complement in children with liver disease . Analysis of 69 cases; Larcher VF et al.; Opsonisation of heat-killed bakers yeast and C3 and C4 concentrations were determined in sera from 69 children with liver disease and 39 controls as simple screening procedures for abnormalities in the complement system . Where significant defects in these moieties were encountered, the activity of the following was measured: total haemolytic complement, C4, C5, total alternative pathway, factor B and D activities were measured . Complement activation was detected by the presence of C3d in EDTA plasma samples . Yeast opsonisation, C3 and C4 concentrations and activities of all complement components measured were significantly (P less than 0.001) reduced in all of 10 children with fulminant hepatic failure (FHF) and six with chronic active hepatitis (CAH) . There was no consistent relationship between complement deficiency and standard tests of liver function . C3 breakdown products were identified in plasma from five patients with CAH and complement deficiency but not in three patients with complement deficiencies associated with FHF . Complement concentration and activity improved in children recovering from FHF and in CAH treated by immunosuppressants . Yeast opsonisation index was raised significantly without parallel increase in C3 or C4 concentration in eight of nine children with biliary atresia before surgery and 11 of 13 with alpha-1-antitrypsin deficiency regardless of age or the presence of active liver disease . Yeast opsonisation indices became normal after successful surgery in patients with biliary atresia. Chem Biol Interact, 1983 Apr-May, 44(1-2), 27 - 39 DNA alkylation by mustard gas in yeast strains of different repair capacity; Kircher M et al.; Reaction of the toxic and mutagenic alkylating agent mustard gas with DNA of the yeast Saccharomyces cerevisiae was analyzed qualitatively and quantitatively . Within the dose range tested (2 X 10(-5)-2 X 10(-3) M) DNA in vivo is alkylated dose-proportionally . DNA alkylation and relative distribution of purine derivatives are not influenced by the cell's sensitivity towards the mutagen . At LD37 (4.4 X 10(-4) M) the wild type contains 44 300 purine derivatives: 9200 3-alkyladenines (20%), 29600 7-alkylguanines (67%) and 5500 diguaninyl derivates (13%) per genome . In sensitive strains the number of derivates per genome at LD37 is reduced according to the dose reduction factor . Alkylation at the position O6 of guanine by mustard gas cannot be shown, the method's limit of detection being 0.3% amongst purine derivates. Can J Biochem Cell Biol, 1983 Apr, 61(4), 171 - 7 Influence of temperature and growth phase on desaturase activity of the mesophilic yeast Candida lipolytica; Ferrante G et al.; Microsomal membranes prepared from Candida lipolytica cells grown at 10 degrees C had a higher lipid content and degree of unsaturation than membranes prepared from cells grown at 25 degrees C . The specific activities of stearoyl-CoA (18:0-CoA), oleoyl-CoA (18:1-CoA), and dioleoyl phosphatidylcholine (18:1-PC) desaturases in microsomes of cells grown at either 25 or 10 degrees C showed maximum values near midlog phase, coinciding with the respective maximum absolute content of linoleic (18:2) in the microsomal preparations . The 18:1-CoA desaturase activity in 10 degrees C cells was nearly double that in 25 degrees C cells, while the 18:0-CoA and 18:1-PC desaturases had considerably lower activities in 10 degrees C cells . An increase in aeration rate (shaking speed, 70 to 130 rpm) resulted in increased proportions of 18:2 (32 to 47%, respectively) in microsomes of cells grown at 25 degrees C and in increased 18:1-CoA desaturase specific activity (83 to 140 pmol X min-1 X mg-1); however, no significant changes occurred in 18:0-CoA or 18:1-PC desaturase activities. Biochim Biophys Acta, 1983 Mar 30, 743(3), 343 - 50 Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast; Wingender-Drissen R et al.; Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts . In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase . The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP . Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase . Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1 . The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor . The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration . The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml) . Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5 . Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM). Biochim Biophys Acta, 1983 Mar 30, 743(3), 451 - 4 Phenylalanyl-tRNA synthetases from yeast cytoplasm and mitochondria . The presence of a carbohydrate moiety in the mitochondrial enzyme and immunological evidence for structural relationship; Gabius HJ et al.; Homogeneous yeast cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) are analysed for structural differences . Only the large subunit of the mitochondrial enzyme is a glycoprotein with nearly 3% carbohydrate by weight . The carbohydrates present are: glucose, N-acetylglucosamine, mannose, galactose and N-acetylneuraminic acid . Removal of the sugar moieties yields an activity increase, but no significant change of sensitivity to proteolytic degradation . Antibodies to both homogeneous enzymes demonstrate a structural similarity for both types of subunit using the highly sensitive immunoblotting technique. Biochemistry, 1983 Mar 29, 22(7), 1709 - 14 Identification and consequences of a guanosine-15 to adenosine-15 change in the yeast mitochondrial tRNASerUCX gene; Miller DL et al.; We have characterized a mutation affecting the yeast mitochondrial tRNASerUCX . The mutation is a single nucleotide substitution located within the structural portion of the tRNASerUCX gene which causes the strain to be respiratory deficient . The substitution is a G leads to A transition located in the dihydrouridine arm . The tRNASerUCX transcripts from the mutant gene are present in the same amount and are the same size as transcripts from the wild-type gene . The mutant tRNASerUCX can be charged in vitro with mitochondrial aminoacyl-tRNA synthetase . Mitochondrial protein synthesis does occur in the mutant, but the amount of cytochrome oxidase subunit I is significantly decreased relative to other mitochondrial translation products. J Mol Biol, 1983 Mar 25, 165(1), 79 - 89 Messenger RNA for ribosomal proteins in yeast; Kim CH et al.; The concentrations and mean lifetimes of the messenger RNAs for four ribosomal proteins of the yeast, Saccharomyces cerevisiae, have been determined by the method of approach to equilibrium labeling, using cloned genes as hybridization probes . In cells growing in a minimal medium with glucose as a carbon source, there are roughly equimolar amounts of the four mRNAs, whose half-lives are very similar: 14 +/- 2 minutes, approximately 10% of a generation time . These results lead to an analysis of the economy of mRNA for ribosomal proteins that suggests that the mRNAs may be substantially underutilized. Nucleic Acids Res, 1983 Mar 25, 11(6), 1943 - 54 Nucleotide sequence of the yeast SUC2 gene for invertase; Taussig R et al.; The yeast SUC2 gene is a structural gene for both the secreted and intracellular forms of invertase . We have determined the nucleotide sequence of the coding region and the 5' and 3' flanking regions . The coding regions for the signal peptide-containing precursor to secreted invertase and for the intracellular invertase begin at different initiation codons within the SUC2 gene but share the same reading frame . The amino acid sequences predicted for the two forms of invertase from the nucleotide sequence are consistent with the properties of the purified enzymes . Potential sites for glycosylation of the secreted invertase are identified. Nucleic Acids Res, 1983 Mar 25, 11(6), 1819 - 37 Expression of the human interferon-gamma cDNA in yeast; Derynck R et al.; Several plasmids which direct the expression of human interferon-gamma (IFN-gamma) cDNA in the yeast Saccharomyces cerevisiae under the transcriptional control of the 3-phosphoglycerate kinase (PGK) promoter have been constructed . Up to 25 x 10(6) units per liter culture of biologically active IFN-gamma are synthesized in yeast transformed with these expression plasmids . The amount of IFN-gamma mRNA in yeast transformed with the IFN-gamma expression plasmids is much lower than the amount of PGK mRNA present when the PGK gene and promoter are inserted on a similar plasmid . The major and three minor sites of transcription initiation have been localized . The major starting point of the IFN-gamma mRNA is at 4 basepairs upstream of the PGK mRNA start . All transcription starts map at an ApG as does PGK mRNA . The yeast IFN-gamma mRNA terminates in a specific segment of the 3' untranslated region of the IFN-gamma cDNA . The polyadenylation site at about 182-184 nucleotides 3' of the IFN-gamma stop codon is preceded by a sequence which is very similar to a sequence proposed to be involved in transcription termination and polyadenylation (49). J Biol Chem, 1983 Mar 25, 258(6), 3539 - 42 Characterization of a cyclic nucleotide phosphodiesterase-deficient mutant in yeast; Uno I et al.; A mutation (pde1) was detected by suppressor activity on the CYR3 mutation which caused cAMP requirement for growth at 35 degrees C by the alteration of cAMP-dependent protein kinase . The pde1 mutant produced a significantly reduced level of cyclic nucleotide phosphodiesterase activity when assayed with 500 microM cAMP . Two cyclic nucleotide phosphodiesterases, I and II, were identified . Approximate molecular weights of these enzymes were 60,000 and 110,000, and the apparent Km values were 100 and 0.4 microM, respectively . The pde1 mutant was deficient in phosphodiesterase I activity . The cells carrying the pde1 mutation accumulated several times over the intracellular cAMP found in wild type cells . Phosphodiesterase I was not essential for growth of yeast cells, but controlled the intracellular cAMP levels in wild type and various mutant strains. Biochim Biophys Acta, 1983 Mar 15, 756(1), 111 - 8 X-ray diffraction study of the zinc(II) binding sites in yeast phenylalanine transfer RNA . Preferential binding of zinc to guanines in purine-purine sequences; Rubin JR et al.; An X-ray diffraction study of a zinc(II) complex of tRNAPhe from yeast reveals the present of five zinc-binding sites on the tRNA molecule . Two of the cooperatively bound Mg2+ in the native tRNA structure are replaced by Zn2+ . The remaining sites involve direct coordination of zinc to the N7 position of tRNA guanine bases, G15, G43 and HG45 . Thus, zinc displays a high specificity for binding to guanine bases in purine-purine sequences. Biochemistry, 1983 Mar 15, 22(6), 1430 - 7 Folding of yeast iso-1-AM cytochrome c; Zuniga EH et al.; We describe a specific modification of iso-1 cytochrome c which results in blocking a single free sulfhydryl group . The derivative differs from the unmodified protein by the introduction of a small, uncharged group, thus maintaining the same charge balance as the native protein . The modified protein, obtained by treatment of iso-1 cytochrome c with iodoacetamide, has an activity indistinguishable from that of the unmodified protein in the lactate dehydrogenase-cytochrome c reductase system from yeast and has the same stability toward denaturation by guanidine hydrochloride . The kinetics of fluorescence changes associated with the guanidine hydrochloride induced folding-unfolding transition for modified iso-1 cytochrome c (iso-1-AM) have been investigated throughout the transition zone by using stopped-flow mixing . The results are compared to those for the yeast isozyme, iso-2 cytochrome c . The main features of the fluorescence-detected folding kinetics are similar, as might be expected for homologous proteins; however, the limiting value of the fraction of fast refolding protein (alpha 2) below the transition zone is smaller for iso-1-AM (approximately 0.7) than for iso-2 (approximately 0.9). Biochemistry, 1983 Mar 15, 22(6), 1423 - 9 Structural intermediates in folding of yeast iso-2 cytochrome c; Nall BT; The kinetic properties of the folding reactions of iso-2 cytochrome c from Saccharomyces cerevisiae have been investigated by stopped-flow and temperature-jump methods . Three different structural probes are compared: (1) absorbance changes in the visible reflecting changes in heme environment, (2) ultraviolet absorbance changes due to the exposure of aromatic groups to solvent, and (3) tryptophan fluorescence attributable principally to the average distance between the tryptophan residue (donor) and the heme (quencher) . In addition, two probes either indicative of or correlated with function, ascorbic acid reducibility and the 695-nm absorbance band, have been used to monitor specifically the rate of formation of the native protein on refolding . The fastest phase observed (tau 3) has a measurable relative amplitude only when monitored by visible absorbance changes, suggesting that this reaction involves changes in heme environment in the absence of significant changes in the heme to tryptophan distance or in the extent to which aromatic groups are exposed to solvent . Different slow phases are observed when complete refolding is monitored by visible or ultraviolet absorbance (tau 1a) as opposed to tryptophan fluorescence (tau 1b), the fluorescence changes being complete on a time scale 4-8-fold faster than for absorbance . A mid-range kinetic phase (tau 2) is detected by all three structural probes . When ascorbic acid reducibility or 695-nm absorbance changes are used to monitor the rate of formation of the native protein, two phases are detected: tau 2 and tau 1a . Taken together these results demonstrate that kinetic phase tau 1b results in the formation of a structural intermediate in folding with fluorescence close to that of the native protein but with distinct absorbance properties. Biochemistry, 1983 Mar 15, 22(6), 1386 - 90 Assignment of imino proton spectra of yeast phenylalanine transfer ribonucleic acid; Roy S et al.; Yeast tRNAPhe has been studied by using proton NMR and nuclear Overhauser effect (NOE) with deuterium substitution . Direct NOE evidence is presented for assignment of imino resonances of 23 of 27 base pairs in this tRNA . Other indirect evidence is presented for tentative assignment of four other base pairs . Almost total assignment also has been made of the important noninternally bonded imino protons and tertiary interactions (however, G18-psi 55 remains unassigned) . The most surprising result has been identification of GC11 at -13.68 ppm; this is the first time a GC base pair has been identified so far downfield . This peak (GC11) is also identified as the resonance of the unique imino proton that exchanges in a time of more than 1 day, as previously described . These identifications of imino proton resonances made it possible to reinterpret the proton solvent exchange rate data previously published on this tRNA and understand them better . The assignments of resonances should pave the way for more detailed solution study of this tRNA and its interaction with biologically relevant molecules. J Biol Chem, 1983 Mar 10, 258(5), 3242 - 50 RNA polymerase I-dependent selective transcription of yeast ribosomal DNA . Identification of a new cellular ribosomal RNA precursor; Swanson ME et al.; Selective transcription of hybrid plasmids containing yeast rDNA was achieved with a template-dependent S100 fraction from whole cell extracts of Saccharomyces cerevisiae . A small region of the yeast rDNA which directs selective initiation in vitro was identified by subcloning . An initiation site was mapped within this region on the basis of the molecular weights of transcripts synthesized in vitro from templates which were cleaved with restriction endonucleases at a series of sites downstream from the site of initiation . The initiation site maps to a position 3.0 kilobase pairs upstream from the sequences which encode the 5' terminus of 18 S rRNA . In vitro initiation from this site is not inhibited by 50 micrograms/ml of alpha-amanitin and is completely abolished when the reactions contain 0.2 M (NH4)2SO4 . Based on these data, selective transcription of yeast rDNA in vitro is RNA polymerase I-dependent . Several S1 nuclease-resistant hybrids are formed between DNA probes labeled at restriction endonuclease sites downstream from the in vitro initiation site and high molecular weight cellular RNA . The 5' terminus of the most abundant rRNA precursor maps approximately 0.7 kilobase pair upstream from sequences which encode the 5' terminus of 18 S rRNA . This corresponds to the 5' terminus of the 35 S rRNA precursor reported by others . The 5' terminus of the largest detectable precursor synthesized in vivo corresponds closely with the initiation site identified in vitro . Based on the data presented here, RNA polymerase I traverses the interspersed 5 S rRNA gene . Since these two ribosomal genes are transcribed in opposite directions, this arrangement of the RNA polymerase I and III promoters may ensure that equivalent amounts of the two gene products are synthesized in vivo. J Biol Chem, 1983 Mar 10, 258(5), 3230 - 41 Purification of yeast RNA polymerases using heparin agarose affinity chromatography . Transcriptional properties of the purified enzymes on defined templates; Hammond CI et al.; A rapid procedure for the simultaneous purification of yeast RNA polymerases I, II, and III is described . The procedure involves direct fractionation of a yeast cell extract by heparin agarose affinity chromatography, followed by glycerol gradient centrifugation and DEAE-Sephadex chromatography . The purification can be completed in 3-4 days using 20-200 g of yeast cells . Two forms each of RNA polymerases I, II, and III are resolved after DEAE-Sephadex chromatography . In the cases of RNA polymerases I and II, these forms differ in subunit structure . The transcriptional properties of the isolated enzymes were determined using hybrid plasmid DNA templates containing yeast ribosomal and glycolytic structural genes . Both forms of RNA polymerases I and II transcribe plasmid DNA templates with low efficiency and no evidence for selective initiation of transcription was found for these enzymes using a wide variety of templates . Both forms of RNA polymerase III transcribe plasmid DNA templates with high efficiency and direct the synthesis of discrete transcripts . Sites for initiation and termination of transcription by RNA polymerase III within defined plasmid DNA templates were determined . The data show that RNA polymerase III-dependent synthesis of discrete transcripts from restriction endonuclease-digested plasmid DNA templates is initiated from selected ends of the templates and terminates at discrete sites downstream from the site of initiation . RNA polymerase III initiates synthesis at many sites within supercoiled plasmid DNA templates. J Biol Chem, 1983 Mar 10, 258(5), 2966 - 72 The zinc-containing high Km cyclic nucleotide phosphodiesterase of bakers' yeast; Londesborough J et al.; The high Km cyclic nucleotide phosphodiesterase of Saccharomyces cerevisiae was purified by an improved procedure . Its amino acid composition is reported . Its pI is 5.85 +/- 0.1 . Sedimentation equilibrium analysis of the native enzyme gave Mr = 88,000 +/- 6,000, whilst gel electrophoresis in the presence of dodecyl sulfate gave a molecular weight of 43,000, indicating that the enzyme is a dimer . Preparations of 94 +/- 4% purity contained about 2.4 atoms of zinc/43,000 daltons . Inactivation of the enzyme by 8-hydroxyquinoline was accompanied by removal of about 2 zinc atoms per monomer . Partially inactivated enzyme regained activity during dialysis against zinc, or, with less effect, cobalt salts . 8-Hydroxyquinoline (Ki = 1.1 mM) and 1,10-phenanthroline (Ki = 0.6 mM) were competitive inhibitors . The enzyme was also inhibited by the nonchelating 1,7-and 4,7-phenanthrolines and by thiols and KCN, but not by NaN3 . These inhibitors probably act by binding to, but not chelating, enzyme-bound zinc. Biochim Biophys Acta, 1983 Mar 9, 728(3), 403 - 8 Solubilization of yeast plasma membranes and mitochondria by different types of non-denaturing detergents; Navarrete R et al.; A comparative study of the solubilization of yeast plasma membranes and mitochondria by different types of non-denaturing detergents has been performed . Zwittergent-14 (3-{tetradecyldimethylammonio}-1-propanesulfonate) at low concentrations (3-4 mM) produced maximum solubilization of both membranes . However, this detergent may inactivate enzymes at high concentrations . Taurodeoxycholate (in the presence of salt) and Triton X-100 were also effective in mitochondria but not in the plasma membranes . Octylglucoside only solubilized these membranes at very high concentrations (20 mM) . CHAPS (3-{cholamidopropyldimethylammonio}-1-propanesulfonate) only achieved partial solubilization even at high concentrations . Our results suggest that Zwittergent-14 at low concentrations is one of the most powerful detergents for the general solubilization of native membrane proteins. Nature, 1983 Mar 3, 302(5903), 84 - 6 Is there left-handed DNA at the ends of yeast chromosomes? Walmsley RM, Szostak JW, Petes TD. Tracts of the alternating copolymer poly(dGdT . dCdA) have been observed in a variety of eukaryotes . Such tracts are of particular interest since homopolymers of this sequence can exist in vitro as left-handed Z form DNA . We have found that the yeast Saccharomyces cerevisiae contains at least 30 poly(GT) tracts at dispersed genomic locations . We show here that one subset of these tracts is located at the ends (telomeres) of the yeast chromosome . In addition, we show that poly(dGdT . dCdA) tracts are added to the ends of the extrachromosomal ribosomal DNA molecules of Tetrahymena when cloned in yeast . These data represent the first reported association between a homopolymeric sequence and a chromosome structure. J Human Stress, 1983 Mar, 9(1), 26 - 31 Life stress and chronic yeast infections; Williams NA et al.; This study investigated the relationships of positive and negative life change to yeast infections in women having a gynecological examination at a university health center . Subjects completed the Life Experiences Survey and a questionnaire about experiences with yeast infections and received, as a routine part of their visitation of the gynecology service, a standard gynecological examination, including a laboratory test for yeast infections . Positive life change was unrelated to any of the variables regarding yeast infections and was only minimally correlated with negative life change . Negative life change, while not related to the presence of current yeast infections, was positively correlated with the reported number of yeast infections during the past year, concern about these infections, and a number of physician visits for yeast infections . Negative life change was also negatively correlated with grade point average . Neither antibiotics nor contraceptive pill use interacted with negative life change to influence experiences with yeast infections. Z Naturforsch {C}, 1983 Mar-Apr, 38(3-4), 212 - 9 Mechanisms of inhibition by mevinolin (MK 803) of microsome-bound radish and of partially purified yeast HMG-CoA reductase (EC.1.1.1.34); Bach TJ et al.; 1) In kinetic studies, mevinolin proved to be a highly specific inhibitor of partially purified yeast HMG-CoA reductase (Ki = 3.5 nM towards HMG-CoA) and of microsomal HMG-CoA reductase from etiolated radish seedlings (Ki = 2.2 nM) . At low concentrations of NADPH, the inhibitor counteracts the sigmoidal response of plant HMG-CoA reductase activity towards the cosubstrate . At higher concentrations of NADPH, the inhibition pattern is of non-competitive type . 2) Our results are extensively compared with that obtained by the use of animal tissue and yeast as an enzyme source in order to discuss model systems probably valid to evaluate properties and regulation of plant as well as yeast HMG-CoA reductase. Orig Life, 1983 Mar, 13(1), 57 - 9 Stabilization of the yeast desaturase system by low levels of oxygen; Volkmann CM et al.; The stability of particulate palmitoyl-CoA desaturase preparations from anaerobically grown yeast cells was increased by exposure to low levels of oxygen . The stabilizing effect of oxygen may be based upon the increased amounts of palmitoleic acid and ergosterol that become available to the cells . These results suggest the evolutionary appearance of this system at a time when atmospheric oxygen was at a low level. Gene, 1983 Mar, 21(3), 193 - 202 Excision sequences in the mitochondrial genome of yeast; de Zamaroczy M et al.; We report an analysis of the sequences used in the excision of the mitochondrial genomes of 22 spontaneous and ten ethidium bromide (EtBr)-induced Saccharomyces cerevisiae petite mutants . In all cases, excision sequences were found to be perfect direct repeats, often flanked on one or both sides by regions of patchy homology . Sequences used in the excision of the genomes of spontaneous petites were always located in the AT spacers and GC clusters of intergenic regions of the genome; the GC clusters corresponded to ori and oris sequences, namely to canonical and surrogate origins of DNA replication, respectively . In the case of the ethidium bromide-induced petites, excision sequences were found not only in intergenic sequences, but also in the introns and exons of mitochondrial genes. Genetika, 1983 Mar, 19(3), 362 - 74 {Metabolic activation of promutagens in the yeast-microsome test . II . Activation of cyclophosphamide and 2-aminofluorene}; Pavlov IuI et al.; The effect of recombinogenic and mutagenic activation of promutagens, cyclophosphamide and 2-aminofluorene was studied in S9 mouse liver preparations . Cyclophosphamide was activated to induce reversions of an ochre mutation and heteroallelic reversion in yeast tester strains r2089-15V-P4 and P3288, respectively . Metabolic activation of this chemical was greatly enhanced by pretreatment of mice with AROGLOR--1254 . 2-aminofluorene was a potent recombinogen after metabolic activation, but proved a poor inducer of reversions of the ochre mutation . For the stationary yeast cells, activated 2-aminofluorene was shown to be not recombinogenic, while for logarithmic cells grown in galactose medium it was moderately recombinogenic, being highly active in this respect for logarithmic cells grown in glucose medium . We recommend to use these compounds as positive controls for exogenous activation in the yeast/microsome test. Proc Natl Acad Sci U S A, 1983 Mar, 80(6), 1496 - 500 Yeast is unable to excise foreign intervening sequences from hybrid gene transcripts; Langford C et al.; To investigate whether transcripts from foreign split genes are correctly processed in yeast cells we have constructed two hybrid genes by inserting into the split yeast actin gene an intron-containing fragment from either the Acanthamoeba actin I gene or the duck alpha D-globin gene . The hybrid genes were inserted into the autonomously replicating yeast plasmid YRp7, which was then used to transform yeast cells . It was found that the yeast but not the foreign intervening sequences were excised from the chimeric transcripts . This indicates that the recognition of intervening sequences or the splicing mechanism of RNA polymerase II transcripts is not universal. Mutat Res, 1983 Mar, 108(1-3), 121 - 31 Influence of misonidazole on survival of wild-type and radiosensitive mutants of yeast; Petin VG; Survival curves were obtained for haploid and diploid yeasts, Saccharomyces cerevisiae, of a wild-type strain and radiosensitive mutants exposed to gamma-rays in oxygenated and hypoxic conditions both in the absence and in the presence of misonidazole . Misonidazole enhanced the radiosensitivity only of hypoxic cells . A correlation between oxygen and misonidazole sensitization was observed . The data confirm that misonidazole mimics the sensitizing effect of oxygen . The degree of liquid-holding recovery of yeast cells was greater when the cells were irradiated in hypoxic conditions with than without misonidazole . Possible reasons for the observed radiation responses are discussed. Cell, 1983 Mar, 32(3), 839 - 52 Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase; Julius D et al.; Alpha factor mating pheromone is a peptide of 13 amino acids secreted by Saccharomyces cerevisiae alpha cells . Nonmating ("sterile," or ste) alpha-cell mutants bearing defects in the STE13 gene do not produce normal alpha factor, but release a collection of incompletely processed forms (alpha factor) that have a markedly reduced specific biological activity . The major alpha-factor peptides have the structures H2N-GluAlaGluAla-alpha factor and H2N-AspAlaGluAla-alpha factor . The ste13 mutants lack a membrane-bound heat-stable dipeptidyl aminopeptidase (DPAPase A) that specifically cleaves on the carboxyl side of repeating -X-Ala- sequences . Absence of DPAPase A and the other phenotypes of a ste13 lesion cosegregate in genetic crosses . The cloned STE13 gene on a plasmid causes yeast cells to overproduce DPAPase A severalfold . A different cloned DNA segment, which weakly suppresses the ste13 defects, causes overproduction of a heat-labile activity (DPAPase B) by about tenfold . Other experiments indicate that DPAPase A action may be rate-limiting for alpha-factor maturation in normal alpha cells. Cell, 1983 Mar, 32(3), 831 - 8 ARS replication during the yeast S phase; Fangman WL et al.; A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1 . Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication . A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase . The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication . The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ . These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase . If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins. Proc Natl Acad Sci U S A, 1983 Mar, 80(5), 1270 - 4 Submitochondrial localization, cell-free synthesis, and mitochondrial import of 2-isopropylmalate synthase of yeast; Hampsey DM et al.; 2-Isopropylmalate synthase (EC 4.1.3.12) of yeast is a mitochondrial enzyme . We now provide evidence showing that a large part of the 2-isopropylmalate synthase activity that is associated with the mitochondria is located in the mitochondrial matrix . In vitro translation of total yeast RNA followed by immunoprecipitation with anti-2-isopropylmalate synthase antibody yields two polypeptides . The larger of these has an apparent molecular weight identical to that of purified 2-isopropylmalate synthase subunit (ca . 65,000) . It is incorporated into isolated yeast mitochondria with no detectable change in molecular weight . The import requires energy . The smaller polypeptide migrates to a position corresponding to a molecular weight of 63,000-64,000 . It is not taken up by mitochondria . Both polypeptides, which also can be obtained by immunoprecipitation of crude extracts, become labeled when in vitro translation is performed in the presence of N-formyl{35S}methionyl-tRNAf . Mutants with no detectable 2-isopropylmalate synthase activity are deficient in either one or both synthase-related polypeptides . These results are discussed in the light of recent evidence for two 2-isopropylmalate synthase-encoding genes in yeast. Biochem Biophys Res Commun, 1983 Feb 28, 111(1), 294 - 300 Similarity of activation of yeast phosphofructokinase by AMP and fructose-2,6-bisphosphate; Nissler K et al.; Phosphofructokinase from yeast is effectively activated by AMP and fructose-2,6-bisphosphate by increasing the affinity of the enzyme to fructose-6-phosphate and the maximum activity toward this substrate . The enzyme is activated by AMP and fructose-2, 6-bisphosphate both at high and at low concentrations of ATP . The half maximum stimulation concentrations of AMP and fructose-2, 6-bisphosphate are about 200 microM and 2 microM, respectively . At saturating concentrations of AMP and fructose-2, 6-bisphosphate similar maximum activities were observed in the dependence of enzyme activity on the concentrations of fructose-6-phosphate . The fructose-6-phosphate affinity is more enhanced by fructose-2, 6-bisphosphate than by AMP. Nucleic Acids Res, 1983 Feb 25, 11(4), 1077 - 97 Yeast killer dsRNA plasmids are transcribed in vivo to produce full and partial-length plus-stranded RNAs; Bostian KA et al.; In vivo transcripts of the L (4.5 kb) and M (1.9 kb) dsRNA plasmids were examined in type I killers of Saccharomyces cerevisiae . Transcripts for both plasmids include full-length (l,m) and partial-length (la,ma) single-stranded species . Both L-dsRNA transcripts (l,la) have in vitro mRNA activity for L-P1, previously shown to be identical to ScV-P1, the 88,000 dalton major capsid protein of the virus-like particles containing L- and M1-dsRNAs . 1, but not 1a, is bound to poly(U)-sepharose and may be polyadenylated . Other L-dsRNA gene products and their transcripts may exist . For M1-dsRNA, both species (m, ma) have in vitro mRNA activity for M1-P1, the 32,000 dalton pre-protoxin encoded by M1-dsRNA . Both m and ma are bound to poly(U)-Sepharose and ma is probably a 5' terminal fragment of m . A functional model for M1-dsRNA killer plasmid structure is presented. J Biol Chem, 1983 Feb 25, 258(4), 2674 - 82 Nucleotide sequence of the yeast alcohol dehydrogenase II gene; Russell DW et al.; The complete nucleotide sequence of the glucose-repressed alcohol dehydrogenase II gene (ADR2) from yeast has been established together with its 5'- and 3'-flanking regions . The limits of the gene have been determined by reverse transcriptase sequencing of the 5'-ends and by S1 nuclease mapping of the 3'-ends of the mature ADR2 mRNA . Comparison of the alcohol dehydrogenase I gene (ADC1) sequence (Bennetzen, J . L., and Hall, B.D . (1982) J . Biol . Chem . 257, 3018-3025) with that of ADR2 indicated four regions of sequence conservation in the 5'-flanking DNAs . One of these conserved regions contains the sequence TCAAG which may function as a yeast cap sequence . The coding sequence of ADR2 is 89% homologous with that of ADC1 and exhibits a bias in its codon utilization . Evidence is presented that the intergenic region at the 3'-end of the ADR2 gene is less than 550 base pairs. J Biol Chem, 1983 Feb 25, 258(4), 2112 - 4 A novel thiol-dependent arsenite-sensitive valyl-tRNA synthetase activity from yeast; Black S; Arsenite strongly inhibits the activation by thiols of a fraction of the valyl-tRNA synthetase in yeast extracts that precipitates in low concentrations of ammonium sulfate . Once activated, however, the enzyme is insensitive to arsenite . It is suggested that arsenite blocks the function of an enzyme-bound hydrogen-transferring agent that mediates reduction of the enzyme and normally serves as part of an oxido-reduction regulatory mechanism . On gel filtration, much of the arsenite-sensitive activity behaves as a complex of about 500,000 molecular weight, whereas the behavior of the arsenite-insensitive activity is consonant with the molecular weight of 130,000 previously reported for yeast valyl-tRNA synthetase. J Biol Chem, 1983 Feb 25, 258(4), 2122 - 5 The oxidation of yeast Complex III . Evidence for a very rapid electron equilibration between cytochrome c1 and the iron-sulfur center; T'sai A et al.; The reoxidation of reduced cytochrome c1 by potassium ferricyanide follows pseudo-first order kinetics with k = 4 x 10(4) M-1 s-1 . However, the reoxidation of this cytochrome in two-electron reduced Complex III does not follow any simple rate law although the overall rate of reaction is essentially unchanged . The observed kinetics can be well fitted with a model in which ferricyanide reacts exclusively with cytochrome c1 together with very rapid electron transfer from the reduced iron-sulfur center to cytochrome c1 . Neither removal of coenzyme Q from the complex nor prior incubation with antimycin A had any effect on the observed kinetics of reoxidation. FEBS Lett, 1983 Feb 21, 152(2), 153 - 6 The primary structure of yeast mitochondrial tyrosine tRNA; Sibler AP et al.; The mitochondrial tyrosine tRNA from Saccharomyces cerevisiae has been sequenced . It has two interesting structural features: (i) it lacks two semi-invariant purine residues in the D-loop which are involved in tertiary interactions in the yeast cytoplasmic tRNAPhe; (ii) it has a large variable loop and therefore resembles procaryotic tRNAsTyr rather than eucaryotic cytoplasmic ones. Biochim Biophys Acta, 1983 Feb 17, 722(2), 349 - 63 Potentiometric studies on yeast complex III; T'sai AL et al.; Potentiometric measurements have been performed on Complex III from bakers' yeast . The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR . A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR) . The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV . An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape . The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C . Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature . The midpoint potential of cytochrome c1 was found to be pH independent . The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers . These two detergents also produced different MCD contributions of the two b cytochromes . A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III . Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species . This transition is mediated by ubiquinone. Biochemistry, 1983 Feb 15, 22(4), 762 - 8 Purification and characterization of a cysteine dioxygenase from the yeast phase of Histoplasma capsulatum; Kumar V et al.; A cysteine dioxygenase, cysteine oxidase (EC 1.13.11.20), has been purified from the cytosolic fraction of yeast phase cells of the dimorphic fungus Histoplasma capsulatum . The cysteine oxidase is an iron-containing dioxygenase with a molecular weight of 10500 (+/- 1500) and is present only in the yeast phase of the fungus . The enzyme is highly specific for L-cysteine, with a Km of 2 X 10(-5) M in vitro . The product of cysteine oxidation is cysteinesulfinic acid, as analyzed by thin-layer chromatography and mass spectroscopy . To our knowledge, this is the first cysteine oxidase isolated from a fungus, and it probably plays an important role in the mycelial to yeast phase transition of H . capsulatum during which redox potential and cysteine levels are crucial factors. Biochemistry, 1983 Feb 15, 22(4), 741 - 5 Melting order of successively longer yeast phenylalanine-accepting transfer ribonucleic acid fragments with a common 5' end; Boyle JA et al.; Temperature-jump methods were used to study the kinetics of the helix to coil transition in three fragments of yeast tRNAPhe that share a common 5' terminus (the 5' end of the mature tRNA) . Correlation of the extrapolated helix dissociation time constants with NMR exchange broadening results allows assignment of the structural basis of the optical melting transition in the fragments . The results confirm nuclear magnetic resonance findings on these fragments: the 5' 1/4 fragment has no helical structure; the 5' 1/2 fragment contains the D stem; and the 5' 3/5 fragment contains the D stem and the anticodon stem . These are the structures expected if sequential folding of the tRNA during biosynthesis were to occur . The D stem is the last helix to melt in the 5' 3/5 fragment . We suggest that structural elements in addition to the four Watson-Crick base pairs of the D-stem helix are responsible for the anomalously high Tm of that hairpin. Eur J Biochem, 1983 Feb 15, 130(3), 517 - 24 Arginyl-tRNA synthetase from brewer's yeast . Purification, properties, and steady-state mechanism; Thiebe R; tRNAArg and arginyl-tRNA synthetase have been purified to homogeneity from brewer's yeast by chromatographic methods . Arginyl-tRNA synthetase is a monomeric enzyme with a molecular weight of 72000 . Two active forms of the enzyme can be found, they are interconvertible . The more stable conformation is probably the natural one . Arginyl-tRNA synthetase seems to recognize arginine very specifically . No evidence for any proof-reading mechanism could be found . The steady-state mechanism is somewhat different from the types found with arginyl-tRNA synthetase from other sources . However, all these results are compatible with a concerted reaction . Simultaneously with the release of AMP or pyrophosphate an allosteric rearrangement occurs . This conversion seems to be determining for the reaction mechanism. Nucleic Acids Res, 1983 Feb 11, 11(3), 707 - 18 Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes; Fournier M et al.; An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described . Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII . A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase . Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions . Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U . In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified . These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide . In this case, guanosine and uridine were modified while adenosine and cytosine were not. Biochem Biophys Res Commun, 1983 Feb 10, 110(3), 884 - 9 Photoaffinity inhibition of peptide transport in yeast; Sarthou P et al.; A photoaffinity label, 4-azidobenzoyltrimethionine has been synthesized . It competitively inhibits trimethionine uptake in the yeast C . albicans . Upon UV irradiation it irreversibly and specifically blocks oligopeptide uptake . These results give the first example of photoinhibition of peptide uptake in yeast. Biochem Biophys Res Commun, 1983 Feb 10, 110(3), 945 - 50 A yeast mitochondrial inner membrane 30K hydrophobic protein: comparison with subunit 32K of the cytochrome BC 1 complex; Alkoutayni M et al.; A yeast mitochondrial inner membrane hydrophobic protein 30K has been isolated and compared to subunit 32K of the yeast cytochrome bc 1 complex . Both proteins are translated on mitochondrial ribosomes, have nearly the same molecular weight and similar aminoacid compositions . Comparison was carried out by immunological techniques with specific antibodies, and by studying 3 yeast strains having mutations in the COB region of the mitochondrial DNA . Results show that the two proteins are not identical. J Biol Chem, 1983 Feb 10, 258(3), 2022 - 6 Purification and characterization of yeast topoisomerase I; Badaracco G et al.; Yeast topoisomerase I (Mr = 76,000) has been purified to 80% homogeneity using a combination of ion exchange, gel filtration, and DNA-cellulose chromatography . The enzyme was characterized with respect to its ability to relax supercoiled DNA and to catenate nicked circular DNA . Yeast topoisomerase I will remove both positive and negative turns in DNA supercoils in the absence of ATP and magnesium ion . The products of the catenating activity of the enzyme were examined on agarose gels and in the electron microscope . These analyses indicate that yeast topoisomerase I will generate large catenated DNA networks which appear to rearrange to multimeric linear structures upon long incubation time. J Biol Chem, 1983 Feb 10, 258(3), 1444 - 9 Phosphofructokinase mutants of yeast . Biochemistry and genetics; Lobo Z et al.; Mutants of Saccharomyces cerevisiae completely lacking the soluble glycolytic enzyme fructose-6-P kinase are described . The mutations are semidominant, do not complement one another, and define a gene PFK1 located 28-cm distal to rna1 on the extended right arm of chromosome XIII . Of 10 independent mutants, 3 can be suppressed by ochre suppressors . All mutants examined synthesize proteins that cross-react to the antibody against the purified yeast P-fructokinase . The enzyme in spontaneous revertants is distinguishable from the wild type enzyme with respect to thermolability and ATP inhibition . The locus PFK1 thus defines the structural gene of the enzyme . The pfk1 mutants are not leaky in vivo . All the glucose consumed by a double mutant lacking both P-fructokinase and 6-P-gluconate dehydrogenase ends up as 6-P-gluconate, yet the pfk1 mutants can glycolyze and grow on glucose in air . The cell mass produced per unit of glucose also remains unchanged . Anaerobically, however, growth does not take place, nor does glycolysis . P-fructokinase is thus a dispensable enzyme for aerobic growth, but indispensable for anaerobic growth . The properties of pfk1 mutants suggest that yeast has an alternative mechanism for the aerobic metabolism of fructose-6-P, presumably through the recently reported particulate P-fructokinase (Lobo, Z., and Maitra, P . K . (1982) FEBS Lett . 137, 279-282). FEBS Lett, 1983 Feb 7, 152(1), 94 - 6 Reconstitution of stellacyanin as a case of direct Cu(I) transfer between yeast copper thionein and 'blue' copper apoprotein; Hartmann HJ et al.; It was of interest to examine whether yeast Cu-thionein could be used to transfer the thiolate bound copper directly into the copper binding site of 'blue' apoproteins which contain free thiol groups . In particular apo-stellacyanin was used in the present study and it was found to be able to accept Cu(I) from yeast Cu-thionein, without any detectable unspecific Cu(II) intermediate, both aerobically and anaerobically. J Biochem (Tokyo), 1983 Feb, 93(2), 661 - 4 Occurrence of acid-labile sulfide in cadmium-binding peptide 1 from fission yeast; Murasugi A et al.; Two kinds of Cd-binding peptides (Cd-BP1 and Cd-BP2) are induced in fission yeast upon addition of CdCl2 to the culture medium (l) . It was also reported that Cd-BP1 and Cd-BP2 consisted of the same components, unit peptides (Cys3, Glu3, Gly1) and Cd atoms, though the respective amounts of components in each molecule were different (1, 2) . Now, we have found that Cd-BP1 contains about 1 mol of acid-labile sulfide per mol, and Cd-BP2 contains no labile sulfide . The existence of the labile sulfide explains the unique physicochemical characteristics of Cd-BP1 . Since acid-labile sulfide has not been found in metallothioneins or other metallothionein-like metal-binding proteins, the occurrence of labile sulfide in Cd-BP1 is the first instance in this field. Infect Immun, 1983 Feb, 39(2), 497 - 504 Ingestion of yeast forms of Sporothrix schenckii by mouse peritoneal macrophages; Oda LM et al.; The ingestion by thioglycolate-elicited mouse peritoneal macrophages of yeast forms of two strains of Sporothrix schenckii was studied . Yeast forms opsonized with concanavalin A (ConA) were extensively phagocytized, and the phagocytic indexes depended on the concentration of ConA and apparently on the number of lectin receptors at the yeast surface as well . Neuraminidase treatment of S . schenckii increased the ingestion of unopsonized yeasts 7.7-fold . The addition of monosaccharides and derivatives partially inhibited phagocytosis . Mannose, rhamnose, and galactose, which are major constituents of S . schenckii surface antigens, reduced the phagocytic indexes by 40 to 50% . Glucosamine, N-acetylglucosamine, and N-acetylneuraminic acid were equally effective as inhibitors of phagocytosis . A mixture of five neutral sugars and glucosamine inhibited phagocytosis by 73% . The inhibitory effect of simple sugars could be amplified by using neuraminidase-treated yeast cells . Pentoses and glucose were inactive or slightly inhibitory . A purified rhamnomannan inhibited phagocytosis of the homologous strain, whereas partially purified peptidopolysaccharides were toxic to peritoneal macrophages . A partially purified galactomannan from S . schenckii was inhibitory (62% inhibition), and a peptidopolysaccharide fraction in which the O-linked carbohydrate chains had been removed neither was toxic to macrophages nor inhibited phagocytosis . Pretreatment of macrophages with simple sugars under conditions inhibiting ingestion or binding of S . schenckii did not affect phagocytosis of latex particles or sensitized sheep erythrocytes . The presence of receptors at the peritoneal macrophages which bind S . schenckii cell surface components is suggested. Biochemistry, 1983 Feb 1, 22(3), 675 - 81 Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation; Baltzinger M et al.; The adenylation of phenylalanine catalyzed by phenylalanyl-tRNA synthetase was investigated in the absence of tRNA, by rapid kinetic measurements using 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS) as a nonspecific fluorescent reporter group . It is shown that each protomer of the enzyme is able to catalyze independently the adenylation of phenylalanine by ATP, as well as the reversion by pyrophosphate, at least in the absence of tRNA . The kinetic rate constants of synthesis and pyrophosphorolysis are respectively found equal to 100 +/- 20 s-1 and 150 +/- 50 s-1 . The symmetric behavior of the enzyme is consistent with a symmetric binding of 2 mol of phenylalanine to the enzyme as shown by equilibrium dialysis experiments . The affinity of phenylalanyladenylate for the enzyme could be characterized by an equilibrium constant of 0.2 x 10(9) M-1. Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 712 - 5 Sterol synergism in yeast; Ramgopal M et al.; Sterol synergism as previously observed {Dahl, C.E., Dahl, J.S . & Bloch, K . (1980) Biochemistry 19, 1462-1467} and defined as a greater-than-additive growth response to pairs of sterols by Mycoplasma capricolum {Dahl, J.S., Dahl, C.E . & Bloch, K . (1981) J . Biol . Chem . 256, 87-91} is now demonstrated in the yeast mutant GL7, which is auxotrophic for sterol and unsaturated fatty acid . Mutant cells growing poorly when provided with cholesterol and oleic acid respond to ergosterol supplements (ergosterol-to-cholesterol ratio, 1:3) by a pronounced increase in growth rates and cell yields . Stigmasterol also elicits a significant synergistic effect, and 7-dehydrocholesterol, a smaller one . Evidence for a metabolic role of ergosterol in yeast membranes is presented . Cells raised on a 1:3 mixture of ergosterol to cholesterol up to midlogarithmic phase subsequently incorporate {1-14C}oleic acid at significantly faster rates into phospholipids than do cells grown on cholesterol alone. Mutat Res, 1983 Feb, 116(2), 119 - 27 Apparent absence of recombinogenic activity of nitropyrenes for yeast; McCoy EC et al.; Nitropyrenes have been shown to be potent bacterial and mammalian mutagens . However, they failed to induce any recombinogenic activity in Saccharomyces cerevisiae D4 even at elevated concentrations and following extended periods of exposure . A plausible explanation for this lack of activity is the absence or the lack of activation of the enzyme required for the activation of nitropyrenes in this test system under the experimental (aerobic) conditions employed. Eur J Biochem, 1983 Feb 1, 130(2), 281 - 6 Intramitochondrial ATP and cell functions: yeast cells depleted of intramitochondrial ATP lose the ability to grow and multiply; Gbelska Y et al.; Cells of the yeast Saccharomyces cerevisiae could be depleted of their intramitochondrial ATP bu culturing on glucose in the presence of antimycin A, which prevents production of ATP in mitochondria, along with bongkrekic acid, which prevents transport of ATP from the cytosol into mitochondria . Alternatively, the depletion could be achieved by culturing respiration-deficient mutants in the presence of bongkrekic acid . The depleted cells of the respiration-deficient mutant did not grow on glucose in a synthetic medium and growth for a few generations was made possible by adding peptone, yeast extract or some amino acids into the medium . The depleted cells did not differ from control cells in their content of amino acids, proteins, nucleic acids and major phospholipids and had preserved the ability to carry on protein and nucleic acid syntheses and to mate to other cells . No conspicuous cytological differences were found between the control and depleted cells . After culturing in a semi-synthetic medium in the presence of bongkrekic acid the cells of the respiration-deficient mutant exhibited almost no cytochrome c in their spectra and their azide-sensitive ATPase activity was drastically reduced . The results suggest that intramitochondrial syntheses of some low-molecular compounds as well as import and/or assembly of some cytoplasmically synthesized mitochondrial proteins into mitochondria may be impaired in cells lacking intramitochondrial ATP and this may be responsible for their inability to grow and multiply. Eur J Biochem, 1983 Feb 1, 130(2), 247 - 51 On the phosphorylation of yeast RNA polymerases A and B; Breant B et al.; In exponentially growing cells, RNA polymerase B is exclusively form BI enzyme with several phosphorylated subunits: B220, B23 and possibly B44.5 . In RNA polymerase A an average of fifteen phosphate groups are distributed on the five phosphorylated subunits: A190 (6), A43 (4), A34.5 (2), A23 (1-2) and A19 (1-2) . Phosphorylation of enzyme A by a yeast protein kinase in vitro adds less than 1 mol phosphate/mol enzyme but occurs essentially at the physiological sites, as shown by a comparison of the peptide patterns obtained by limited proteolysis of subunits 32P-labelled in vivo and in vitro . No evidence was found in favor of a modulation of RNA polymerase activity in vitro or in vivo via phosphorylation. Cell, 1983 Feb, 32(2), 409 - 15 Control of yeast cell type by the mating type locus: positive regulation of the alpha-specific STE3 gene by the MAT alpha 1 product; Sprague GF Jr et al.; The mating type locus (MAT) determines the three yeast cell types, a, alpha, and a/alpha . It has been proposed that alleles of this locus, MATa and MAT alpha, encode regulators that control expression of unlinked genes necessary for mating and sporulation . Specifically, the alpha 1 product of MAT alpha is proposed to be a positive regulator of alpha-specific genes . To test this view, we have assayed RNA production from the alpha-specific STE3 gene in the three cell types and in mutants defective in MAT alpha . The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants . Using the cloned STE3 gene as a probe, we find that alpha cells produce STE3 RNA, whereas a and a/alpha cells do not . Furthermore, mat alpha 1 mutants do not produce STE3 RNA, whereas mat alpha 2 mutants do . These results show that the STE3 gene, required for mating only by alpha cells, is expressed only in alpha cells . They show also that production of RNA from the STE3 gene requires that alpha 1 product of MAT alpha . Thus alpha 1 positively regulates at least one alpha-specific gene by increasing the level of that gene's RNA product. Arch Biochem Biophys, 1983 Feb 1, 220(2), 467 - 76 Purification and characterization of intact cytochrome b5 from yeast microsomes; Yoshida Y et al.; A method for purification of detergent-solubilized cytochrome b5 to gel electrophoretic homogeneity from yeast (Saccharomyces cerevisiae) microsomes is described . The purified preparation shows the same absorption spectra as the trypsin-solubilized cytochrome (Y . Yoshida, H . Kumaoka, and R . Sato J . Biochem . 75, 1211-1219 (1974)) in the visible and Soret regions . The detergent-solubilized cytochrome is an amphipathic protein having a monomeric molecular weight of about 18,000 and exists as a hexa- or heptameric aggregate (Mr 122,000) in aqueous media . In the presence of low concentrations of Triton X-100, it interacts effectively with the intact form of NADH-cytochrome b5 reductase purified either from yeast microsomes or from rabbit liver microsomes . Upon trypsin digestion, it is converted to a heme-containing, hydrophilic fragment (Mr 13,000) which retains the spectral characteristics of the original cytochrome, does not form aggregates, and interacts with the reductase only poorly . It is concluded that the preparation purified in this study represents the intact form of yeast cytochrome b5 consisting of a hydrophilic, heme-containing moiety (Mr 13,000) and a hydrophobic, membrane-binding tail (Mr 5000). J Bacteriol, 1983 Feb, 153(2), 791 - 9 Yeast mutant defective in phosphatidylcholine synthesis; Greenberg ML et al.; The Saccharomyces cerevisiae opi3-3 mutant was shown to be defective in the synthesis of phosphatidylcholine via methylation of phosphatidylethanolamine . The opi3-3 mutant was isolated on the basis of an inositol excretion phenotype and was not auxotrophic for choline . Inositol, but not choline, stimulated growth of the mutant . The opi3-3 mutation was recessive and was genetically linked to the ino4 locus . When grown in the absence of exogenous choline, the opi3-3 mutant had a phospholipid composition consisting of 2 to 3% phosphatidylcholine compared with 40 to 50% in wild-type strains . In addition, the mutant accumulated elevated amounts of two intermediates, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine . The incorporation of label from {methyl-14C}methionine into phosphatidylcholine was reduced 80 to 90% in the mutant compared with wild-type strains . However, label was recovered in the intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine . The mutant is believed to be defective in the third and possibly the second methylation reaction in the formation of phosphatidylcholine from phosphatidylethanolamine . The first methylation reaction appeared to be occurring at normal or even elevated levels . Based upon incorporation of choline into phosphatidylcholine, it is concluded that the opi3-3 mutant has no defect in the synthesis of phosphatidylcholine from exogenous choline . Furthermore, phosphatidylcholine represents over 25% of the phospholipid composition of the mutant when it is grown in the presence of exogenous choline. Cell, 1983 Feb, 32(2), 391 - 6 Unequal excision of complementary strands is involved in the generation of palindromic repetitions of rho- mitochondrial DNA in yeast; Sor F et al.; In the rho- mutants of yeast, the mitochondrial genome is made up of a small segment excised from the wild-type mitochondrial DNA . The segment is repeated either in tandem or in palindrome to form a series of multimeric DNAs . We have asked how the palindromic organization arises . From several palindromic rho- mitochondrial DNAs, we have isolated the restriction fragments that contained the head-to-head or tail-to-tail junction of the repeating units, and have determined their nucleotide sequences . We found that the palindromes were not symmetrical right up to the junction points: at the junction, there was always an asymmetrical sequence of variable length . At both ends of this junction sequence, we found inverted oligonucleotide sequences that were variable in each mutant and that were present in the wild-type DNA . At the moment of excision, a single-strand cut seems to occur at each of these short inverted repeats, in such a way that the two complementary strands of the genome are cut unequally and the single-stranded overhangs become the junction sequences between the palindromic repeating units . This scheme may account for the complex structures of many rho- mitochondrial DNAs. Cell, 1983 Feb, 32(2), 379 - 89 Two intron sequences in yeast mitochondrial COX1 gene: homology among URF-containing introns and strain-dependent variation in flanking exons; Hensgens LA et al.; The DNA sequences of two optional introns in the gene for subunit I of cytochome c oxidase in yeast mitochondrial DNA have been determined . Both contain long unassigned reading frames (URFs) . These display regions of amino acid homology with six other URFs, two of which encode proteins involved in mitochondrial RNA splicing . Such conserved regions may thus define functionally important domains of proteins involved in RNA processing . This homology also implies that these URFs had a common ancestral sequence, which has been duplicated and dispersed around the genome . Comparison of the flanking exons in the long strain KL14-4A with their unsplit counterpart in D273-10B reveals clustered sequence differences, which lead in D273-10B to codons rarely used in exons . These differences may be linked to the loss or absence of one of the optional introns. J Bacteriol, 1983 Feb, 153(2), 644 - 51 Regulation of yeast trehalase by a monocyclic, cyclic AMP-dependent phosphorylation-dephosphorylation cascade system; Ortiz CH et al.; Mutation at the GLC1 locus in Saccharomyces cerevisiae resulted in simultaneous deficiencies in glycogen and trehalose accumulation . Extracts of yeast cells containing the glc1 mutation exhibited an abnormally high trehalase activity . This elevated activity was associated with a defective cyclic AMP (cAMP)-dependent monocyclic cascade which, in normal cells, regulates trehalase activity by means of protein phosphorylation and dephosphorylation . Trehalase in extracts of normal cells was largely in a cryptic form which could be activated in vitro by ATP . Mg in the presence of cAMP . Normal extracts also exhibited a correlated cAMP-dependent protein kinase which catalyzed incorporation of label from {gamma-32P}ATP into protamine . In contrast, cAMP had little or no additional activating effect on trehalase or on protamine phosphorylation in extracts of glc1 cells . Similar, unregulated activation of cryptic trehalase was also found in glycogen-deficient strains bearing a second, independently isolated mutant allele, glc1-2 . Since trehalase activity was not directly affected by cAMP, the results indicate that the glc1 mutation results in an abnormally active protein kinase which has lost its normal dependence on cAMP . Trehalase in extracts of either normal or mutant cells underwent conversion to a cryptic form in an Mg2+-dependent, fluoride-sensitive reaction . Rates of this reversible reduction of activity were similar in extracts of mutant and normal cells . This same, unregulated protein kinase would act on glycogen synthase, maintaining it in the phosphorylated low-activity D-form . The glc1 mutants provide a novel model system for investigating the in vivo metabolic functions of a specific, cAMP-dependent protein kinase. Cell, 1983 Feb, 32(2), 525 - 36 Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease; Peebles CL et al.; Splicing of transfer RNA precursors containing intervening sequences proceeds in two distinct stages: endonucleolytic cleavage, followed by ligation . We have physically separated endonuclease and ligase activities from extracts of yeast cells, and we report properties of the partially purified endonuclease preparation . The endonuclease behaves as an integral membrane protein: it is purified from a membrane fraction from which it can be solubilized with nonionic detergents, and the activity of the endonuclease in the membrane fraction is stimulated by nonionic detergents . The endonuclease cleaves precursor tRNAs at two sites to excise the intervening sequence precisely . Both the extent and the accuracy of cleavage are enhanced by the presence of spermidine; the degree of stimulation varies with the pre-tRNA substrate . The cleavage products possess 5'-hydroxyl and 2',3'-cyclic phosphodiester termini . The cyclic phosphodiester termini can be opened to 2'-phosphates by a cyclic phosphodiesterase activity in the preparation. Nature, 1983 Jan 27, 301(5898), 296 - 301 Rearranged mitochondrial genes in the yeast nuclear genome; Farrelly F et al.; We have found a contiguous DNA sequence in the yeast nuclear genome with extensive homology to non-contiguous yeast mitochondrial DNA sequences . Closely linked to this nuclear sequence in some, but not all, yeast strains is a tandem pair of transposable (Ty) elements . Certain features of the content and organization of this nuclear DNA sequence suggest that it may have originated from petite mitochondrial DNA which integrated into the nuclear genome. Biochim Biophys Acta, 1983 Jan 26, 742(2), 419 - 25 Involvement of arginine residues in glutathione binding to yeast glyoxalase I; Schasteen CS et al.; Yeast glyoxalase I was inactivated by arginine-specific reagents . Inactivation by 2,3-butanedione, phenylglyoxal and camphorquinone 10-sulfonic acid followed pseudo first-order kinetics with the rate dependent upon modifier concentration . Extrapolation to complete inactivation showed modification of approx . two of the ten total arginyl residues in the native enzyme, with approx . one residue protected by glutathione (GSH) as determined by {ring-14C}phenylglyoxal incorporation . GSH protected the enzyme from inactivation, whereas methylglyoxal, glutathione disulfide (GSSG) and dithiothreitol afforded partial protection . The hemimercaptal of methylglyoxal and GSH and the catalytic product, S-lactoylglutathione provided substantial protection from inactivation . A methyl ester placed on the glycyl carboxyl moiety of GSH abolished all protective capability which suggests that this functionality is responsible for binding to the enzyme . These results provide the first evidence concerning the molecular binding mode of GSH to an enzyme . Arginyl residues are proposed as anionic recognition sites for glutathione on other GSH-utilizing enzymes. Nucleic Acids Res, 1983 Jan 25, 11(2), 403 - 20 Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene; Larkin JC et al.; Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59 . A recessive cryR1 allele has also been cloned, using the integration excision method . The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval . The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo . The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed. J Biol Chem, 1983 Jan 25, 258(2), 946 - 52 Pure yeast RNA polymerase B (II) initiates transcription at specific points on supercoiled yeast DNA; Lescure B; Pure yeast RNA polmymerase B (II) can selectively initiate abortive transcription on a supercoiled recombinant plasmid DNA carrying yeast DNA in the presence of low concentrations of ribonucleoside triphosphates and Mn2+ . Five major products ranging between 60 and 150 nucleotides were characterized by hybridization . Three of them originate from the vector pBR322 and two from the yeast DNA insert . Based on a RNA primer extension reaction with recombinant M13 DNAs as template, a method allowing the mapping of the short abortive RNA products has been developed . An initiation site within the yeast DNA insert has thus precisely been mapped . The DNA sequence in this region was determined and showed several relevant features . The in vitro initiation site is preceded by a potential TATATATA box at -40 base pairs and at -105 by the sequence GTTAATCT similar to the consensus sequence GCTCAATCT usually found around 80 base pairs upstream from the cap site . Large blocks of alternated purine pyrimidine residues are found in this region as for several known yeast promotors . The 5' end of the RNA initiated from this site contains several potential signals for the initiation of translation . The possibility that a B to Z transition of DNA could be important for the interaction of the RNA polymerase with its template is discussed. Biochim Biophys Acta, 1983 Jan 20, 739(1), 122 - 31 Reverse transcription of yeast double-stranded RNA and ribosomal RNA using synthetic oligonucleotide primers; Brizzard BL et al.; The ability of the four oligodeoxyribonucleotide primers oligo(dT)12-18, oligo(dA)12-18, oligo(dG)12-18 and oligo(dC)12-18 to act as primers for avian myeloblastosis virus reverse transcriptase on denatured yeast double-stranded (ds) RNA templates was investigated . Oligo(dT) and oligo(dA) were found to prime the synthesis of 1.1 and 1.0 kb reverse transcripts, respectively, using denatured M dsRNA as a template . The oligo(dT)- and oligo(dA)-primed cDNAs of M dsRNA hybridized to the region of the M dsRNA that encoded the killer toxin and to each other . Addition of oligo(dT) to reverse transcription reactions of denatured L dsRNA produced a 4.3 kb cDNA . During the course of this investigation oligo(dC) was observed to be a highly efficient primer for reverse transcription of yeast 18 S ribosomal RNA . Oligo(dC) primed the synthesis of a 1.0 kb transcript of 18 S rRNA which hybridized to the large Eco RI fragment of the 18 S rRNA gene . Reverse transcription of double-stranded RNA and 25 S ribosomal RNA was found to occur to some extent in the absence of added oligonucleotide primer. Nature, 1983 Jan 13, 301(5896), 167 - 9 Evolutionary divergence of the mRNA transcription initiation mechanism in yeast; Russell PR; The promoters of eukaryotic genes are being increasingly defined through the identification of consensus DNA sequences, by mutational analysis, and by in vitro and in vivo studies of transcription . Whereas the TATA sequence (Goldberg-Hogness box) has been largely conserved among protein encoding genes (transcribed by RNA polymerase II) of eukaryotes, there is some evidence that other structural and functional determinants of mRNA transcription are not conserved between species . I report there an in vivo comparative analysis of the transcription initiation systems of the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharmyces pombe (which can both be transformed by identical plasmids) . I have found no instance in which a gene is transcribed in the same fashion in both yeasts . Instead, I have found that the in vivo transcription starting points for many different yeast genes are determined by the cell in which it is transcribed rather than its gene structure alone . The evidence also suggests that the divergence of the transcription initiation system may partly involve the mechanism or structure which determines the distance from the TATA consensus sequence to the site of transcription initiation. Biochim Biophys Acta, 1983 Jan 12, 742(1), 1 - 8 Kinetics of yeast cytochrome c peroxidase compound I formation with modified substrates (peroxybenzoic acids); Frew JE et al.; Th