Yeasts

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.; The kinetics of formation of Compound I of yeast cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) with a series of peroxybenzoic acids were studied . Reactivity is affected not only by protein ionization, as in the reaction with H2O2, but also by substrate ionization . The reactivity of negatively charged substrates is markedly lower than that of uncharged species, implying that electrostatic factors profoundly influence substrate binding . The rate constants for neutral peroxybenzoic acids carrying electron-withdrawing substituents increase with increasing peroxy acid pKa . This behaviour suggests that, as previously discussed for reactions of turnip peroxidases, formation of peroxy anion by ionization of substrate within the active site is kinetically important . The results support the mechanism of cytochrome c peroxidase Compound I formation which has been proposed by Poulos and Kraut (J . Biol . Chem . 225 (1980), 8199-8205) on the basis of enzyme structural studies.

Biochemistry, 1983 Jan 4, 22(1), 130 - 6
Role of mono- and divalent metal cations in the catalysis by yeast aldolase; Kadonaga JT et al.; The rate of deuterium exchange between {1-(S)-2H}dihydroxyacetone 3-phosphate and the solvent catalyzed by native and metal-substituted yeast aldolases has been measured . In the presence of 0.1 M potassium acetate at 15 degrees C, pH 7.3, the deuterium exchange reaction catalyzed by native yeast aldolase has a kcat of 95 s-1 . In contrast to the 7-fold activity enhancement by 0.1 M potassium ion (relative to 0.1 M sodium ion) of the cleavage of D-fructose 1,6-bisphosphate catalyzed by native yeast aldolase, a negligible (1.1-fold) activation by 0.1 M potassium ion is observed in the rate of dedeuteration of {1(S)-2H}dihydroxyacetone 3-phosphate . The order of reactivity of the yeast metalloaldolases in the deuterium exchange roughly parallels that seen in the fructose bisphosphate cleavage reaction . These findings suggest that the carbonyl groups of enzyme-bound D-fructose 1,6-bisphosphate and dihydroxyacetone phosphate are both polarized by the active site divalent metal cation . A mechanistic formulation consistent with the results of this and the previous paper is presented.

Biochemistry, 1983 Jan 4, 22(1), 122 - 9
Polarization of substrate carbonyl groups by yeast aldolase: investigation by Fourier transform infrared spectroscopy; Belasco JG et al.; The infrared spectrum of the complex of D-fructose 1,6-bisphosphate bound to yeast aldolase displays three spectral features between 1700 and 1800 cm-1 . One of these (at 1730 cm-1) corresponds to the carbonyl group of enzyme-bound D-fructose 1,6-bisphosphate and/or dihydroxyacetone phosphate . The frequency of this band, which is unaffected by the removal of the intrinsic zinc ion from the enzyme, demonstrates that this carbonyl group is not significantly polarized when the substrate binds to the enzyme . In contrast, the spectral band assigned to the carbonyl group of enzyme-bound D-glyceraldehyde 3-phosphate (at 1706 cm-1) appears at a frequency 24 cm-1 lower than when this substrate is in aqueous solution . This shift indicates considerable polarization of the carbonyl group when D-glyceraldehyde 3-phosphate is bound at the active site . The third spectral feature (at 1748 cm-1), which is observed only in the presence of potassium ion, probably corresponds to an enzymic carboxyl group in a nonpolar environment.

Z Allg Mikrobiol, 1983, 23(3), 147 - 57
Genetic studies on the yeast Saccharomycopsis lipolytica . Inactivation and mutagenesis; Barth G et al.; Spontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized . Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica . The frequencies of such mutants were mainly arranged between 1 X 10(-7) and 5 X 10(-6) mutants per cell . But one class of glucosamine resistant mutants (GAMRA) occurred more frequently . Among the resistant mutants different types of dominant and recessive resistant mutants could be observed . UV light was used for inactivation of cells and induction of mutants from S . lipolytica . Comparing four haploid strains only small differences were detected in sensitivity to UV light . UV light at a dosage of 135 J/m2 was applied to increase the mutant frequencies in three haploid strains . Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, allylalcohol, glucosamine, 2-deoxyglucose or nystatin resistant mutants, hitherto not described for S . lipolytica, were isolated and partially characterized.

Mikrobiologiia, 1983 Jan-Feb, 52(1), 64 - 7
{Transport and accumulation of biotin by cells of various fungal and yeast strains}; Naumova ES et al.; Transport of 14C-biotin was studied in cells of different biotin-prototrophous yeast and mold fungal strains . An inverse correlation was established between the capability of the fungi to synthesize biotin and the exogenous vitamin transport: 14C-biotin did not penetrate into the cells of strains which excreted great quantities of the vitamin . It is likely that a higher level of biotin synthesis in certain fungi is caused by a peculiar transport system, which results in a one-way permeability of their cell membrane for biotin . Biotin is eliminated from the cell and cannot repress its own synthesis . Active transport of biotin in the studied prototrophous organisms occurs against a concentration gradient and does not depend on the presence of glucose in the medium . There are apparently other energy sources for this process.

Nucleic Acids Symp Ser, 1983, (12), 133 - 6
Thermal and Mg2+ dependent behavior of pseudouridines at 39th and 55th of yeast tRNAPhe; Nagamatsu K et al.; Pseudouridine psi 55 alone and both psi 55 and psi 39 in yeast tRNAPhe are selectively modified with fluorescent reagent of 4-bromomethyl-7-methoxycoumarin (BMC) . The change of fluorescence intensity was measured as a function of temperature and Mg2+ concentration . Fluorescent quenching shows the stacked and unstacked forms of Y base, dependent on Mg2+ concentration . In contrast, Mg2+ had no effect on psi 55-BMC in T psi C loop at 20 degrees C . Fluorescence on titrating Mg2+ exhibited a kind of Mg2+-induced structural collapse at the corner of L-structure . The melting of psi 55-BMC takes place at 70 degrees C in 10mM Mg2+ . At very low Mg2+ concentration, melting takes place at 35 degrees C . The melting of psi 39-BMC, located near the anticodon loop, was observed before the unfolding of the whole structure of tRNAPhe . A conformational transition of the anticodon loop takes place at a lower temperature and it is also expected in the quenching experiment of Y base.

Experientia Suppl, 1983, 46, 209 - 12
Transport of nutrients in yeast protoplasts; Kotyk A; Protoplasts were prepared with snail-gut juice from Saccharomyces cerevisiae and Candida utilis . Transport of D-xylose (A), trehalose (B), 2-deoxy-D-glucose (C), L-leucine (D), L-proline (E), inorganic phosphate (F) and H+ ions (G) was studied with special emphasis on the first species . Transport of A which is not sensitive to glucose stimulation in its synthesis was not affected by protoplast formation . Similarly unaffected were transports of B, D, E and F in their "residual" form in starved cells . However, transport of these substances after glucose-stimulated synthesis was practically fully suppressed by protoplast formation . This may be connected with the virtually complete inhibition of the proton pump (G) in protoplasts with the implication that B, D, E and F are transported in conjunction with protons but only those that are immediately produced by the proton pump (glucose consumption, oxygen utilization and membrane potential were not substantially altered in protoplasts) . Transport of C was stimulated nearly two-fold in protoplasts . Uranyl ions (0.1 mM) had a pronouncedly lower inhibitory effect on all the transports studied in protoplasts.

Acta Biochim Pol, 1983, 30(3-4), 345 - 53
Yeast ribosome core particles deficient in acidic proteins L44 and L45 and their activity in reconstitution experiments; Palen E et al.; The yeast ribosome core particles partly depleted of acidic proteins, L44 and L45, were isolated and their activity was examined using a highly purified protein synthesizing system . It was shown that both the phenylalanine polymerization reaction and the elongation factor 2-dependent GTP hydrolysis were stimulated by the addition of the extracted acidic protein fraction . The results obtained clearly indicate a functional role of these proteins in the elongation step of eukaryotic protein synthesis . The essential parameters of ribosome reconstitution experiments are discussed.

EMBO J, 1983, 2(12), 2169 - 72
Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane; Hase T et al.; The nucleotide sequence of the yeast chromosomal gene coding for the 70-kd protein of the mitochondrial outer membrane was determined . The deduced amino acid sequence of the protein agrees with the experimentally determined size and amino acid composition of the purified protein and correctly predicts the fragments obtained by cleaving the protein at its single tryptophan residue . The deduced NH2-terminal sequence features an uninterrupted stretch of 28 uncharged amino acids flanked on both sides by basic amino acids . By sequencing a truncated version of the gene it was found that the corresponding polypeptide product lacks the 203 carboxy-terminal amino acids of the authentic 70-kd protein . As shown in the accompanying paper, this protein fragment still becomes attached to the mitochondrial outer membrane in vivo.

Z Allg Mikrobiol, 1983, 23(8), 513 - 5
Development of breeding stocks of the yeast Saccharomycopsis lipolytica by methods of moderate inbreeding; Kurischko C et al.; Sporulation parameters of genetically labelled strains, derived from a wild strain of the alkane-utilizing yeast Saccharomycopsis lipolytica were improved by a breeding program using brother-sister crosses . Sporulation frequency, the number of four-spored asci and viability of ascospores could be significantly enhanced . To date a number of genetically well-defined strains is available that have good sporulation parameters and show a 1:1 segregation pattern of markers suitable for genetic analysis.

Mol Gen Genet, 1983, 192(3), 361 - 5
Stability of a cloned gene in yeast grown in chemostat culture; Walmsley RM et al.; A study has been made of the stability of LEU2, a cloned chromosomal gene of Saccharomyces cerevisiae, when reintroduced into yeast on a number of plasmid vectors which permit a chromosomal or episomal location for the gene in either high or low copy number . Glucose-limited continuous culture was employed to ensure that there was no selection for the inserted gene . Both the rate of segregation of plasmid minus cells and the effect of the plasmid on host growth rate were found to determine plasmid stability which, in many cases, could be predicted by simple mathematical models . The presence or absence of the endogenous 2 mu plasmid of yeast was found to have an important influence on the stability of 2 mu-based vectors . This led to the discovery that, for the host strain used, the presence of 2 mu sequences represented a selective advantage for the cells.

Mol Gen Genet, 1983, 192(1-2), 101 - 3
The rna2 mutation of yeast affects the processing of actin mRNA as well as ribosomal protein mRNAs; Teem JL et al.; The temperature sensitive rna2 mutation of Saccharomyces cerevisiae causes a rapid and dramatic decrease in the abundance of most ribosomal protein mRNAs We and others have recently shown that the processing of ribosomal protein mRNAs is defective at the nonpermissive temperature, suggesting that inefficient mRNA processing might be responsible for the decline in ribosomal protein mRNA levels . Actin is the only known intron-containing non-ribosomal protein yeast nuclear gene We show here that the processing of actin mRNA is also defective at the nonpermissive temperature in rna2-containing strains . The observation supports the notion that all intron-containing genes are affected in a similar fashion by the rna2 mutation.

EMBO J, 1983, 2(11), 2047 - 52
Determination of functional domains in intron bI1 of yeast mitochondrial RNA by studies of mitochondrial mutations and a nuclear suppressor; Schmelzer C et al.; The sequence of intron 1 in the cob gene in mtDNA (bI1) of the yeast strain 777-3A has been determined . Furthermore, we have performed a systematic search for complementary sequence stretches within this intron RNA, and within the RNA of intron 5 gamma of the oxi3 gene (aI5 gamma) which shares distinctive sequences with bI1 . Possible secondary structure models derived from this analysis show nearly identical core structures for bI1 and aI5 gamma RNA with conserved sequence stretches in prominent positions . These core structures are similar to those previously reported for RNAs of introns having very limited sequence homology with bI1 and aI5 gamma . In two mutants which are defective in bI1 excision from cob pre-mRNA, nucleotide sequence alterations in bI1 have been determined . One mutation (G5049) apparently affects the stability of a hybrid stretch in the proposed secondary structure of bI1 RNA whereas the other one (M1301), a deletion of one A in a run of five As, affects a sequence which is conserved in bI1 and aI5 gamma and is involved in the formation of a distinct secondary structure . Out of seven revertants of M1301, three were found to have restored the wild-type bI1 sequence AAAAA, three others had the related sequence AAAAG which is functionally indistinguishable from wild-type, whereas one revertant had a nuclear mutation which suppresses the splicing defect exerted by the mitochondrial mutation M1301 . This nuclear suppressor (SUP-101) is allele specific and dominant . The possible role of the sequence affected by M1301 in terms of a recognition site for a nuclear gene product will be discussed.

Mol Gen Genet, 1983, 191(3), 512 - 8
Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis . Deletion mapping and restriction mapping studies; Underbrink-Lyon K et al.; Yeast mitochondrial DNA codes for a complete set of tRNAs . Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves . Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus . Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain . A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs . By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map . Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.

Mol Gen Genet, 1983, 191(3), 434 - 41
Transcription initiation in eukaryotes: analysis of heterologous in vitro systems utilizing components from mammalian and yeast cells; Bitter GA; The properties of heterologous in vitro transcription systems utilizing components from mammalian and yeast cells have been investigated . Purified yeast RNA polymerase II, when supplemented with a full complement of mammalian transcription factors, does not promote specific transcription initiation on cloned mammalian class II genes . Similarly, a complete mammalian transcription system does not initiate specific transcription on cloned yeast class II genes . These results indicate evolutionary divergence in function of yeast and mammalian class II genes and the associated transcription apparatus . The functional differences observed in this study are corroborated by previously reported structural differences between yeast and mammalian RNA polymerase II and class II genes . In contrast, the mechanism of eukaryotic class III gene transcription appears to be evolutionarily conserved . Thus, a mammalian transcription extract specifically transcribes the cloned yeast 5S rRNA gene . This system synthesizes without processing the 130 base 5S rRNA precursor, and this primary transcript may be processed to the mature 120 base RNA using a partially purified yeast processing activity . An homologous yeast class III transcription system has also been developed . This yeast system contains all components necessary for proper synthesis, processing and splicing of yeast tRNA precursors . Using the homologous yeast transcription system, a template in which the 5' flanking region and first 5 base pairs of the 5S rRNA gene had been deleted is utilized to synthesize a 120 base transcript, but the efficiency of transcription is reduced to about 10% that of the wild type gene . Thus, the yeast 5S rRNA gene has an internal transcription control region, but the immediate 5' flanking sequence have effects on the level of transcription.

Mol Gen Genet, 1983, 191(3), 366 - 71
Identification and physical characterization of yeast maltase structural genes; Chow T et al.; Each of at least five unlinked MAL loci (MAL1 through MAL4 and MAL6) on the yeast genome controls the ability to synthesize an inducible alpha-D-glucosidase (maltase) . A subcloned fragment of the coding sequence of the MAL6 maltase structural gene was used as a hybridization probe to investigate the physical structure of the family of MAL structural genes in the genomes of different Saccharomyces strains . MAL+ strains, each carrying a genetically defined MAL locus, were crossed with a MAL- strain and the segregation behavior of the functional locus and of sequences complementary to the maltase structural gene at that locus analyzed . The maltase structural gene sequences of each MaL locus were detected by Southern blot hybridization using BamH1 digests of genomic DNA of the meiotic products . This restriction enzyme was previously shown to cleave outside the confines of the MAL 6 locus . The results of such experiments indicate that each MAL locus encompasses at least one maltase structural gene sequence homologous to that of MAL6, that yeast strains that lack functional MAL loci may or may not contain the corresponding maltase structural gene sequence, that the MAL1 maltase structural gene sequence or one of its alleles can be detected in all laboratory yeast strains examined and that each MAL locus can be identified as a characteristic BamH1 fragment of genomic DNA which includes a maltase structural gene . Yeast strains vary in the number of maltase structural gene sequences that they carry . By using the approach described in this report, the ones corresponding to the different functional MAL loci and residing within a BamH1 generated restriction fragment can be identified.

Biomed Biochim Acta, 1983, 42(4), 343 - 9
A rapid procedure for the preparation of highly purified pyruvate decarboxylase from brewer's yeast; Sieber M et al.; A rapid purification procedure for pyruvate decarboxylase (E.C . 4.1.1.1.) from fresh cells of brewer's yeast (Saccharomyces carlsb.) is reported . The preparation of a crude enzyme (30-45 U/mg) by the use of fractionation steps with protamine sulfate, acetone, and ammonium sulfate takes about 6-7 h . A stable pyruvate decarboxylase (70-85 U/mg) was obtained from such preparations after purification on CM Sephadex C 50 after another 2-3 h . Stability and structural properties are compared for enzymes prepared from fresh and dried yeast.

Comp Biochem Physiol B, 1983, 75(4), 693 - 8
Purification and properties of enolase from carp (Cyprinus carpio) . Comparison with enolases from mammals' muscles and yeast; Pietkiewicz J et al.; The enolase (2-phospho-D-glycerate hydrolyase E.C . 4.2.1.11) from carp muscle was obtained, the specific activity--88 U/mg of protein . Km for 2-phosphoglycerate was 0.313 mM and for phosphoenolpyruvate--0.76 mM . The enzyme is active only in the presence of divalent metal ions, Mg2+ being the best activator . The phosphate and fluoride decreased the activity of enzyme . The molecular weight of the dimeric form of enzyme was found to be 93,000 . The enzyme is immunologically different from pig muscle enolase.

Arch Immunol Ther Exp (Warsz), 1983, 31(1), 27 - 32
Immunosuppressive properties of yeast extract; Wojskowicz J et al.; Autolysate of yeast extract exhibits immunosuppressive properties . It inhibits phytohemagglutinin stimulated incorporation of 13C thymidine to rabbit lymphocytes . When administered to mice after immunization with sheep erythrocytes, it suppresses the appearance in the spleen of IgM antibody-producing cells (PFC) . It also prolongs the survival time of skin allotransplants in mouse if given before and after transplantation.

Adv Exp Med Biol, 1983, 163, 75 - 83
Chemical synthesis of folylpolyglutamates, their reduction to tetrahydro derivatives, and their activity with yeast C1-THF synthase; Rabinowitz JC; Pteroyldi- through -pentaglutamic acids were synthesized through the use of the carbodiimide method, rather than the anhydride method or solid phase synthesis . The compounds were converted to the tetrahydro derivatives by enzymatic reduction . The Km values of the folate coenzymes for yeast C1-THF synthase were determined . The determination of the values for the formyltetrahydrofolate synthetase activity of the multifunctional enzyme was possible through the use of newly devised assay based on the fluorescence properties of the pteridine derivatives . The value for the tetraglutamyl coenzyme derivative was approximately 1000-fold lower than that of the monoglutamyl coenzyme, tetrahydrofolate.

Mol Gen Genet, 1983, 191(1), 165 - 6
Two-spored asci produced by interrupted sporulation: a novel approach to linkage analysis in yeast; Srivastava PK et al.; Genetical analysis of two-spored asci formed by interrupted sporulation offers a novel procedure for mapping of centromere-linked genes in Saccharomyces cerevisiae . Unlike the two-spored asci encountered under normal sporulation conditions, these asci are produced by a nonrandom mechanism . They fall into three categories (+ +), (+ -) and (- -) with respect to any marker . The percentage of (+ -) asci varies directly as a function of centromere-linkage of a gene . It is observed that almost 100% asci are of the (+ -) type in case of very tightly linked genes like trp-1 and cdc-10, while in case of markers unlinked to the centromere, e.g, trp-5 and met-8, the (+ -) asci constitute 50% of the total number of asci . Other markers with varying degrees of linkage, e.g . ura-3 and lys-1 show corresponding numbers of (+ -) asci between 50% and 100% of the total asci . These findings are in contrast to the results expected from a random abortion of two spores, in which case the (+ -) asci would constitute 67% of the total number of asci irrespective of the degree of centromere linkage of a marker . The linkage-dependent segregation of markers in these new kind of two-spored asci permits a rapid and accurate estimate of centromere linkage of a gene.

J Cell Sci, 1983 Jan, 59, 183 - 201
Rate of cell cycle initiation of yeast cells when cell size is not a rate-determining factor; Lord PG et al.; The control of cell proliferation under steady-state conditions in the budding yeast, Saccharomyces cerevisiae, is well described by either the tandem or sloppy size control models, both of which suggest that differences in cycle time between individual cells or between parents and daughters is largely due to differences in birth size . These models have been investigated further under conditions in which cell size has not been a rate-determining factor for cell cycle initiation . Two approaches have been used . The first involves the growth of cells in low concentrations of hydroxyurea (HU), which has the effect of prolonging the duration of DNA synthesis . This leads to a lengthening of the budded period, which in turn leads to daughter cells being larger at division than the normal cell cycle initiation size of daughters in steady-state populations . The second approach involves the accumulation of cells at the key control point of the cycle, called start, using the pheromone alpha-factor . Since growth is unaffected, all cells eventually become larger than the volume at which they would normally initiate the cell cycle . The kinetics of proliferation were followed after release from alpha-factor arrest . The results from both approaches were broadly consistent with the predictions of both models . However, abolition of birth-size differences between parents and daughters in the presence of HU did not lead to a complete disappearance of differences in either cycle time or proliferation kinetics . Furthermore, following release from alpha-factor arrest, the rate of cell cycle initiation of parent cells was slower than in steady-state culture and the daughters' cells behaved as if comprising two separate populations . These discrepancies suggest that besides a size difference, there are additional physiological differences between parent and daughter cells.

Mol Cell Biochem, 1983, 51(2), 161 - 4
A comparative study of bovine, porcine and yeast superoxide dismutases; Marmocchi F et al.; The Cu,Zn superoxide dismutases from bovine and porcine erythrocytes and from yeast have been investigated with the aim to identify structural differences in relation to possible functional variability in this highly homologous class of protein . The isoelectric points of the bovine, porcine and yeast proteins were found to be 4.8, 5.8 and 4.5 respectively . According to these values the net protein charge, as evaluated by gel electrophoresis, varied more significantly for the porcine protein than for the other two proteins tested . The catalytic constants were found to be higher at pH = 7.6 than at pH 10.0 for all the three enzymes . This relative increase was much more pronounced in the case of the porcine enzyme . The KM value at pH = 10.0 was also significantly higher for the porcine enzyme . Since the spectroscopic properties of the active sites were identical for the three proteins, these results point to modulation effects by positively charged amino acid residues on the superoxide dismutase activity of these proteins, in a way that the resultant net charge of the protein seems to be as important as specific residues.

Mol Cell Biochem, 1983, 51(2), 123 - 7
NADPH/NADP+ ratio: regulatory implications in yeast glyoxylic acid cycle; Satrustegui J et al.; The utilization by yeast of two carbon sources is carried out through the operation of the glyoxylic acid cycle . Kinetic acid from the isocitrate transforming enzymes suggest that the flow of isocitrate through the glyoxylic acid cycle depends upon the inhibition of the isocitrate decarboxylating enzymes . Both isocitrate dehydrogenases are inhibited by a mixture of glyoxylate + oxaloacetate, but for the reasons described in the text we consider that this inhibition is of no physiological significance . On the other hand, we have found that NADPH is a competitive inhibitor of NADP-isocitrate dehydrogenase with respect to NADP+, with a KI similar to its KM . It also produces an additive effect on the NADH-produced inhibition of NAD-isocitrate dehydrogenase . We propose NADPH as the compound that channels the utilization of isocitrate into the glyoxylic acid cycle . This is supported by the finding of an increased NADPH/NADP+ ratio in acetate grown yeast with respect to glucose grown cells.

Mol Gen Genet, 1983, 189(1), 148 - 56
Participation of transcriptional and post-transcriptional regulatory mechanisms in the control of arginine metabolism in yeast; Messenguy F et al.; In yeast, as in other organisms, amino acid biosynthetic pathways share a common regulatory control . The manifestation of this control is that derepression of the enzymes belonging to several amino acid biosynthetic pathways follows amino acid starvation or tRNA discharging . The arginine anabolic and catabolic pathways are, in addition, regulated specifically by arginine in opposite ways by common regulators . We have measured the mRNA levels for four genes subject to the general amino acid control: HIS4, ARG3, ARG4 and CPAII and compared them to the corresponding enzyme levels . Similarly we have measured the mRNA levels for two genes subject to the arginine specific regulation: ARG3 and CAR1, the former gene belongs to the arginine anabolic pathway and the latter to the arginine catabolic one . HIS4, ARG4 and CPAII enzyme and messenger amounts are perfectly coordinated in all the conditions of general repression or derepression tested . However, arginine does not reduce the level of the ARG3 mRNA enough to explain the reduction of ornithine carbamoyltransferase activity nor does it increase the level of the CAR1 mRNA enough to explain the extent of induction of arginase . Coordination of enzyme and ARG3 mRNA is achieved only when the specific control is eliminated . The half-lives of the ARG3 and CAR1 messengers are enhanced in mutants leading to constitutive expression of ornithine carbamoyltransferase and arginase . These data suggest that the control that coordinates the synthesis of all the amino acids in the yeast cell operates at the level of transcription while the arginine specific regulatory mechanism seems to operate at a post-transcriptional level.

J Gen Microbiol, 1983 Jan, 129 (Pt 2), 63 - 73
Photosensitivity in thiopyronine-resistant yeast mutants; Anderson JM et al.; In yeast, thiopyronine (TP) acts both as a growth inhibitor (in the dark) and a photodynamic sensitizer (in the light) . We have isolated a large number of TP-resistant mutants of Saccharomyces cerevisiae and characterized them by genetic and photobiological techniques . Resistance to the growth-inhibitory (dark) effects of TP can arise by mutations in at least four separate genes . Growth-inhibitory and photodynamic effects of TP can be genetically separated, as evidenced by the finding that resistance mutants affected in three of the genes were not altered in their photodynamic response to the dye . In contrast, a mutation in the fourth gene simultaneously conferred high resistance to both the growth-inhibitory and photosensitizing effects of TP, suggesting that a mutation had occurred at a step common to the expression of both effects . Preliminary data suggest that this mutation reduces the ability of the cells either to take up, or bind, TP.

Cell, 1983 Jan, 32(1), 89 - 98
A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast; Donahue TF et al.; We have made deletions of the HIS4 5' noncoding region in vitro and inserted these deletions into the yeast genome by transformation . Deletions that extend from -588 to -235 have no detectable effects on either promoter or regulatory functions . Deletions that extend to -138 affect promoter function, but are still regulated by the general control of amino acid biosynthesis . A deletion that extends to -136 cannot derepress HIS4 mRNA in response to the general control . This deletion removes all copies of the sequence 5'-TGACTC-3', which appears at positions -194, -182 and -138 in strains without the deletion . The importance of at least one copy of this repeat for regulation of HIS4 is shown by the reappearance of this sequence in revertants of the -136 deletion that have regained the regulatory response . The fact that deletion of this sequence leads to the inability to derepress suggests that HIS4 is under positive control.

Cell, 1983 Jan, 32(1), 67 - 76
Mutation in the a block of the yeast tRNAleu3 gene that allows transcription but abolishes splicing and 5'-end maturation; Mattoccia E et al.; A three-base substitution mutant of the yeast tRNALeu3 has been constructed . The mutation, introduced through the use of a heptadecanucleotide as a site-specific mutagen, is localized in the anterior portion of the promoter and results in the inability to form a D stem . The mutant is active in transcription, but maturation of the 5' terminus and splicing are abolished . The results are discussed in the light of a recently proposed model for initiation of transcription of eucaryotic tRNA genes.

Proc Natl Acad Sci U S A, 1983 Jan, 80(1), 228 - 32
Instability of dicentric plasmids in yeast; Mann C et al.; Dicentric plasmids containing either two copies of centromere 4 or one copy of centromere 4 and one copy of centromere 3 in the yeast plasmid vector YRp17 were constructed in vitro and introduced into yeast cells by DNA transformation . The resulting colonies were heterogeneous for a mixed population of rearranged plasmids . The rearrangements always involved deletion of one or both centromere sequences originally present on the plasmid . Heterogeneity was due to the continued production of deleted plasmids from a pool of unrearranged dicentric plasmids maintained within some of the yeast cells in the colony . The RAD52 gene product is known to be required for the repair of DNA double-strand breaks in yeast . Transformation of rad52 mutant yeast cells with dicentric plasmids gave rearranged plasmids similar to those observed with RAD+ yeast cells, but the transformation frequency was only 5-10% compared to transformation with monocentric plasmids . Also, the ratio of unrearranged dicentric plasmid to deleted plasmids was greatly reduced in the rad52-transformed cells . These observations are consistent with a model in which centromeric DNA sequences can interact independently with the yeast cell spindle apparatus . Occasional movement of centromeres to opposite poles may result in mechanical breakage of plasmid sequences . Plasmids deleted for one or both centromere sequences can be obtained from these broken molecules and are resistant to further rearrangement.

EMBO J, 1983, 2(10), 1765 - 70
Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast . Discoordinate synthesis of the 11-kd subunit in response to increased gene copy number; Van Loon AP et al.; In wild-type yeast cells, steady-state concentrations of subunits of the ubiquinol-cytochrome c reductase complex (complex III) and the levels of their translatable mRNAs change coordinately in response to the need for mitochondrial function . Despite this, re-introduction of the cloned gene for one of the subunits (11 kd) into cells by transformation with a free-replicating plasmid results in the discoordinate synthesis of this subunit only, without effects on either the synthesis or degradation of the other subunits . The overproduced subunit is associated with the mitochondrial fraction, yet does not interfere with mitochondrial function, as judged by the growth of transformed cells on nonfermentable media . Quantitative analysis of both mRNA and protein levels suggests that both translational controls and elevated turnover of excess protein contribute to a partial compensation for the effects of increased gene dosage in transformed cells . These contain approximately 30 copies of the cloned gene and 15-30 times the normal level of its mRNA . Nevertheless, synthesis of the 11-kd protein is only 6- to 8-fold higher than normal, and steady-state levels are increased only 5- to 10-fold . These findings imply that synthesis of the various subunits of complex III is not tightly coupled and that for the 11-kd subunit at least, the level of mRNA is likely to be the most important means of regulating protein level . Fine-tuning may be additionally achieved by control of translation and degradation of excess protein which is not assembled in the complex.

Methods Enzymol, 1983, 101, 202 - 11
One-step gene disruption in yeast; Rothstein RJ; The one-step gene disruption techniques described here are versatile in that a disruption can be made simply by the appropriate cloning experiment . The resultant chromosomal insertion is nonreverting and contains a genetically linked marker . Detailed knowledge of the restriction map of a fragment is not necessary . It is even possible to "probe" a fragment that is unmapped for genetic functions by constructing a series of insertions and testing each one for its phenotype.

Acta Biochim Pol, 1983, 30(2), 165 - 73
Phosphoprotein phosphatase from yeast and its ribosomal substrate; Palen E et al.; Phosphoprotein phosphatase was isolated from yeast postribosomal supernatant and partly characterized . The enzyme preferentially dephosphorylated phospho-casein and acidic ribosomal proteins L44 and L45, the eukaryotic analogues of bacterial proteins L7 and L12 . The evidence suggests that this enzyme is not a catalytic subunit of the multifunctional phosphoprotein phosphatase present in most eukaryotic organisms.

Anal Biochem, 1983 Jan, 128(1), 47 - 53
A rapid procedure for the isolation of yeast mitochondrial DNA suitable for restriction fragment analysis; Hwang-Lee L et al.; A method for the rapid isolation of mitochondrial DNA from the yeast Saccharomyces cerevisiae is described . Cells are first disrupted by vortexing with glass beads and the mitochondrial DNA is then extracted directly from the cell lysate by poly-L-lysine-kieselguhr-exchange chromatography . The method is unique from most other published procedures in that there is no requirement for the isolation of either a crude or purified mitochondrial preparation . Mitochondrial DNA isolated by this procedure is shown to yield restriction endonuclease fragment patterns identical to those obtained from DNA isolated by other previously reported procedures.

J Biochem (Tokyo), 1983 Jan, 93(1), 189 - 96
pH-Induced conformational change of ATPase inhibitor from yeast mitochondria . A proton magnetic resonance study; Fujii S et al.; The ATPase inhibitor from yeast, Saccharomyces cerevisiae, was found to strongly inhibit the ATPase activity when it was preincubated in an acidic pH region . This paper describes the conformational changes of the inhibitor in the acidic pH region, as studied by 1H NMR spectroscopy . In the pH range from 7.43 to 4.48, two conformations were detected . With lowering of the pH in this pH region, one conformation increased at the expense of the other . In the two conformations, His 39 had different pKa values which were determined at 23 degrees C to be 6.08 +/- 0.04 and 5.91 +/- 0.03 from the pH-titration curves of His 39 . The exchange between the two conformations was reversible with pH and the rate of the conformational change was estimated to be slower than 22 s-1 at 23 degrees C as estimated by the line-widths (7.0 Hz) of the peaks from the C2-proton of His 39 in the two conformations . The equilibrium constant between the two conformations was dependent on the temperature . From the equilibrium constants in the temperature range from 23 to 45 degrees C, the apparent delta H'a of the conformational change was calculated to be 14 kcal/mol at pH 6.95 . It was confirmed by 1H NMR spectra that at pH 6.95 the structure of the inhibitor was reversible with temperature at least below 80 degrees C . The reversibility is consistent with the high thermal stability of the protein.

Biokhimiia, 1983 Jan, 48(1), 3 - 10
{Reverse electron transfer in mitochondria of the yeast Endomyces magnusii grown on sucrose}; Zviagil'skaia RA et al.; Some specific features of cytosol-mitochondria interactions during the growth of the Endomyces magnusii yeast on sucrose media were investigated . Under the given experimental conditions exogenous (cytoplasmic) NADH is the main source of reducing equivalents . Using low temperature EPR spectroscopy, it was demonstrated that exogenous NADH can effectively induce reverse electron transfer in the respiratory chain of the yeast . Fluorimetric assays suggest that End . magnusii mitochondria can utilize the reducing equivalents formed by reverse electron transfer for the reductive amination of alpha-ketoglutarate to form glutamate . The intramitochondrial localization of aminotransferases was postulated.

Eur J Biochem, 1983 Jan 1, 129(3), 653 - 61
Is cytochrome b really the antimycin-binding component of the cytochrome b--c1 complex of yeast mitochondria; Chevillotte-Brivet P et al.; 1 . The antimycin binding sites were found to be independent of cytochrome(s) b synthesis in pseudo-wild-type revertants of cytochrome b mutants . These revertants, whose primary mutation is located in the introns of the cob-box gene of mitochondrial DNA, have modified contents of cytochromes b-562 and b-565 and fully functional respiratory chain . 2 . Missense mutations in three genetic loci allocated to the exons of the cytochrome b gene, abolish the strong affinity binding of antimycin . 3 . It is proposed that the antimycin-binding component is not cytochrome b itself, but interacts with it in such a way that an alteration of the cytochrome b structure affects the antimycin-binding site.

Cell, 1983 Jan, 32(1), 77 - 87
Analysis of a yeast nuclear gene involved in the maturation of mitochondrial pre-messenger RNA of the cytochrome oxidase subunit I; Faye G et al.; We have analyzed the mitochondrial RNA of a yeast nuclear pet mutant with no cytochrome oxidase activity . The product of the gene affected in this mutant appears to be necessary for the correct maturation of the mitochondrial pre-mRNA of the cytochrome oxidase subunit I . It does not affect, however, the overall splicing of cytochrome b pre-mRNA or the intron excision of the 21S ribosomal RNA precursor . This gene has been isolated by genetic complementation in yeast, and its DNA sequence has been determined . It is transcribed, as detected by S1 mapping experiments, and could encode a protein of 436 amino acids.

Proc Natl Acad Sci U S A, 1983 Jan, 80(1), 151 - 5
Modulator sequences mediate oxygen regulation of CYC1 and a neighboring gene in yeast; Lowry CV et al.; Three transcripts from Saccharomyces cerevisiae--CYC1 mRNA (transcribed from the iso-I cytochrome c gene) and two RNAs of unknown function, designated tr-1 and tr-2-were identified by reverse Southern blot analysis and found to be regulated in response to oxygen . CYC1 mRNA and tr-1 accumulation occurred only in the presence of oxygen while tr-2 appeared only under anaerobic conditions . tr-2 was transcribed from a region approximately 1 kilobase 5' from the CYC1 coding sequence and in the opposite direction . tr-1 showed homology to the same region as tr-2 but was transcribed from elsewhere in the genome . Expression of tr-2 and CYC1 was observed to be normal in cells transformed with centromeric plasmids carrying the two genes . Mutant transforming plasmids were constructed in which a 400-base-pair region between tr-2 and CYC1 was either deleted or inverted . The deletion led to low-level nearly unregulated expression of both the CYC1 and tr-2 genes, suggesting that sequences upstream from both genes are important for their expression and regulation . The inversion mutation produced a reversed pattern of CYC1 regulation in which the mRNA was present in anaerobically grown cells but absent in the presence of oxygen, mimicking wild-type tr-2 regulation and suggesting that the CYC1 transcription unit is under the control of the translocated tr-2 modulator sequences . Models for the function of these modulators are discussed.

Biomed Biochim Acta, 1983, 42(9), 1067 - 77
Modification of yeast phosphofructokinase with pyridoxal 5'-phosphate; Reuter R et al.; Modification of yeast phosphofructokinase (E.C . 2.7.1.11) with pyridoxal 5'-phosphate leads to a decreased enzyme activity . ATP at higher concentrations protects the enzyme against inactivation, while fructose 6-phosphate has no effect . At relatively low concentrations of pyridoxal phosphate the inhibition of activity by ATP was decreased or even abolished . AMP still activates the modified enzyme pointing to a separate binding site in the regulatory centre . The dissociation of the phosphopyridoxyl enzyme in products with apparent sedimentation coefficients of about 4-6 S gives evidence that lysine might be involved in the stabilization of the quaternary structure of yeast phosphofructokinase . The incorporation of pyridoxal phosphate in the 17 S form of yeast phosphofructokinase as obtained by a partial proteolysis does not show differences from the 20 S, proteolytically unmodified enzyme.

Acta Microbiol Pol, 1983, 32(1), 73 - 85
Taxonomic study of four yeast strains assimilating methanol; Nowakowska-Waszczuk A et al.; The new isolated yeasts were very good producers of biomass from methanol . Their taxonomic studies were based on classical classification, GC content of DNA, proton magnetic resonance spectrum of the cell wall mannans and mannan and glucan and chitin contents in the cell walls . The isolates could not be identified with any species described in literature . Considering their special features and some relation to the known species, the isolated yeasts were classified as follows: C-16 as Candida bimundalis var . chlamydospora, C-4 as Candida melinii var . melibiosica, D-3 as Candida silvicola var . melibiosica and M-1 as Torulopsis candida var . nitratophila.

Comp Biochem Physiol A, 1983, 75(4), 609 - 13
Influence of Hansenula anomala yeast intake on the liver and kidney metabolism of the trout (Salmo gairdneri); Sanchez-Muniz FJ et al.; The nutritional and metabolic effects of the use of Hansenula anomala yeast as the only protein source were studied in the trout . Food intake, weight and length increments significantly decreased in trout fed on yeast diet . Hepatosomatic index did not suffer any change . Soluble protein content in liver and kidney rose significantly . Liver alkaline phosphatase activity increased significantly . Liver lactate dehydrogenase (LDH) activity did not change in the experimental conditions . Glutamic-oxalacetic transaminase (GOT) activity appeared as the most affected parameter in both organs, increasing significantly in those trout fed on yeast diet.

Bioorg Khim, 1983 Jan, 9(1), 43 - 51
{Chemico-enzymatic synthesis of the structural gene of yeast valine tRNA}; Berlin IuA et al.; Structural gene coding for the yeast tRNA Val1 has been synthesized and cloned in the pBR322 DNA.

J Biol Chem, 1982 Dec 25, 257(24), 14657 - 66
Glycoprotein synthesis in yeast . Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing; Byrd JC et al.; Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with {2-3H}mannose and gel filtration analysis of the products released by endoglycosidase H . Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled . The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides . No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure . In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and {14C}Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and {14C}Man8GlcNAc2 . Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis . Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue . The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R . E., Zhang, W.-j., and Ballou, C . E . (1982) J . Biol . Chem . 257, 5730-5737) . It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins . The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).

J Biol Chem, 1982 Dec 25, 257(24), 14745 - 52
Energy transduction by the reconstituted b-c1 complex from yeast mitochondria . Inhibitory effects of dicyclohexylcarbodiimide; Beattie DS et al.; A purified cytochrome b-c1 complex isolated from yeast mitochondria has been reconstituted into proteoliposomes . The reconstituted comp}lex catalyzed antimycin A-sensitive electron transfer from different analogues of coenzyme Q to cytochrome c . The reconstituted complex was also capable of energy conservation as indicated by uncoupler-stimulated rates of electron transfer, electrogenic proton ejection, and reversed electron flow from cytochrome b to coenzyme Q2 in the presence of antimycin A driven by a valinomycin-induced K+-diffusion potential (negative inside) . Close to four protons were ejected per two electrons transported through the reconstituted b-c1 complex with ferricyanide as an artificial and impermeable electron acceptor.l The H+/2e- ratio decreased to two in the presence of the proton-conducting agent, carbonyl cyanide m-chlorophenylhydrazone . The same processes were studied in parallel in energy-conserving site 2 of rat liver mitochondria with similar results . In the reconstituted b-c1 complex, dicyclohexylcarbodiimide (DCCD) blocked the function of the electrogenic proton translocating device in the forward direction of proton ejection as well as in the backwards direction, measured as reversed electron flow from cytochrome b to coenzyme Q2 driven by a K+-diffusion potential . The primary effect of DCCD is localized on the proton ejection process, as the low proton conductance of the proteoliposome membrane was totally preserved after DCCD treatment.

J Biol Chem, 1982 Dec 25, 257(24), 14579 - 81
Covalent phosphorylation of the Mg2+-dependent ATPase of yeast plasma membranes; McDonough JP et al.; Phosphorylation by {gamma-32P}ATP of proteins associated with the plasma membrane of Saccharomyces cerevisiae has been studied both in vivo and in vitro . Although at least nine proteins are labeled in vivo, there is only one major protein labeled in vitro . This species with an apparent molecular weight of 114,000 has been identified as the plasma membrane Mg2+-ATPase . Phosphorylation of this enzyme occurs exclusively on serine residues . This is the first report that the proton-translocating ATPase of fungal plasma membranes is subject to phosphorylation by a protein kinase.

Science, 1982 Dec 24, 218(4579), 1323 - 5
Yeast mating pheromone activates mammalian gonadotrophs: evolutionary conservation of a reproductive hormone?
Loumaye E, Thorner J, Catt KJ.
alpha-Factor, a tridecapeptide mating pheromone of yeast (Saccharomyces cerevisiae), has extensive sequence homology with the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) . Both synthetic and natural preparations of alpha-mating factor were found to bind specifically to rat pituitary GnRH receptors and to stimulate the release of luteinizing hormone from cultured gonadotrophs . The ability of the yeast pheromone to reproduce the biological actions of GnRH in the mammalian pituitary gland indicates that the structural and functional properties of GnRH-related peptides may have been highly conserved during evolution.

Philos Trans R Soc Lond B Biol Sci, 1982 Dec 24, 300(1099), 185 - 94
Synthesis and possible role of carbohydrate moieties of yeast glycoproteins; Tanner W et al.; The pathways for protein N- and O-glycosylation in yeast cells are summarized . Evidence is presented that the terminal glucosyl residues of the dolichyl-PP-oligosaccharide intermediate are responsible for decreasing the Km for the peptide to be N-glycosylated . A liposomal model system is introduced that allows the study of a dolichyl phosphate (Dol-P) dependent transmembrane transport of mannosyl residues . The results obtained so far suggest that the mannosylation of Dol-P and the transmembrane translocation of Dol-P-Man are catalysed by the enzyme more or less simultaneously . However, only about 8-10% of the enzyme molecules incorporated into the liposomes seem to carry out the 'coupled' reaction . The glycosylation of carboxypeptidase Y is not required for this protein to reach the vacuole, its target organelle . In the presence of low concentrations of tunicamycin, however, yeast cells do stop growth . This does not seem to be due to the inhibition of secretion of glycoproteins like external invertase . It is postulated that protein glycosylation is crucial for a cell cycle event during the G1 phase.

Biochemistry, 1982 Dec 21, 21(26), 6950 - 6
X-ray crystallographic and nuclear magnetic resonance spectral studies of the products from the yeast inorganic pyrophosphatase-Co(NH3)4PP reaction . Investigation of the pyrophosphatase reaction mechanism; Haromy TP et al.; Yeast inorganic pyrophosphatase catalyzes the hydrolysis of P1,P2-bidentate Co(NH3)4 pyrophosphate {Co(NH3)4PP} to the cis, bis(phosphate) complex Co(NH3)4(Pi)2, which is not stable at neutral pH and over a period of 24 h converts to HPO4(2-) and a mixture of bidentate Co(NH3)4(PO4) and monodentate Co(NH3)4(H2O)(HPO4) . Concurrent with this process is the reduction and subsequent release of Co(H2O)6(2+) from the cobalt tetraammine bis(phosphate) complex and/or the cobalt tetraammine monophosphate complex . Bidentate tetraammine phosphatocobalt (III), hexaaquocobalt(II), orthophosphate, and two free water molecules cocrystallize {Co(NH3)4PO4 . Co(H2O)6(2+) . HPO4(2-) . 2H2O} from the reaction mixture in the triclinic space group Pi (Z = 2) with cell dimensions a = 6.849 (1) A, b = 11.693 (2) A, c = 12.630 (2) A, alpha = 65.60 (1) degree, beta = 88.98 (1) degree, and gamma = 73.04 (1) degree . The structure was solved by the heavy atom technique and refined to an R index of 0.040 by using 3077 intensities measured up to a 2 theta limit of 155 degrees . 31P NMR studies of the equilibrium mixture reveal that the equilibrium constant is a sensitive function of solution pH and temperature . Unlike the Co(NH3)4PP complex, there is evidence indicating that the Mg(H2O)4PP complex is degraded to monodentate Mg(H2-O)5PO4 in the enzyme active site.

Nucleic Acids Res, 1982 Dec 20, 10(24), 8341 - 9
Procedure for C2 deuteration of nucleic acids and determination of A psi 31 pseudouridine conformation by nuclear Overhauser effect in yeast tRNAPhe; Roy S et al.; Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA . Deuterated tri-ethyl orthoformate was condensed with 5(4)-amino imidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine . C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated yeast tRNAPhe was isolated . This C2 deuterated tRNAPhe was used to identify A psi 31 and U8-A14 . A psi 31 was found to be bonded through N1H . The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.

Nucleic Acids Res, 1982 Dec 20, 10(24), 8297 - 305
Nuclear Overhauser effect study of yeast tRNAVal 1: evidence for uridine-pseudouridine base pairing; Schejter E et al.; The proton NMR spectrum of yeast tRNAVal 1 has been studied using nuclear Overhauser effect (NOE), including comparison of NOE patterns between purine C8 deuterated and nondeuterated samples . Studies of the downfield region enable us to reliably assign many resonances in the acceptor and D stems . Prominent among these reliable assignments is that of the unusual base pair U psi, which is made here for the first time . Other identifications include GU2, U8-A14, the three AU base pairs of the acceptor stem, and N1 and N3 protons of psi 55.

J Biol Chem, 1982 Dec 10, 257(23), 14110 - 5
Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase; Uno I et al.; The CYR3 mutant of yeast, Saccharomyces cerevisiae, partially accumulated unbudded cells and required cAMP for the best growth at 35 degrees C . The CYR3 mutation was partially dominant over the wild type counterpart and suppressed by the bcy1 mutation which is responsible for the deficiency of the regulatory subunit of cAMP-dependent protein kinase . The molecular weights of cAMP-dependent protein kinase and its catalytic and regulatory subunits were 160,000, 30,000, and 50,000, respectively . No significant differences in the molecular weights of cAMP-dependent protein kinase and the subunits were found between the wild type and CYR3 mutant strains . However, the cAMP-dependent protein kinase activity of CYR3 cells showed significantly higher Ka values for activation by cAMP at 35 degrees C than those of wild type and a clear difference in the electrophoretic mobility of the regulatory subunit was found between the wild type and CYR3 enzymes . The CYR3 mutation was suppressed by the IAC mutation which caused the production of a significantly high level of cAMP . The results indicate that the CYR3 phenotype was produced by a structural mutation in the CYR3 gene coding for the regulatory subunit of cAMP-dependent protein kinase in yeast.

Biochim Biophys Acta, 1982 Dec 6, 709(1), 46 - 52
Studies on yeast sulfite reductase . VI . Use of the effects of ionic strength as a probe for enzyme structure and mechanism; Kobayashi K et al.; The effects of ionic strength on the structure of yeast sulfite reductase (EC 1.8.1.2) were investigated . Gel filtration and sedimentation analysis revealed that this enzyme dissociated at ionic strength below 0.1 into two components having molecular weights of 300000 and sedimentation coefficients of 7-9 S . The secondary structure and the surroundings of the prosthetic groups were not appreciably affected by ionic strength . This enzyme showed CD spectra in the near-ultraviolet region considered to be due to tyrosine residues . On lowering the ionic strength, they decreased, together with the disappearance of the difference spectra in the same region, indicating that some tyrosine residues became exposed to the environment at low ionic strength . SH groups were also exposed at low ionic strength and reacted with DTNB and PCMB more easily to cause the loss of NADPH-sulfite reductase activity . These essential SH groups as well as some tyrosine residues may exist in the region of contact between the two components constituting the enzyme molecule.

Biochim Biophys Acta, 1982 Dec 6, 709(1), 38 - 45
Studies on yeast sulfite reductase . V . Effects of ionic strength on enzyme activities; Kobayashi K et al.; Yeast sulfite reductase (EC 1.8.1.2) has the unique property that it is inactivated at low ionic strength . The effects of ionic strength on various enzyme activities were investigated and the characterization of the inactivation was carried out . The multiple activities shown by this enzyme were all nullified by exposing the enzyme to low ionic strength, except for reduced methyl viologen-sulfite reductase activity, which was increased rather than decreased . The Km values for NADPH and sulfite were not significantly affected by ionic strength . When the enzyme was reduced with NADPH at low ionic strength, the height of the peak at 455 nm was decreased to one-half of the fully-reduced level, while the height of the peak at 587 nm was unchanged . These results, together with the experiment using an FMN-depleted apoenzyme, indicate that the inactivation at low ionic strength was caused by the interception of the electron flow between FAD and FMN . The inactivation at low ionic strength was reversible, but the restoration of the activity was dependent on the incubation period at low ionic strength and the protein concentration . The kinetics and the effect of glycerol and/or 2-mercaptoethanol on the inactivation and the restoration showed that further change, including oxidation of SH groups, should occur in the enzyme through prolonged incubation at low ionic strength . Changes in the enzyme structure related to these results are described in the subsequent paper.

Arch Microbiol, 1982 Dec, 133(2), 155 - 61
Microtubule function and its relation to cellular development and the yeast cell cycle in Wangiella dermatitidis; Jacobs CW et al.; The microtubule inhibitor nocodazole (methyl-5-{2-(thienylcarbonyl)-1H-benzimidazol-2-yl}-carbamate) prevented nuclear migration and nuclear division in yeasts and developing multicellular forms of the polymorphic fungus Wangiella dermatitidis . It did not prevent yeast bud formation during at least two or three budding cycles, and caused yeasts to accumulate as premitotic forms with one to three buds . The effects of the drug suggested that at least three control pathways were involved in the yeast cell cycle; that the nocodazole block point was separate from the execution points of two temperature-sensitive mutations which lead to multicellularity; and that microtubules were controlling neither the yeast budding process nor the development of multicellular forms.

Antonie Van Leeuwenhoek, 1982 Dec, 48(5), 471 - 83
Sterigmatosporidium gen . n., a new heterothallic basidiomycetous yeast, the perfect state of a new species of Sterigmatomyces fell; Kraepelin G et al.; Sterigmatosporidium gen . n . is described as a new basidiomycetous yeast-like fungus with the single species Sterigmatosporidium polymorphum sp . n . for which a Latin diagnosis and a preliminary life cycle are presented . The mean character distinguishing the new genus from the imperfect genus Sterigmatomyces is the development of a dikaryotic mycelium with clamp connections producing sexual spores in ramified whorls and lateral chlamydospores as well as blastospores . The dikaryotic phase could be induced by crossing compatible haploid clones of the heterothallic fungus, which are similar to Sterigmatomyces but not identical with any known species.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7127 - 31
Replacement of anticodon loop nucleotides to produce functional tRNAs: amber suppressors derived from yeast tRNAPhe; Bruce AG et al.; The method of anticodon loop replacement has been used to make derivatives of yeast tRNAPhe . By constructing tRNAs with a CUA anticodon, complementary to the amber (UAG) terminator, functional amber suppressor tRNAs were produced . The activity of these tRNAs was assayed in a mammalian cell-free protein synthesizing system . The level of suppression reflects the efficiency of codon recognition . tRNAs were constructed with either A, C, U, or G on the 3' side of the CUA anticodon . The tRNAs containing the purines were efficient amber suppressors, whereas those containing pyrimidines were inefficient.

Cell, 1982 Dec, 31(2 Pt 1), 429 - 41
Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes {HOK}, {NEX} and {EXL} are on one of these dsRNAs; Sommer SS et al.; {HOK}, {NEX} and {EXL} are non-Mendelian genes affecting the K1 and K2 killer systems of Saccharomyces cerevisiae . T1 fingerprints of L double-stranded RNA from {HOK}-, {NEX}- and {EXL}-containing cells and their heat-cured derivatives indicate that: there are three distinct double-stranded RNAs, L-A, L-B and L-C; {HOK}, {NEX} and {EXL} are all located on L-A; there are three functional variants of L-A that produce the {HOK} {NEX}, {HOK} {EXL} or {EXL} phenotypes; L-A is compatible with L-B or L-C; and there are additional sequences present in lower copy number . Although their fingerprint patterns are unrelated, solution hybridization shows that L-B and L-C share sequence homology . Strains carrying L-A as the major double-stranded RNA or only L-B or only L-C all have similarly sedimenting (160S) virus-like particles with RNA polymerase activity . Virus-like particles from strains with L-A all have proteins of 81,000 and 180,000 daltons that are absent from isogenic strains cured of L-A . Virus-like particles from strains with only L-B or only L-C both have major proteins of 77,000 and 73,000 daltons.

Int J Radiat Biol Relat Stud Phys Chem Med, 1982 Dec, 42(6), 591 - 600
Heavy ion effects on yeast cells: induction of canavanine-resistant mutants; Kiefer J et al.; The induction of forward mutations (resistance to canavanine) by heavy ion bombardment was investigated in wild type haploid yeast Saccharomyces cerevisiae . Accelerated ions of argon, titanium, nickel, krypton, xenon, lead and uranium with specific energies between 1.7 and 9.25 MeV/u were obtained from the UNILAC machine at the Gesellschaft fur Schwerionenforschung, Darmstadt/Germany . LET-values ranged from 1200 to about 15 000 keV/microns . There was no unequivocal dependence of mutation induction cross section on either LET or Z*2/beta 2, but also a prominent influence of ion specific energy . This is explained by the action of long-ranging delta-electrons.

Hoppe Seylers Z Physiol Chem, 1982 Dec, 363(12), 1473 - 81
Phenylalanyl-tRNA synthetases from cytoplasm and mitochondria of yeast and hen liver: comparison of their structural and catalytic properties; Gabius HJ et al.; Amino acid compositions and tryptic maps of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases from yeast and hen liver, respectively, demonstrate the similarity of these enzymes, although they are clearly not identical . Moreover, similarity is noted for catalytic properties like stoichiometries of complex formation with tRNAPhe and negative cooperativity of tRNAPhe binding, triggered by substrates . Analysis of the kinetics at saturating and subsaturating substrate concentrations indicates the contribution of the transfer of phenylalanine from the adenylate to tRNAPhe to the rate-determining step in aminoacylation and subunit interactions in the tetrameric enzymes . Furthermore, the locations of substrate-binding sites appear rather constant within species and in interspecies comparison . Subtle differences at certain sites, although homology exists, are exemplified by a special regulatory effect on activity by other amino acids only in the case of the cytoplasmic enzyme from hen liver . The results support the idea of a common ancestry by gene duplication for the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases in fungi and animals, respectively.

Cell, 1982 Dec, 31(2 Pt 1), 337 - 46
Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system; Edwards JC et al.; We have developed an in vitro transcription system for yeast mitochondrial rRNA genes . Using highly purified yeast mitochondrial RNA polymerase and bacterial plasmids carrying DNA segments containing the mitochondrial rRNA sites of transcriptional initiation, we have been able to demonstrate correct initiation of transcription in vitro . By directly sequencing the transcription products, we show that transcription in vitro of both the 14S and 21S rRNAs is initiated at precisely the same site as it is in vivo . Transcription of the rRNA genes is highly sensitive to ionic strength and RNA polymerase concentration . Additional factors or modified conditions may be necessary to permit accurate transcription of mitochondrial protein genes.

Proc Natl Acad Sci U S A, 1982 Dec, 79(24), 7674 - 8
Insertion of a repetitive element at the same position in the 5'-flanking regions of two dissimilar yeast tRNA genes; Sandmeyer SB et al.; The regions 5' proximal to many yeast tRNA genes exhibit a high frequency of DNA sequence polymorphisms . DNA sequence analysis of polymorphic variants of SUQ5, a tRNA Ser UCA gene, and SUP2, a tRNA Tyr gene, shows that in each case one sequence variant of the tRNA gene is 346 base pairs longer than the other . The longer variants appear to have arisen from the shorter ones by the insertion of nearly identical copies of a 341-base pair sigma element into a site 16 base pairs upstream from the 5' ends of the tRNA-coding regions . The sequences of the two copies of the sigma element differ at only five positions . The element has a number of properties that are typical of many transposable elements: (i) there is a perfect eight-base-pair inverted repeat at its ends, (ii) these ends are flanked by a five-base-pair direct repeat of a sequence that occurs only once in the target DNA, (iii) there are approximately 20 copies of the element in the yeast genome, and (iv) there is considerable strain-to-strain variation in the sizes of the restriction fragments on which these copies lie . The presence of the sigma element has no gross effect on the phenotype of a SUP2 ochre suppressor . Analysis of the SUQ5 and SUP2 sequences favors the hypothesis that sigma is a transposable element with a novel type of insertion specificity, which is primarily based on the presence of a tRNA-coding region a fixed distance from the insertion site, rather than on the immediate target sequences.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7410 - 4
A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site; Guarente L et al.; We have identified the promoter region of the GAL10 gene (whose product is UDP-galactose epimerase) of Saccharomyces cerevisiae; this promoter mediates galactose induction of transcription in conjunction with the product of the GAL4 regulatory gene . This identification was achieved by excising a 365-base-pair fragment of GAL10 leader DNA with a GAL10 proximal endpoint greater than 100 base pairs upstream of the transcriptional start site and substituting it in place of the upstream activation site of the CYC1 (iso-1-cytochrome c) promoter {Guarente, L . & Ptashne, M . (1981) Proc . Natl . Acad . Sci . USA 78, 2199-2203} . The hybrid promoter is composed of DNA encoding CYC1 mRNA start sites and the GAL segment upstream of these sites . This promoter is regulated in a manner analogous to GAL10; i.e., it is induced by galactose and responds to mutations in the GAL4 and GAL80 regulatory loci . The activity of the hybrid promoter requires sequences in the region of the CYC1 mRNA start sites but does not require a precise spacing between these sequences and the GAL segment . The transposed GAL segment appears not to contain sequences that mediate glucose repression . Thus, the picture of the GAL10 promoter that emerges is one of an upstream activation site that responds to the GAL4 product plus galactose, and a region of transcription initiation that may contain sequences that mediate glucose repression . Experiments employing strains inducible (GAL80) or constitutive (gal80) for GAL10 expression indicate that an additional component of glucose repression is inducer exclusion.

J Bacteriol, 1982 Dec, 152(3), 1295 - 7
Facilitated diffusion of 6-deoxy-D-glucose in bakers' yeast: evidence against phosphorylation-associated transport of glucose; Romano AH; 6-Deoxy-D-glucose, a structural homomorph of D-glucose which lacks a hydroxyl group at carbon 6 and thus cannot be phosphorylated, is transported by Saccharomyces cerevisiae via a facilitated diffusion system with affinity equivalent to that shown with D-glucose . This finding supports the facilitated diffusion mechanism for glucose transport and contradicts theories of transport-associated phosphorylation which hold that sugar phosphorylation is necessary for high-affinity operation of the glucose carrier.

Gene, 1982 Dec, 20(3), 323 - 37
Isolation and characterization of nuclear genes coding for subunits of the yeast ubiquinol-cytochrome c reductase complex; van Loon AP et al.; Nuclear genes coding for the Mr 17 000, 14 000 and 11 000 subunits of the ubiquinol-cytochrome c reductase complex (complex III) in yeast have been isolated from a clone bank of yeast nuclear DNA by use of a mRNA hybridization-competition assay . This is based on our observations that levels of mRNAs for these subunits are much reduced during glucose repression and in cytoplasmic petite mutants and the procedure should be of general application for the isolation of other inducible or repressible genes coding for mRNAs present at low levels in the cell . A first characterization of the clones is presented . The genes are not closely linked in the genome and those coding for Mr 14 000 and 11 000 subunits are present in unique genomic environments, which suggests that there are only single copies of each in the nuclear genome.

Biochemistry, 1982 Nov 23, 21(24), 6081 - 8
Nuclear overhauser effect study of yeast aspartate transfer ribonucleic acid; Roy S et al.; Nuclear Overhauser effect studies are described for yeast tRNAAsp in 0.1 M NaCl, pH 7.0 . A primary aim is to develop a general method for attacking the problem of assignment in transfer ribonucleic acids (tRNAs) . Previously, we have demonstrated the utility of the nuclear Overhauser effect (NOE) between protons on adjacent base pairs combined with C8 deuterium substitution, by assigning the imino protons of the dihydrouridine stem and the two reverse-Hoogsteen base pairs T54-A58 and U8-A14 . Here, we extend that approach to other parts of the molecule . We also describe several NOE-connected patterns for, e:g., m5CG and psi 55 N3H imino protons which may be of general utility . For the first time, a purine-15-pyrimidine-48 base pair (in this case A15-U48) has been assigned . A total of 13 of 25 base pairs from all parts of the molecule and several noninternally bonded imino protons have now been assigned unambiguously . This is a general method for assigning resonances in tRNA and perhaps in all double-stranded nucleic acids . This, and the distance information inherent in NOE measurements, should make NMR more generally applicable to nucleic acids.

Biochim Biophys Acta, 1982 Nov 19, 708(3), 326 - 9
A nuclear magnetic relaxation study of conformational changes induced by substrate and temperature in bovine liver thiosulfate sulfurtransferase and yeast hexokinase; Blicharska B et al.; Nuclear magnetic relaxation studies have been performed on thiosulfate sulfurtransferase (EC 2.8.1.1) and hexokinase (EC 2.7.1.1) . Observation of proton spin-lattice relaxation times T1 indicates that structural transitions occur in these enzymes in the range 0-40 degrees C and that there are different temperature-dependent forms of thiosulfate sulfurtransferase and hexokinase . Thermal transitions between these forms are affected by the binding of the substrates . The results may be due to changes in the interactions between the structural domains into which the single polypeptide chains of thiosulfate sulfurtransferase and hexokinase are folded.

Eur J Biochem, 1982 Nov 15, 128(2-3), 489 - 94
Wild-type and mutant forms of homoisocitric dehydrogenase in the yeast Saccharomycopsis lipolytica; Gaillardin CM et al.; Homoisocitric dehydrogenase (EC 1.1.1.155) has been purified 525-fold from the yeast Saccharomycopsis lipolytica with a yield of 25% . The preparation was judged to be homogeneous by electrophoresis under denaturing and non-denaturing conditions and by isoelectric focusing; it consisted of a single protein with molecular weight of 48000 . In the presence of homoisocitric acid, a higher molecular weight was observed, suggesting a dimeric structure for the native enzyme . Complementing mutants devoid of homoisocitric dehydrogenase activity mapped at two closely linked loci (lys9 and lys10) . Lys10 mutants displayed NAD-reducing activity, whereas lys9 mutants retained some carboxylating activity . Our results are best explained by the assumption that the active enzyme is a dimer of identical subunits involved in successive dehydrogenation and decarboxylation steps.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6981 - 7000
Nuclear magnetic resonance studies on yeast tRNAPhe I . Assignment of the iminoproton resonances of the acceptor and D stem by means of Nuclear Overhauser Effect experiments at 500 MHz; Heerschap A et al.; Resonances of the water exchangeable iminoprotons of the acceptor and D stem of yeast tRNAPhe have been assigned by means of Nuclear Overhauser Effects (NOE's) . Assignments were made for spectra recorded from tRNA dialysed against a buffer with 110 mM sodium and 5 mM magnesium ions and against a buffer with 430 mM sodium and no magnesium ions . Remarkable is the assignment of a resonance at 13.6 - 13.7 ppm to the iminoproton of C11G24 . This assignment as well as those of G1C72, G3C70, U7A66, U12A23 and C13G22 are different from those made previously on the basis of less direct evidence . NOE experiments performed at 45 degrees C support the view that the D stem together with the tertiary interaction U8A14 is one of the most stable parts of the molecule in the presence of magnesium ions . A comparison of the spectra recorded under the two different buffer conditions shows that an excess of 320 mM sodium ions is not capable to force the tRNA in the same conformation as 5 mM magnesium ions can do.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6903 - 18
Structural and functional analysis of separated strands of killer double-stranded RNA of yeast; Thiele DJ et al.; The two strands of the M double-stranded RNA species from a killer strain of Saccharomyces cerevisiae have been separated, and the 3'-terminal sequences of these strands have been determined . The positive strand programs the synthesis of the putative killer toxin precursor (M-p32) in a rabbit reticulocyte in vitro translation system . Only the negative strand hybridizes to the positive polarity transcript (m) synthesized in vitro by the virion-associated transcriptase activity . Secondary structural analysis of the extreme 3'-terminus of the negative strand using S1 nuclease is consistent with the presence of a large stem and loop structure previously proposed on the basis of RNA sequence data . This structure, and a similar structure at the corresponding 5'-terminus of the positive strand, may have functional significance in vivo.

J Biol Chem, 1982 Nov 10, 257(21), 13075 - 80
Import of proteins into mitochondria . Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria; Daum G et al.; Import of in vitro-synthesized cytochrome b2 (a soluble intermembrane space enzyme) was studied wih isolated yeast mitochondria . Import requires an electrochemical gradient across the inner membrane and is accompanied by cleavage of the precursor to the corresponding mature form . This conversion proceeds via an intermediate form of cytochrome b2, which can be detected as a transient species when mitochondria are incubated with the cytochrome b2 precursor for short times or at low temperatures . Conversion of the precursor to the intermediate form is energy-dependent and catalyzed by an o-phenanthroline-sensitive protease located in the soluble matrix . The intermediate is subsequently converted to mature cytochrome b2 in a reaction which is o-phenanthroline-insensitive and requires neither an energized inner membrane nor a soluble component of the intermembrane space . Whereas mature cytochrome b2 is soluble, the intermediate formed by isolated mitochondria is membrane-bound and exposed to the intermembrane space . The same intermediate is detected as a transient species during cytochrome b2 maturation in intact yeast cells (Reid, G . A., Yonetani, T., and Schatz, G (1982) J . Biol . Chem . 257, 13068-13074) . The in vitro studies reported here suggest that a part of the cytochrome b2 precursor polypeptide chain is transported to the matrix where it is cleaved to a membrane-bound intermediate form by the same protease that processes polypeptides destined for the matrix space or for the inner membrane . In a second reaction, the cytochrome b2 intermediate is converted to mature cytochrome b2 which is released into the intermembrane space . The binding of heme is not necessary for converting the intermediate to the mature polypeptide.

J Biol Chem, 1982 Nov 10, 257(21), 13068 - 74
Import of proteins into mitochondria . Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells; Reid GA et al.; The import of cytochrome b2 and cytochrome c peroxidase into mitochondria was investigated by pulse-chase experiments with intact yeast cells combined with subcellular fractionation . Import and processing of the precursors of these intermembrane space proteins is blocked by uncouplers of oxidative phosphorylation, indicating that an "energized" inner membrane is required . Cytochrome b2 is processed in two steps . The first step involves energy-dependent transport across both mitochondrial membranes and cleavage by a matrix-located protease to yield an intermediate which is smaller than the precursor, but larger than the mature protein . The second step involves conversion of the intermediate to the mature form . Whereas the precursor and the mature form are soluble, the intermediate is membrane-bound and exposed to the intermembrane space . The maturation of cytochrome c peroxidase is much slower than that of cytochrome b2 . Proteolytic processing rather than import is rate-limiting since cytochrome c peroxidase precursor labeled during a 3-min pulse is already found attached to the outer face of the mitochondrial inner membrane . Import of cytochrome b2 and probably also of cytochrome c peroxidase thus involves energy-dependent transport to the matrix and cleavage by a matrix-localized protease . Maturation of cytochrome b2 proceeds in the sequence: soluble precursor leads to membrane-bound intermediate form leads to soluble mature form.

J Biol Chem, 1982 Nov 10, 257(21), 13056 - 61
Import of proteins into mitochondria . Yeast cells grown in the presence of carbonyl cyanide m-chlorophenylhydrazone accumulate massive amounts of some mitochondrial precursor polypeptides; Reid GA et al.; Cytoplasmically synthesized precursors of mitochondrial polypeptides have previously been observed in trace amounts after pulse labeling of yeast spheroplasts or after in vitro translation of yeast mRNA (Maccecchini, M . L., Rudin, Y., Blobel, G., and Schatz, G . (1979) Proc . Natl . Acad . Sci . U . S . A . 76, 343-347) . Some of these precursors are shown here to accumulate in large amounts (up to 150 micrograms/g of cell protein) during growth of a cytoplasmic petite (rho-) mutant in the presence of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation . Cytochrome c1 precursor accumulated under these conditions is unstable; it is degraded with a half-life of about 10 min . In contrast, the F1-ATPase beta-subunit precursor is degraded considerably more slowly and, following removal of the uncoupler, can be post-translationally imported into mitochondria where it is processed to the mature polypeptide.

J Biol Chem, 1982 Nov 10, 257(21), 13028 - 33
Import of proteins into mitochondria . Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria; Daum G et al.; Yeast mitochondria were fractionated into inner membrane, outer membrane, matrix, and intermembrane space . Identity and purity of each fraction were monitored by enzyme assays, dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological detection of characteristic mitochondrial polypeptides . Cytochrome b2 and cytochrome c peroxidase were found to be components of the intermembrane space . The most reliable marker of the outer membrane was a major 29,000-dalton polypeptide component . The availability of submitochondrial fractions provides a basis for studying import of precursor polypeptides into isolated yeast mitochondria.

Biochim Biophys Acta, 1982 Nov 9, 708(2), 225 - 32
Susceptibility to proteinases of yeast enzymes selectively modified by fatty acids; Burlini N et al.; To investigate a possible correlation between selective modification and degradation of enzymes, the susceptibility to intracellular yeast proteinases A and B of yeast enzymes treated with fatty acids was tested . Enzymes used were glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-phosphoglycerate kinase (EC 2.7.2.3), which are sensitive to the denaturing modification caused by fatty acids, and alcohol dehydrogenase (EC 1.1.1.1) which is insensitive . Proteinases and substrate enzymes were all pure preparations . Without modification by fatty acids, at neutral pH, the three enzymes are remarkably resistant to degradation by both proteinases . Treatment with myristic or oleic acid definitely enhances the susceptibility to proteolysis of the sensitive glucose-6-phosphate dehydrogenase and 3-phosphoglycerate kinase, whereas it leaves negligible that of the insensitive alcohol dehydrogenase . The selective effect of fatty acids on the degradation is pH-dependent: with proteinase A it was lost at acidic pH . Since intracellular levels of free fatty acids near or even higher than 1 mM were actually measured in yeast cells, it is possible that free fatty acids, in some cellular conditions, affect yeast enzyme composition . However, the control of specific enzyme degradation in yeast is still an open question.

Biochemistry, 1982 Nov 9, 21(23), 5787 - 94
Proteolipid of adenosinetriphosphatase from yeast mitochondria forms proton-selective channels in planar lipid bilayers; Schindler H et al.; Proteolipid isolated from yeast mitochondrial adenosinetriphosphatase by butanol extraction is reincorporated into lipid vesicles from which planar membranes are formed . The proteolipid permits electric conductance through the membrane . This conductance occurs through membrane channels which are highly selective for protons . Proton channels in the membrane are directly observed at high proton concentrations in the aqueous phases . Channels open and close independently from each other; their open-state conductances and lifetimes are monodisperse but influenced by the applied voltage (12 pS and 3 s, respectively, at pH 2.2 and 100 mV) . Proton channels do not occur in single proteolipid molecules; the conducting structure consists of at least two polypeptide chains since channels form in a (reversible) bimolecular reaction of nonconducting forms of proteolipid . The number of proton channels at a constant proteolipid concentration changes in sharp transitions and by orders of magnitudes upon critical changes of membrane composition and pH . These transitions are caused by transitions of proteolipid organization in the membrane from a dispersed state (equilibrium between channel-forming "dimers" and a large pool of "monomers") to a state of almost complete aggregation of proteolipid which stabilizes large proton-conducting structures (probably associates of channel-forming dimers) . This self-association of isolated proteolipid into structures containing proton-selective channels suggests that the six proteolipids in the adenosinetriphosphatase complex exist as a self-associating entity containing most likely three proton channels.

Biochim Biophys Acta, 1982 Nov 8, 692(2), 202 - 9
Effect of phophatidylcholine and phosphatidylethanolamine enrichment on the structure and function of yeast membrane; Trivedi A et al.; The phospholipid composition of yeast plasma membrane was manipulated by two different methods: (i) by using two auxotrophic strains KA101 (cho1) and MC13 (Cho+) which required phospholipid bases for growth and (ii) by supplementing Saccharomyces cerevisiae (3059) cells with high concentration of choline or ethanolamine . It was possible to enrich the plasma membrane with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) by both methods . The uptake of amino acids, e.g., glycine, glutamic acid, leucine, lysine methionine, phenylalanine, proline and serine, was significantly reduced in PC- or PE-enriched cells . However, the extent of reduction in transport was variable among different strains . A fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS), was used to monitor the structural changes induced by altered phospholipid composition . It was observed that the relative fluorescence intensity of bound ANS was decreased as a consequence of PC or PE enrichment . The decrease in fluorescence was probably associated with reduced number of available binding sites (n) and increased apparent dissociation constant (Kd) . Furthermore, our results also suggest that a critical level of PE or PC is required for proper functioning of yeast membrane.

FEBS Lett, 1982 Nov 8, 148(2), 267 - 70
Separation and chemical differentiation of alpha and beta subunits in yeast phosphofructokinase; Tijane MN et al.; The two types of subunits alpha and beta constitutive of yeast phosphofructokinase have been separated by ion-exchange chromatography under denaturating conditions . Amino acid analysis and peptide mapping were performed on the isolated subunits . The frequence of most of the amino acids significantly differs between the two types of polypeptide chains . Moreover, tryptic peptide maps of alpha and beta subunits are clearly not superimposable . These chemical differences seem sufficient to account for the distinct catalytic and regulatory functions of beta and alpha subunits in the yeast phosphofructokinase reaction.

Biochem J, 1982 Nov 1, 207(2), 323 - 29
Role of the N-terminus of glutathione in the action of yeast glyoxalase I; Douglas KT et al.; A number of S-substituted glutathiones and the corresponding N-substituted S-substituted analogues have been found to be linear competitive inhibitors of yeast glyoxalase I at 26 degrees C over the pH range 4.6-8.5 . N-Acetylation of S-(p-bromobenzyl)glutathione weakens binding by 13.7-fold . N-benzoylation by 25.6-fold, N-trimethylacetylation by 53.3-fold and N-carbobenzoxylation by 7.8-fold, indicating a minor steric component in the binding at the N-site . The Ki-weakening effect of N-substitution of glutathione depends on the chemical nature of the S-substituent, indicating flexibility in the glutathione and/or glyoxalase I contributions to the binding site for glutathione derivatives . The effect of N-acylation on Ki is in accord with a charge interaction of the free enzyme with S-blocked glutathione in a region of reasonably high dielectric constant . There is a slight pH effect on Ki for S-(m-trifluoromethylbenzyl)glutathione but not for S-(p-bromobenzyl)glutathione.

Med Hypotheses, 1982 Nov, 9(5), 481 - 8
Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: XI . A theoretical interpretation of the sequential methylation of yeast phenylalanine tRNA; Hancock CD et al.; Theoretical studies on the mechanisms of methylation of tRNA was reported in a previous paper dealing with the induction of genic activity of proposed "embryonic" tRNA methylases . The mechanism described only the first methylation of a tRNA that had a known structure, namely yeast phenylalanine tRNA . In this paper, a complete scheme of methylation for the molecule is reported in detail using molecular model building . The mechanism uses only one specific uridine residue with which S-adenosyl-L-methionine complexes for all required methylation sites.

Biochimie, 1982 Nov-Dec, 64(11-12), 1073 - 9
The primary structures of three yeast mitochondrial serine tRNA isoacceptors; Martin R et al.; Yeast mitochondria contain several isoaccepting species of serine-tRNA . The relative amount of these isoacceptors varies according to the conditions used to grow the yeast cells . In order to gain insight into the structural differences among these isoacceptors, the three mitochondrial tRNAsSer, which are present in derepressed yeast cells, have been sequenced . The primary structure of tRNASer1 differs considerably from that of tRNASer2; these two isoacceptors have only 39 nucleotides in common . In contrast, tRNASer3 differs from tRNASer2 by only one post-transcriptional modification: the psi residue in position 28 of tRNASer2 is replaced by a normal U in tRNASer3 . Unlike tRNASer2 and tRNASer3, the primary sequence of tRNASer1 shows two unusual structural features: it has a D in position 14 instead of the "universal" A14 of the standard tRNA cloverleaf and it contains two G residues between the D-stem and the anticodon-stem . Considering their respective anticodons, tRNASer1 should recognize the two serine codons A-G-C and A-G-U, whereas both tRNASer2 and tRNASer3 should recognize all four serine codons of the U-C-N series.

Cell, 1982 Nov, 31(1), 193 - 200
There are at least two yeast viral double-stranded RNAs of the same size: an explanation for viral exclusion; Field LJ et al.; The yeast virus ScV is similar to other double-stranded RNA fungal viruses, which persist indefinitely without harm to their host cells . A single large viral double-stranded RNA (L) of 4.8 kilobase pairs codes for the major viral capsid polypeptide . Some strains have a second, satellite virus, with a genomic RNA (M) of 1.9 kilobase pairs, that codes for an extracellular toxin (killer toxin) responsible for killing sensitive cells . Sensitive cells lack a functional resistance gene on M . There are a number of different varieties of this satellite virus with different toxin and resistance specificities . Two of these, k1 and k2, are well characterized . A cytoplasmic genetic element causes the loss of the k2 virus . We show that this element is actually one of the k1 viruses; there are two k1 viruses with L genomic RNAs of the same size but different sequence, which account for some of the previously observed 3'-end heterogeneity of L.

Biofizika, 1982 Nov-Dec, 27(6), 1017 - 21
{Large-scale structural changes of yeast phosphoglycerate kinase molecule upon substrate binding}; Timchenko AA et al.; Changes of dimensions and form of the yeast phosphoglyceratekinase molecule upon ATP and 3-phosphoglycerate binding have been investigated by diffuse X-ray scattering . It has been shown that simultaneous sorption of both substrates is accompanied by an essential decrease both of the radius of gyration and the extent of asymmetry of the phosphoglyceratekinase molecule.

J Microsc, 1982 Nov, 128 Pt 2, 157 - 66
Reliable use of resistance evaporation of Pt and C for high resolution freeze-fracturing and a crystal surface image complementary to the E-face of yeast plasma membranes; Steere RL; Freeze-fracture specimens of bakers yeast plasma membranes faces were prepared in both a modified Denton DFE-2-freeze-etch module and a modified Balzers BAF-301 freeze-etch unit . Each unit was equipped with a liquid nitrogen cooled shroud, resistance evaporators with PT-C and C sources 7 cm from the specimens and with a resistance monitor to control PT-C shadow film thickness . Optical diffraction patterns of specimens prepared in these units have fourth, fifth or sixth order spots . Therefore, on the basis of optical diffraction patterns, resolution of yeast plasma membrane specimens prepared in these units is equivalent to or better than that obtained by others with an ultrahigh vacuum system equipped with specially redesigned electron guns . A new image with tube-like particles in hexagonal arrays, each surrounded by six substructure particles, nearly perfect high-resolution complement to the hexagonal array of ring-like depressions and the six surrounding subunit depressions of the E-face, has been revealed on the surfaces of cubic crystals (presumably ice) which formed in the gap between the P- and E-faces within fissures that occurred when the samples were frozen in liquid Freon 22 . When the samples were subsequently freeze-fractured at 77 K at a chamber vacuum of 13 microPa in which the specimens were protected from surface contamination by a liquid nitrogen cooled shroud, these crystals remained attached to the P-face but pulled away from the E-face against which they had apparently made molecular contact.

Mutat Res, 1982 Nov, 105(5), 313 - 8
Mutagenesis in cdc7 strains of yeast . The fate of premutational lesions induced by ultraviolet light; Njagi GD et al.; cdc7-1 cells bearing UV-revertible mutations are virtually immutable by all means so far tested . By mating UV treated cdc7-1 cells with untreated cdc7+ cells carrying the same revertible alleles it is possible to rescue premutational lesions as revertants and to study their fate in cdc7-1 cells . If storage intervenes between treatment and mating there is a rapid decline in revertants rescued . This is not related to the death of cdc7-1 cells with storage nor does it reflect a progressive loss in their ability to mate.

Exp Cell Res, 1982 Nov, 142(1), 69 - 78
Control of the yeast cell cycle by protein synthesis; Popolo L et al.; The increased synthesis of ribosomal RNA (rRNA) is correlated with enhanced cell proliferation, and it has been suggested that rRNA metabolism may have a regulatory role in the progression of the cell cycle . Alternatively, it might be the ensuing more active protein synthesis that drives the cell cycle progression . We have found that treatment with low doses of cycloheximide dissociates rRNA and protein synthesis . In fact, after the addition of cycloheximide the protein synthesis rate is strongly inhibited, whereas the rate of rRNA synthesis is unaffected for some time . The progression of the cell cycle, monitored as analysis of DNA distribution by flow cytometry and as bud emergence, is quickly and largely inhibited, thus indicating that a sustained rRNA metabolism is not sufficient to allow continuous cycle progression . The effects of cycloheximide on the daughter and mother duplication times, on the mean cell volume, and on the volume at budding were also analyzed . The results suggest that protein synthesis, rather than rRNA synthesis, may have a key role in the control of cell cycle progression in Saccharomyces cerevisiae.

Cell, 1982 Nov, 31(1), 201 - 13
Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes; Celniker SE et al.; An enzyme system prepared from Saccharomyces cerevisiae carries out the replication of exogenous yeast plasmid DNA . Replication in vitro mimics that in vivo in that DNA synthesis in extracts of strain cdc8, a temperature-sensitive DNA replication mutant, is thermolabile relative to the wild-type, and in that aphidicolin inhibits replication in vitro . Furthermore, only plasmids containing a functional yeast replicator, ARS, initiate replication at a specific site in vitro . Analysis of replicative intermediates shows that plasmid YRp7, which contains the chromosomal replicator ARS1, initiates bidirectional replication in a 100 bp region within the sequence required for autonomous replication in vivo . Plasmids containing ARS2, another chromosomal replicator, and the ARS region of the endogenous yeast plasmid 2 microns circle give similar results, suggesting that ARS sequences are specific origins of chromosomal replication . Used in conjunction with deletion mapping, the in vitro system allows definition of the minimal sequences required for the initiation of replication.

Cell, 1982 Nov, 31(1), 183 - 92
Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus; Strathern JN et al.; A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching . Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off . Stationary phase cultures do not exhibit the cut . Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut . The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process . An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching . Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.

Proc Natl Acad Sci U S A, 1982 Nov, 79(22), 6827 - 31
Isolation and preliminary characterization of the GAL4 gene, a positive regulator of transcription in yeast; Laughon A et al.; The GAL4 locus encodes a positive regulator of the inducible galactose and melibiose genes of yeast . Using the yeast plasmid vector YEp13, we have cloned GAL4 by complementation of a gal4 mutation . Restriction endonuclease mapping of subclone DNA has delimited the region sufficient for complementation to a 3.2-kilobase segment of DNA . The GAL4 mRNA is 2.8 kilobases long, sufficient to encode a protein as large as 105,000 daltons . The concentration of the GAL4 transcript is about 0.1 per cell and is almost identical in galactose-induced and noninduced cells . This result is consistent with a previously proposed model in which the activity of the GAL4 protein and not the transcription of the GAL4 gene is modulated by galactose induction.

FEBS Lett, 1982 Nov 1, 148(1), 49 - 53
An obligatory role of protein glycosylation in the life cycle of yeast cells; Arnold E et al.; In the presence of 2-4 micrograms tunicamycin/ml, yeast cells stop growth after the cell number has increased 1.5-1.7-fold . The cells are arrested in G1: the rate of DNA synthesis greatly decreases and the budding index drops from 0.6 to 0.1 RNA synthesis is inhibited concomitantly, whereas protein synthesis is little affected . It is postulated that for G1/S phase transition to occur one or more proteins have to be glycosylated.

Nucleic Acids Res, 1982 Oct 11, 10(19), 5893 - 904
Yeast RNA polymerase I binds preferentially to A+T-rich linkers in rDNA; Gabrielsen OS et al.; Restriction fragments of yeast rDNA retained by purified RNA polymerases on nitrocellulose filters were analysed by gel electrophoresis . The EcoRI fragment B was preferentially retained by RNA polymerase I, but not by RNA polymerase III . The in vivo initiation sites for both polymerases are located within this fragment . Further analysis indicated that the preferred binding site for RNA polymerase I is highly AT-rich regions rather than a true promoter . The reported selective in vitro transcription of rDNA by purified yeast RNA polymerase I could then be explained by this preferential binding.

Nucleic Acids Res, 1982 Oct 11, 10(19), 5869 - 78
The structure of the gene coding for the phosphorylated ribosomal protein S10 in yeast; Leer RJ et al.; From previous studies on cloned yeast ribosomal protein genes we obtained evidence that a large number of them contain an intron {Bollen et al . (1982) Gene 18, 29-38} . In the temperature-sensitive rna2-mutant transcription of these genes leads to the accumulation of precursor RNAs at the restrictive temperature . These precursor mRNAs are several hundreds of nucleotides longer than the respective mature mRNAs . The split character of one of these ribosomal protein genes, viz . the gene coding for the major phosphorylated small-subunit protein S10, was further established by sequence analysis . The intervening sequence interrupts the coding sequence after the second codon and has a length of 352 nucleotides . Genomic Southern hybridizations with a DNA fragment carrying part of the S10-gene revealed that this gene is duplicated on the yeast genome . The molecular weight of S10 as deduced from the sequence analysis was estimated to be 31462 dal . Comparison of the N-terminal aminoacid sequence of the yeast ribosomal protein S10 with that of ribosomal protein S6 from rat liver revealed a striking homology between both proteins . Moreover, at the C-terminal end of the yeast ribosomal protein the sequence Arg-Ala-Ser-Ser-Leu-Lys is present which is very similar to the phosphorylation site of the rat liver protein S6.

Biochim Biophys Acta, 1982 Oct 5, 707(2), 280 - 8
Kinetic studies of ferrochelatase in yeast . Zinc or iron as competing substrates; Camadro JM et al.; Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) has been studied in yeast mitochondrial membranes with special reference to zinc-chelatase and iron-chelatase activities . Using physiological substrates (protoporphyrin IX, Fe(II) and Zn(II), anaerobic conditions of incubation and direct spectrophotometric assay, apparent Km values smaller than those previously described were found for the membrane-bound enzyme . Fe(II) but not Fe(III) was a strong competitive inhibitor of zinc-chelatase activity, while Zn(II) was a slight competitive inhibitor of iron-chelatase activity . These results could point to modes of control of ferrochelatase activity in yeast . We suggest that reduced supply of Fe(II) may explain the in vivo accumulation of zinc-protoporphyrin in yeast cells incubated under 'resting' conditions.

Biochim Biophys Acta, 1982 Oct 5, 707(2), 267 - 72
Specific modification of a single cysteine residue in both bovine liver glutamate dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase . Difference in the mode of modification by pyrene maleimide; Rasched I et al.; Pyrene maleimide is shown to be a 'half of the sites' reagent for glutamate dehydrogenase and for glyceraldehyde-3-phosphate dehydrogenase . The modified residues are identified as cysteine-115 for glutamate dehydrogenase and cysteine-149 for glyceraldehyde-3-phosphate dehydrogenase . The two enzymes react differently with pyrene maleimide . Whereas the hydrophobic environment of cysteine-115 directs the modification of glutamate dehydrogenase, the high reactivity of cysteine-149 determines the specific modification of glyceraldehyde-3-phosphate dehydrogenase . Glutamate dehydrogenase activity is unaltered by the modification: glyceraldehyde-3-phosphate dehydrogenase activity in inhibited.

Biochimie, 1982 Oct, 64(10), 941 - 7
Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide; Foucaud B et al.; Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett . 40, 29-32) . Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label . The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site . The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants . In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified . Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.

Biochem J, 1982 Oct 1, 207(1), 73 - 80
Multiple binding of thallium and rubidium to potassium-activated yeast aldehyde dehydrogenase . Influences on tertiary structure, stability and catalytic activity; Bostian KA et al.; Univalent cation activators of aldehyde dehydrogenase have dual effects, both interpreted as cation-induced or -stabilized conformation changes . These two processes are differentiated by the time scales of their associated changes in activity . Using Tl+ as an activator, under certain conditions, the slower change in activity saturates at a Tl+ concentration which is only 0.1 Ks for the faster change . This, together with evidence for cation-induced rather than cation-stabilized conformation changes, is used to propose separate binding sites for cations responsible for the two activation processes . Equilibrium dialysis indicates 4 binding sites per active site for Rb+ or 6 sites for Tl+ . At least one of the additional sites for Tl+ is an inhibitory site which has been differentiated from the activator sites on the basis of steady-state and pre-steady-state kinetic data.

J Inorg Biochem, 1982 Oct, 17(2), 121 - 9
Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes; Feldman I et al.; Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg2+, Mn2+, Ca2+, Cd2+, and Zn2+ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20 degrees C . At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn2+ or Ca2+ was present in the buffered isozyme solution before the glucose titration, whereas Mg2+ caused the glucose binding to become noncooperative . Zn2+ and Cd2+ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4% . Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II . Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca2+, Mg2+, or Mn2+, as well as in buffer alone, at both pH's . At the lower pH, Ca2+ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn2+ increased it only about 40% and Mg2+ not at all . Further, Ca2+ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg2+ and Mn2+ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase . However, at pH 6.5 the Ca2+ effect on the cooperativity was negligible, while Mg2+ and Mn2+ decreased the coefficient from 1.6 in buffer to about 1.4 . The biological implications of these diverse metal ion effects are discussed.

Int J Radiat Biol Relat Stud Phys Chem Med, 1982 Oct, 42(4), 369 - 83
Effect of gamma-radiation on the structure and function of yeast membrane; Khare S et al.; A decrease in the influx of several amino acids was observed following gamma-irradiation . At low dose (2.5 Gy), which does not affect cell survival, a stimulation in the uptake was visible; moreover, sulphydryl loss and lipid peroxidation were also evident . With further increase in the dose of radiation, a parallel increment in the loss of sulphydryl groups and production of malonaldehyde was observed . Radioprotectors like L-cysteine and dithiothreitol were shown to shield the radiation-induced loss of sulphydryl and damage to transport and survival . Reduced glutathione, on the other hand, exhibited protection at the level of sulphydryl damage only . N-ethylmaleimide, a well known hypoxic cell radiosensitizer, enhanced the radiosensitivity with respect to survival; it, however, had no effect on amino acid transport . Oxygen enhancement of radiation damage to transport and cell survival and the radioprotection by sodium formate under these circumstances, and more so by anoxia, were demonstrated . The results indicate that the manifestation of damage to membrane structure and function precedes any observable loss of survival.

Eur J Biochem, 1982 Oct, 127(2), 391 - 6
Biosynthesis of ascorbate in yeast . Purification of L-galactono-1,4-lactone oxidase with properties different from mammalian L-gulonolactone oxidase; Bleeg HS et al.; An enzyme from Saccharomyces cerevisiae which catalyzes the reaction: L-galactonolactone + O2 leads to L-ascorbate + H2O2 has been purified 466-fold from the mitochondrial fraction of a yeast homogenate . The enzyme has several properties that are different from the L-galactonolactone oxidase described by Nishikimi et al . {Arch . Biochem . Biophys . 191, 479-486 (1978)} . By gel filtration in the presence of sodium deoxycholate an apparent Mr of 70 000 was obtained for the active enzyme . Polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave an Mr of 74 000, whereas sodium dodecylsulfate/polyacrylamide gel electrophoresis showed only one protein band corresponding to an Mr of 18 000 . A tetrameric structure of the enzyme is thereby suggested . The substrate specificity is confined to the aldonoacid lactones L-galactono-, D-altrono-, L-fucono-, D-arabino- and D-threono-1,4-lactones . Competitive inhibition was demonstrated with L-gulono- and D-galactono-1,4-lactones . p-Chloromercuriphenyl sulfonate, iodoacetamide, N-ethylmaleimide, sulfite and sulfide were all inhibitory to the enzyme . No effect was seen when cyanide, azide, EDTA, alpha, alpha'-bipyridyl or bathocuproine disulfonate was added . An apparent Km of 0.3 mM with L-galactonolactone as a substrate was found . The Km for oxygen was 0.18 mM . The pH/activity curve exhibited a maximum around pH 8.9 and a shoulder at pH 6.5 . Evidence of a covalently bound flavin coenzyme and involvement of an iron-sulfur cluster was obtained from difference spectra of oxidized minus substrate-reduced enzyme with peaks or shoulders of the oxidized enzyme at 475, 445, 410, 375 and 350 nm . In sodium dodecylsulfate/polyacrylamide gels the enzyme subunit(s) had a bright yellow fluorescence after fixation in 7% acetic acid or 5% formaldehyde . The galactonolactone oxidase is stable with 50% activity being lost in 6 months at + 5 degrees C.

Cell, 1982 Oct, 30(3), 933 - 43
Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor; Kurjan J et al.; We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells . A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor . The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids . The putative precursor begins as a signal sequence for secretion . The next segment, of approximately 60 amino acids, contains three potential glycosylation sites . The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.

Eur J Biochem, 1982 Oct, 127(3), 605 - 8
Phosphorylation and inactivation of yeast fructose-bisphosphatase in vivo by glucose and by proton ionophores . A possible role for cAMP; Mazon MJ et al.; Addition of glucose to yeast cells causes a phosphorylation and an inactivation of the gluconeogenic enzyme fructose-bisphosphatase {Mazon, M.J., Gancedo, J.M., and Gancedo, C . (1982) J . Biol . Chem . 257, 1128-1130} . We report here that the addition of the proton ionophores 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone to yeast cells produces the same effect as that of glucose . Both glucose and ionophores produced: (a) phosphorylation and inactivation of fructose-bisphosphatase, (b) an immediate rise in the intracellular concentration of cAMP, (c) an instant inhibition of the transport of amino acids driven by the membrane potential . It is proposed that the effect of glucose on fructose-bisphosphatase involves as a first step the depolarization of the plasma membrane resulting in an increase of the intracellular concentration of cAMP . This in turn would stimulate phosphorylation of fructose-bisphosphatase.

Mol Biochem Parasitol, 1982 Oct, 6(4), 253 - 64
Identification of maxicircle DNA sequences in Leishmania Tarentolae that are homologous to sequences of specific yeast mitochondrial structural genes; Simpson L et al.; Sequences homologous to the yeast mitochondrial structural genes for cytochrome oxidase subunits I and II, ATPase 6 and cytochrome b were identified on the kinetoplast DNA maxicircle molecule by low stringency hybridization of maxicircle blots with heterologous probes derived from mitochondrial DNA of yeast petite mutants . No hybridization was observed with the yeast ATPase 9 gene probe . The relative extent of base sequence mismatch was determined by melting of the heterologous hybrids . Candidates for the transcripts of these presumptive structural genes were proposed with reference to the transcriptional map of the maxicircle of Leishmania tarentolae . These results provide the first indication that maxicircle DNA specifies information for a limited number of conserved mitochondrial gene products similar to those already described for other eukaryotic cells.

J Biol Chem, 1982 Sep 25, 257(18), 10536 - 9
A structurally modified yeast tRNAPhe with six nucleotides in the anticodon loop lacks significant phenylalanine acceptance; Nishikawa K et al.; The deletion of nucleoside 37 from yeast tRNAPhe was accomplished in a three-step procedure that involved (i) specific depurination of this purine under acidic conditions and removal of the carbohydrate moiety with aniline-2-aminopyridine, (ii) removal of phosphate monoesters from the 5'-half-molecule with alkaline phosphatase, and (iii) resealing of the anticodon loop with T4 RNA ligase . The course of the resealing reaction was monitored by polyacrylamide gel electrophoresis and found to be complete within a few hours when incubated at 37 degrees C in the presence of 215 units/ml of RNA ligase . Although the half-molecules used to prepare the modified tRNA had substantial phenylalanine acceptance when assayed at low ionic strength, the modified species itself was essentially devoid of phenylalanine acceptance . We conclude that the anticodon loop of tRNAPhe is involved in the activation of this tRNA and that both the presence of specific nucleotides in this loop and their ability to assume an appropriate spatial or conformational arrangement may be important for enzyme recognition.

J Biol Chem, 1982 Sep 25, 257(18), 11181 - 5
In vitro translation of mRNA for yeast citrate synthase; Alam T et al.; The citrate synthase of yeast was purified to homogeneity and shown to have a subunit molecular weight of 52,000 . Antibodies were prepared to it in rabbits . Translation of total yeast mRNA using a reticulocyte lysate showed that {3H}leucine was incorporated into a protein precipitable by rabbit anti-yeast citrate synthase with a molecular weight of about 54,000 . No incorporation of {35S}methionine into an anti-citrate synthase precipitable protein could be detected in a similar experiment . In vivo labeling of cells with 35SO4 did not result in the labeling of citrate synthase.

J Biol Chem, 1982 Sep 25, 257(18), 10644 - 9
AMP deaminase as a control system of glycolysis in yeast . Mechanism of the inhibition of glycolysis by fatty acid and citrate; Yoshino M et al.; The role of fatty acid and citrate on the interaction of the AMP deaminase (EC 3.5.4.6) reaction with glycolysis was investigated using permeabilized yeast cells . (a) Linolenate and citrate inhibited glycolytic flux and the recovery of the adenylate energy charge; however, linolenate remarkably retarded the depletion of the total adenylate pool, which was not at all affected by the addition of citrate . (b) Linolenate inhibited AMP deaminase activity in situ, resulting in the subsequent decrease in ammonium production, which reduced the activity of 6-phosphofructokinase (EC 2.7.1.11), whereas linolenate itself had no ability to inhibit the phosphofructokinase activity in the presence of excess ammonium concentration . (c) Citrate inhibited the activity of phosphofructokinase in situ in the presence and absence of ammonium ion, followed by an inhibition of glycolysis; however, AMP deaminase activity was not inhibited by citrate . The inhibition of glycolysis by fatty acids can be accounted for by the lowered activity of phosphofructokinase as a result of the decreased level of ammonium ion through the inhibition of the AMP deaminase reaction by these ligands, whereas the effect of citrate on glycolysis is a direct inhibition of phosphofructokinase without affecting the activity of AMP deaminase . Fatty acid and citrate, a principal metabolic product of fatty acid oxidation, can be responsible for the control of glycolysis in two different manners.

Biochemistry, 1982 Sep 14, 21(19), 4545 - 50
Isolation of seventeen proteins and amino-terminal amino acid sequences of eight proteins from cytoplasmic ribosomes of yeast; Otaka E et al.; Seventeen ribosomal proteins of Saccharomyces cerevisiae were isolated from small ribosomal subunits and disodium ethylenediaminetetraacetate treated 80S ribosomes by chromatography on a column of carboxymethylcellulose and/or by filtration through Sephacryl S-200 . The isolated proteins are YS4, YS7, YS8, YS9, YS10, YS12, YS14, YS18, YS23, YS29, YL11, YL13, YL16, YL17, YL22, YL38, and YL40 {nomenclature according to Otaka & Osawa (1981) {Otaka, E., & Osawa, S . (1981) Mol . Gen . Genet . 181, 176-182}} . The purification procedures and the amino acid compositions of these proteins are presented . Amino-terminal amino acid sequences of YS4, YS6, YS11, YS15, YS16, YS22, YL10, and YL31 have been determined and compared with those from rat liver {Wittmann-Liebold, B., Geissler, A . W., Lin, A., & Wool, I . G . (1979) J . Supramol . Struct . 12, 425-433} and Halobacterium cutirubrum {Matheson, A . T., Moller, W., Amons, R., & Yaguchi, M . (1980) in Ribosomes: Structure, Function and Genetics (Chambliss, G., et al., Eds.) pp 297-332, University Park Press, Baltimore, MD; M . Yaguchi, unpublished experiments}.

Nucleic Acids Res, 1982 Sep 11, 10(17), 5223 - 38
A yeast transcription system for the 5S rRNA gene; van Keulen H et al.; A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed . Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established . The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA . The in vitro transcription extract does not transcribe yeast tRNA genes . The extract does increase the transcription of tRNA genes packaged in chromatin.

Nucleic Acids Res, 1982 Sep 11, 10(17), 5149 - 60
The reactivity of sulfhydryl groups of yeast DNA dependent RNA polymerase I; Bull P et al.; The number of reactive cysteine residues of yeast RNA polymerase I was determined and their function was studied using parachloromercury benzoate (pCMB), dithiobisnitrobenzoate (DTNB) and N-ethyl-maleimide (NEM) as modifying agents . By treatment with DTNB about 45 sulfhydryl groups react in the presence of 8M urea . Under non-denaturing conditions only 20 sulfhydryl groups are reactive with pCMB and DTNB . Both reagents completely inactivate the enzyme and this effect can be reversed by reducing agents . The sedimentation coefficient and the subunit composition are not affected when the enzyme is inactivated . Two of the most reactive sulfhydryl groups are necessary for activity . The modification of these groups is partially protected by substrates and DNA, suggesting that they are involved either in catalysis or in the maintenance of the conformation of the active site . Experiments with 14C-NEM indicate that the most reactive groups are located in subunits of 185,000, 137,000 and 41,000 daltons.

Nucleic Acids Res, 1982 Sep 11, 10(17), 5197 - 208
Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling; Pedone F et al.; Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling . Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates . For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription . P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation . However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero . P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity . Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density . Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription.

J Biol Chem, 1982 Sep 10, 257(17), 9919 - 21
Effects of lipid fluidity on quenching characteristics of tryptophan fluorescence in yeast plasma membrane; Esfahani M et al.; Fluorescence characteristics of tryptophan residues in yeast plasma membrane indicate that the residues are buried . The fluorescence is fully quenchable by iodide with similar quenching kinetics at temperatures from 8 to 37 degrees C in oleate-enriched membranes and from 25 to 37 degrees C in palmitelaidate-enriched membranes . Substantial increases in lipid microviscosity in palmitelaidate-enriched membranes reduce the fraction of quenchable tryptophan fluorescence by about 40% and increase the effective quenching constant 3-fold . These observations indicate that at above 25 degrees C, proteins in this membrane undergo transient conformational changes and that freedom of conformational changes of the proteins is regulated by lipid microviscosity.

FEBS Lett, 1982 Sep 6, 146(1), 59 - 64
Covalent attachment of aspartic acid to yeast aspartyl-tRNA synthetase induced by the enzyme; Lorber B et al.; Aspartic acid can be covalently linked to yeast aspartyl-tRNA synthetase and to other proteins, in the absence of tRNA, under conditions where the synthetase activates the amino acid into aspartyl-adenylate, i.e., in the presence of ATP and MgCl2 . The linkage between aspartic acid and the protein is acid and alkali resistant; thus it is likely a peptide-like amide bond formed between the activated carboxylate group of aspartic acid and the primary amine function of the side chain of lysine residues.

Gene, 1982 Sep, 19(2), 225 - 30
Cloning of cDNA to a yeast viral double-stranded RNA and comparison of three viral RNAs; Bobek LA et al.; We have constructed recombinant DNA clones containing small complementary DNA (cDNA) sequences homologous to portions of a 4.8-kb yeast viral double-stranded RNA (dsRNA) (L1) that codes for the viral capsid polypeptide . Neither the viral dsRNA nor its in vitro transcript is polyadenylated; hence the cDNAs were synthesized by reverse transcriptase on the in vitro mRNA transcript made by the viral transcriptase, using sheared salmon sperm DNA as a random primer . This is the first reported cloning of cDNA homologous to a viral double-stranded RNA . This method should be of general utility for dsRNA viruses, since all have a capsid-associated transcriptase activity . The lengths of the overlapping cDNA inserts varied from 100 to 800 bp . About 40% of them mapped to the 5' end of the in vitro transcript, and these have been ordered . At least 1485 bp of this end of L1 is represented in the cloned cDNAs characterized . Using the cloned cDNAs as probes, we have shown that the L dsRNAs of two viral subtypes are similar at the transcription initiation site and dissimilar elsewhere.

Cell, 1982 Sep, 30(2), 617 - 26
Location and structure of the var1 gene on yeast mitochondrial DNA: nucleotide sequence of the 40.0 allele; Hudspeth ME et al.; Alleles of the var1 locus on yeast mitochondrial DNA specify the size of var1 ribosomal protein . We report the nucleotide sequence of a var1 allele that determines the smallest var1 protein . It contains an open reading frame of 396 codons, which we identify as the structural gene for var1 protein . The var1 protein specified by this allele has an amino acid composition in close agreement with that predicted by the DNA sequence . The var1 coding region is highly unusual: it is 89.6% AT and contains a 46 bp GC-rich palindromic cluster that accounts for 38% of the total GC residues . Our results strongly suggest that like mammalian mitochondria but unlike those from Neurospora, yeast mitochondria use AUA as a methionine codon . Comparison with the sequence of a var1 allele specifying a larger protein suggests that some size polymorphism of var1 protein results from in-frame insertions of a variable number of AAT (Asn) codons.

Z Naturforsch {C}, 1982 Sep, 37(9), 845 - 8
Yeast aminopeptidase II: rapid purification using affinity chromatography of the periplasmic enzyme; Knuver J et al.; Periplasmic aminopeptidase II of yeast was isolated from protoplast supernatants by ammonium sulfate precipitation, DEAE-Sephacel chromatography and affinity chromatography on immobilized bestatin . This isolation method is more rapid than conventional procedures and, in addition, avoids contact of the enzyme with yeast proteinases . Thus the preparation obtained is likely to represent the native periplasmic from of aminopeptidase II.

Gene, 1982 Sep, 19(2), 163 - 72
The yeast frameshift suppressor gene SUF16-1 encodes an altered glycine tRNA containing the four-base anticodon 3'-CCCG-5'; Gaber RF et al.; The SUF16 frameshift suppressor locus encodes a glycine tRNA . The SUF16-1 suppressor tRNA is inferred by DNA sequence analysis to contain the four-base anticodon sequence 3'-CCCG-5' in place of the wild-type anticodon 3'-CCG-5' . SUF16-1 mediates translation of the four-base messenger RNA (mRNA) sequence 5'-GGGU-3' but apparently fails to act at the sequence 5'-GGGG-3' . A molecular model is presented that accounts for the observed specificity of tRNA-mediated frameshift suppression in Saccharomyces cerevisiae.

Mol Biol (Mosk), 1982 Sep-Oct, 16(5), 948 - 55
{Construction of hybrid plasmids containing the yeast replicator}; Larionov VL et al.; In Saccharomyces cerevisiae strain 6-1G-P188 about 10 per cent of rRNA genes exist as extrachromosomal copies of rDNA repeating units . These extrachromosomal copies can be isolated as covalently closed molecules with lengths around 3mu . We have constructed a set of hybrid plasmids containing the bacterial vector pBR325, the LEU2 gene of yeast encoding beta-isopropylmalatedehydrogenase and various EcoRI restriction fragments of the 3mu DNA . We have tested the ability of our hybrid plasmids to transform LEU2 strain DC5 to leucine prototrophy . One of the plasmids Rcp21/11 transforms DC5 at the frequency comparable with that obtained with YEp13, containing the 2mu DNA replication origin . The 2400 bp EcoRI-B fragment of the 3mu DNA in Rcp21/11 carries a gene for 5S rRNA and two spacers . Our results on transformation experiments allow un to suggest that this EcoRI fragment also carries the 3mu DNA replication origin . Yeast transformants containing this plasmid are highly unstable but during the prolonged growth in selective conditions the stabilization of the LEU+ phenotype is observed being most likely a result of integration of Rcp21/11 into the yeast chromosome.

Eur J Biochem, 1982 Sep 1, 126(3), 631 - 7
Activity of cyclic-AMP phosphodiesterase in permeabilised cells of Bakers' yeast; Londesborough J; Yeast cyclic-AMP phosphodiesterases were assayed in situ, using cells permeabilised with cytochrome c, to get information about the kinetics of these enzymes at the high concentrations of macromolecules occurring in vivo . Protamine treatment was not suitable, because it perturbed the intracellular localisations of both the (Mg-dependent) low-Km enzyme and the (EDTA-insensitive) high-Km enzyme . The pH-dependence of Km and V for EDTA-insensitive activity in situ agreed well with the behaviour of pure high-Km enzyme, except that near pH 8 Hofstee plots were bent slightly upwards both for activity in situ and with crude broken-cell preparations . Hofstee plots of Mg-dependent activity in situ were distinctly concave, and could be resolved mathematically into two activities, one (accounting for about 30% of the Mg-dependent V) with a Km close to the value in vitro of 0.2 microM, and the other with an apparent Km of 3 microM . The 3 microM Km activity probably represents a fraction of the low-Km enzyme that is particle-bound at the high protein concentrations occurring in situ.

Proc Natl Acad Sci U S A, 1982 Sep, 79(18), 5621 - 5
Movement of yeast transposable elements by gene conversion; Roeder GS et al.; We have constructed yeast strains in which Ty (transposon yeast) elements at the HIS4 locus are genetically marked with the yeast URA3 gene . By isolating and analyzing Ura- derivatives of these strains, we have detected a variety of Ty-mediated recombination events . In this paper, we describe events in which the DNA sequence of the Ty element at the HIS4 locus is replaced by the DNA sequence of a different Ty element . These replacements occur without alterations in the flanking DNA sequence and without chromosomal aberrations . We believe that these events result from gene conversion between the Ty element at HIS4 and a Ty element at a different site in the yeast genome . Gene conversion can occur between Ty elements that differ by large insertion and substitution mutations . These recombination events result not only in the movement of Ty sequences but also in alterations in expression of the adjacent HIS4 gene . Different Ty elements at the same site in the HIS4 regulatory region can result in His-, His+, and cold-sensitive His+ phenotypes . Several Ty elements render expression of the HIS4 gene subject to control by genes at the mating type locus.

Proc Natl Acad Sci U S A, 1982 Sep, 79(17), 5342 - 6
Tandem gene amplification mediates copper resistance in yeast; Fogel S et al.; Resistance to copper's toxicity in yeast is controlled by the CUP1r locus . This gene was cloned by transforming sensitive recipients (cup1(8)) with a collection of hybrid DNA molecules, consisting of random yeast DNA fragments inserted into the vector YRp7 . Four resistant transformants were studied in detail . Autonomously replicating or integrated by homologous recombination into chromosomal sites, the corresponding plasmids and several subclones confer resistance on sensitive recipients carrying the natural variant allele, cup1(8) . Tetrad analysis and genetic mapping established that integration occurs typically at the cup1(8) site located 28 centimorgans distal to thr1, a chromosome VIII marker . Restriction endonuclease cleavage and electrophoretic mobility studies revealed that the CUP1r locus consists of a tandem array of repetitive units . Each unit is 1.95 kilobases in length and contains single sites for Kpn I and Xba I and two Sau3A sites . The sensitive allele represents one repeat and the resistant allele embraces 15 tandemly arrayed repeat units . Progressive selections in higher copper concentrations establish strains with markedly enhanced resistance . Resistance, we propose, is mediated by a gene amplification mechanism based on unequal sister chromatid exchange.

Biochemistry, 1982 Aug 31, 21(18), 4285 - 90
Binding of fluoride by yeast enolase; Bunick FJ et al.; The kinetics of fluoride binding by yeast enolase have been examined by direct measurement of equilibrium fluoride (F) concentrations with an ion-specific electrode . Mg2+ and inorganic phosphate (Pi) affect the binding of F, and evidence is presented for an ordered binding mechanism in which phosphate and F interact with conformational and catalytic Mg2+ species, with the overall formation of a quaternary complex . A maximum of four atoms of F were bound per enzyme dimer, and the data point to the nonequivalent binding of pairs of F atoms . The dissociation constants were 5.0 X 10(-4) M and 8.2 X 10(-5) M for the first and second pairs of F atoms, respectively . The first pair of binding sites was filled when the ratio of phosphate ions/enolase dimer exceeded 2 and appeared to involve the pair of conformation Mg2+ ions . The binding of the second pair of F atoms followed the binding of catalytic Mg2+ in the presence of Pi and, furthermore, appeared to exhibit positive cooperativity with respect to F . The data suggest, also, that the binding of Pi may involve sequential addition of Pi pairs to the different Mg2+ species on the enzyme . F binding was at a maximum between pH 5.5 and pH 6.0, consistent with an involvement of the monovalent form of Pi . In the absence of added Pi, (MgF)+ appeared to be the preferred ligand . Addition of the enzyme substrate 2-phosphoglycerate led to the release of bound F . These findings are consistent with the known patterns of inhibition of enzymatic activity by F.

Nature, 1982 Aug 26, 298(5877), 815 - 9
Reversion of a promoter deletion in yeast; Scherer S et al.; Promoter function in yeast has been examined by obtaining revertants of a his3 promoter deletion in vivo . Events which provide a new promoter for the his3 gene include insertion of the transposable element Ty1, rearrangements of the plasmid vector, and chromosomal mutations . A role for dicentric chromosomes as a source of the plasmid rearrangements is discussed.

Biochim Biophys Acta, 1982 Aug 23, 706(1), 111 - 6
Interaction of the AMP deaminase-ammonium system with glutamate dehydrogenase in yeast; Yoshino M et al.; NH+4 produced as a result of the activation of AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was utilized effectively to form glutamate from 2-oxoglutarate by the action of NADP-glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase (deaminating), EC 1.4.1.4) under in situ conditions in yeast cells: the decrease in total adenylates stoichiometrically corresponded to the production of NH+4 plus glutamate . Reducing equivalents, NADPH, for the synthesis of glutamate can be supplied by the pentose phosphate pathway . The addition of spermine, an activator of AMP deaminase without changes in glutamate dehydrogenase activity, resulted in an increase in ammonium concentration, which can enhance the formation of glutamate from 2-oxoglutarate . A close correlation of NADP-glutamate dehydrogenase with AMP deaminase activity was observed under various growth conditions . The interaction of the AMP deaminase-ammonium system with glutamate dehydrogenase as an ammonium-assimilating reaction may participate in the control of the cellular NH+4 level, which can correlate with glycolysis.

Biochemistry, 1982 Aug 17, 21(17), 3921 - 6
Specific interaction of anticodon loop residues with yeast phenylalanyl-tRNA synthetase; Bruce AG et al.; Thirteen different yeast tRNAPhe variants with single nucleotide changes in positions 34-37 in the anticodon region were prepared by an enzymatic procedure described previously . Aminoacylation kinetics using purified yeast phenylalanyl-tRNA synthetase revealed that the level of aminoacylation was very different for different sequences inserted . The low level of aminoacylation was the result of a steady state between a slow forward reaction rate and spontaneous deacylation of the product . Aminoacylation kinetics performed at higher synthetase concentrations revealed that substitution at position 34 in tRNAPhe decreased the Km nearly 10-fold but only had a small effect on Vmax . Similar substitutions at positions 35, 36, and 37 had a lesser effect . These data suggest a sequence-specific contact between the anticodon of yeast tRNAPhe and the cognate synthetase.

J Biol Chem, 1982 Aug 10, 257(15), 9119 - 27
Urea carboxylase and allophanate hydrolase are components of a multifunctional protein in yeast; Sumrada RA et al.; Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide . We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci and 2) pleiotropic mutations each resulting in the loss of both activities are found in both loci . These and other observations led to the hypothesis that urea degradation might be catalyzed by a multifunctional polypeptide . Waheed and Castric (1977) J . Biol . Chem . 252, 1628-1632), on the other hand, purified urea amidolyase from Candida utilis and reported it to be a tetramer composed of nonidentical 70- and 170-kilodalton subunits . To resolve the differing views of urea amidolyase structure, we purified the protein using rapid methods designed to avoid proteolytic cleavage . Application of these methods resulted in the isolation of a single, inducible and repressible, 204-kilodalton species . We observed no evidence for the existence of nonidentical subunits . A similar inducible, high molecular weight species was also detected in C . utilis . These biochemical results support our earlier hypothesis that urea degradation is carried out in yeast by an inducible and repressible protein composed of identical, multifunctional subunits.

Biochim Biophys Acta, 1982 Aug 6, 717(2), 203 - 9
Purification and identification of inactive forms of repressible and constitutive acid phosphatase in yeast; Schweingruber AM et al.; Acid phosphatase (EC 3.1.3.2, orthophosphoric-monoester phosphohydrolase, (acid optimum) from the budding yeast Saccharomyces cerevisiae was purified from repressed and derepressed cells . Without Triton X-100 in the extraction buffer only the constitutive or repressible active enzyme eluted from a Sepharose CL-6B column, the last step of the purification procedure . When Triton X-100 was included in the extraction buffer, an additional protein peak eluted prior to the active acid phosphatase . The material from this new peak, a glycoprotein, had no acid phosphatase activity but cross-reacted with antibodies raised against repressible acid phosphate . The tryptic fingerprints of the inactive proteins are very similar to the ones of the corresponding active enzymes . We conclude that this new glycoprotein represents an inactive form of repressible and constitutive acid phosphatase . The fact that inactive acid phosphatase can be recovered only in the presence of Triton X-100 indicates that it is membrane-bound.

Can J Microbiol, 1982 Aug, 28(8), 942 - 4
A cyclohexanecarboxylic acid utilizing yeast: isolation, identification, and nutritional characteristics; Higuchi K et al.; A yeast capable of utilizing cyclohexanecarboxylic acid as sole carbon and energy source, strain KUY-6A, was isolated from soil by enrichment cultures . Taxonomical studies indicated that strain KUY-6A was Trichosporon cutaneum . Strain KUY-6A grew on a number of carboxylic acids . Among the cyclic compounds tested, cyclohexanecarboxylic acid was the best substrate . Cyclopentanecarboxylic acid, cycloheptanecarboxylic acid, cyclopentanone, cyclohexanone, and cyclopentanol also supported growth . In addition, the organism used the monocarboxylic acids, butyric, valeric, and caproic; the dicarboxylic acids succinic, glutaric, adipic, pimelic, and suberic; and the aromatic acids, benzoic and o-, m-, and p-hydroxybenzoic . The yeast did not require any vitamins for growth, although thiamine gave slight stimulation . The cell dry weight yield was 0.75 g from 1 g cyclohexanecarboxylic acid used.

Biochem J, 1982 Aug 1, 205(2), 453 - 6
Metal-ion-promoted binding of triazine dyes to proteins . The interaction of Cibacron Blue F3G-A with yeast hexokinase; Hughes P et al.; Bivalent metal ions, particularly Zn2+ and other members of the first-row transition series, promote irreversible inactivation of yeast hexokinase by Cibacron Blue F3G-A at a site competitive with both ATP and D-glucose . Difference spectroscopy indicates that the protein-dye dissociation constant is decreased from 250 micrometers in the absence of metal ions to less than 100 micrometers in the presence of appropriate concentrations of metal ions, with specificity displayed in the sequence of Zn2+ greater than Cu2+ greater than Ni2+ greater than Mn2+ . Quantitative inactivation of yeast hexokinase leads to the incorporation of approx . 1 mol of Cibacron Blue F3G-A/mol of subunit of mol . wt . 51 000 in both the presence and the absence of metal ion . These results suggest the formation of a highly specific ternary complex involving enzyme, dye and metal ion at the active-site region of the enzyme, and correlate well with the known effects of metal ions in promoting the binding of hexokinase to immobilized Cibacron Blue F3G-A.

Mol Cell Biol, 1982 Aug, 2(8), 985 - 92
Sensitivity to the Yeast Plasmid 2mu DNA is conferred by the nuclear allele nibl; Holm C; Two strains of Saccharomyces carlsbergensis that lacked the plasmid 2mu DNA responded differently when the plasmid was introduced into them . In one strain, cells lacking 2mu DNA ("cir0") produced the normal "smooth" colony morphology, but cells bearing 2mu DNA ("cir+") produced heterogeneous "nibbled" colonies . In the second strain, both cir+ and cir0 strains exhibited a smooth colony morphology . Crosses between these strains revealed that a single recessive nuclear gene, called nibl, conferred the nibbled colony morphology in the presence of 2mu DNA . By a series of backcrosses, nibl was introduced into a Saccharomyces cerevisiae background . nibl caused a nibbled colony morphology in this background just as it did in S . carlsbergensis . nibl was mapped to the left arm of chromosome XVI . Twelve independent smooth revertants were isolated from two nibl {cir+} strains . Seven were analyzed, and all were found to be chromosome VII disomes . Chromosome VII disomy and suppression of the nibbled phenotype cosegregated in crosses . Thus, chromosome VII disomy can suppress the nibbled phenotype . The results of other experiments (C . Holm, Cell 29:585-594, 1982) indicate that the nibbled colony morphology is the result of lethal sectoring and that the lethality is caused by a high copy number of 2mu DNA . I suggest, therefore, that the product of the nibl gene may play a role in controlling the copy number of 2mu DNA . Possible models for the suppression of the nibbled phenotype by chromosome VII disomy are discussed.

J Lipid Res, 1982 Aug, 23(6), 803 - 10
Effect of detergents on sterol synthesis in a cell-free system of yeast; Hata S et al.; In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated . Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined . Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol . Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41) . The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture . The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.

Eur J Biochem, 1982 Aug, 126(1), 77 - 81
Interference of ligands on the phenylalanyl-tRNA synthetase from yeast; Thiebe R; The present paper reports a study of the mutual interactions between the substrates, the intermediate, and the products of the aminoacylation reaction, when bound to the phenylalanyl-tRNA synthetase from yeast . The following conclusions can be drawn . a) tRNAPhe displaces Phe-tRNAPhe from the synthetase by lowering the affinity of the enzyme for the aminoacylated tRNA . b) Phe-tRNAPhe and Phe-AMP compete for the catalytically active site of the enzyme . c) Chemically synthesized Phe-AMP, when added to the synthetase, primarily forms a low-affinity complex with the enzyme . The transformation of this complex into the high-affinity catalytic complex is a very slow process . These findings confirm a previous study, based on steady-state kinetics . A schematic representation of the aminoacylation process is given . It summarizes the present and previous results and illustrates a rather complex 'flip-flop' mechanism.

Cell, 1982 Aug, 30(1), 305 - 10
Periodic transcription of yeast histone genes; Hereford L et al.; Periodic transcription of yeast histone genes has been demonstrated by DNA excess filter hybridization of in vivo pulse-labeled RNA isolated from synchronous cell cultures . Using strains carrying cell division cycle (cdc) mutations, we show that both activation and termination of transcription are determined by temporally separable (cell cycle) events . Activation of histone mRNA synthesis occurs late in G1, at a point prior to initiation of DNA replication . Cessation of histone mRNA synthesis, however, is dependent upon the entry of cells into S . These results suggest a simple model for the control of histone gene transcription in which changes in chromatin that must precede the initiation of DNA replication also bring about activation of histone mRNA synthesis . Cessation of synthesis would occur once this region had been replicated and the chromatin restored to its prereplicative state.

Mutat Res, 1982 Aug, 105(1-2), 59 - 63
A correlation between ethidium bromide uptake and petite mutagenesis during the yeast cell cycle; Cottrell SF; The intracellular uptake of radioactively labeled ethidium bromide is shown to exhibit a periodic increase at the beginning of each cell cycle in synchronously growing cultures of the yeast, Saccharomyces cerevisiae . These peak rates of ethidium bromide uptake coincide in time with both new rounds of bud formation and the initiation of nuclear DNA synthesis during each cell cycle . Moreover, the peak rates of ethidium bromide uptake are also shown to be correlated in time with the maximum rate of petite mutagenesis induction by this drug during successive cell cycles.

J Inorg Biochem, 1982 Aug, 17(1), 15 - 28
Circular dichroism (CD) studies on yeast enolase: activation by divalent cations; Collins KM et al.; The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes . This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure . There was no effect of magnesium ion on the far ultraviolet spectrum . Evidently magnesium ion has no effect on the secondary structure . Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity . The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions . Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes . The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups . Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme . The "catalytic" and "inhibitory" sites did not appear to be very CD active.

Fed Proc, 1982 Aug, 41(10), 2653 - 5
Construction of yeast strains containing genetically marked transposons; Roeder GS et al.; Haploid yeast cells contain approximately 35 Ty (transposon yeast) elements . To facilitate the study of these elements, we have constructed yeast strains in which one of these transposons carries a genetic marker . The elements that we have modified are Ty912 and Ty917, elements originally detected at the HIS4 locus in spontaneously occurring his4- mutants . The strain constructions took place in three stages: 1) cloning of the mutant HIS4 genes containing the Ty elements; 2) introduction of a HindIII restriction fragment containing the yeast URA3 gene into the cloned transposons; and 3) replacement of the chromosomal HIS4 gene with the modified genes constructed in vitro . These strains will be extremely useful in the study of Ty transposition and other Ty-promoted DNA rearrangements.

Eur J Biochem, 1982 Aug, 126(2), 319 - 25
Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast; Sharma CB et al.; Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III) . The test conditions are described in detail . All three enzymes were solubilized from yeast membranes with detergents . Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively . The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+ . Enzyme II was stimulated almost to the same extent also by Ca2+ . The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM . The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose) . The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1 . All reactions were inhibited by nucleoside diphosphates.

Biochim Biophys Acta, 1982 Jul 26, 705(2), 163 - 6
Characterization of the proteolytic activity firmly attached to yeast phoshoenolpyruvate carboxykinase; Beck I et al.; Incubation of partially purified yeast phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) with 5% mercaptoethanol and 0.01% sodium dodecyl sulfate at 37 degrees C results in degradation of the enzyme . The degradation can be partially prevented by addition of proteinase B inhibitor 2 or phenylmethylsulfonyl fluoride, an inhibitor of proteinase B and carboxypeptidase Y . The degradation can be completely inhibited by addition of proteinase B inhibitor 2 together with pepstatin, and inhibitor of proteinase A . Thus it appears that proteolytic activities are firmly attached to phosphoenolpyruvate carboxykinase and are identical with the yeast proteinases A and B . The latter conclusion was supported by experiments using the pure yeast proteinases.

J Biol Chem, 1982 Jul 25, 257(14), 8432 - 41
Specific transcription of homologous class III genes in yeast-soluble cell-free extracts; Klekamp MS et al.; Cell-free extracts prepared from whole yeast cells carry out selective and accurate transcription, in vitro, of purified yeast class III genes . Both 5 S rRNA and tRNA genes are specifically transcribed by DNA-dependent RNA polymerase III present in these whole cell extracts . These extracts also appear to carry out nucleolytic processing of the in vitro synthesized transcripts . Optimal conditions for specific class III gene transcription in vitro are defined . Initial fractionation of the yeast extract has indicated that multiple chromatographically separable factors (fractions) are required, in addition to RNA polymerase III, for specific in vitro transcription of class III genes.

Science, 1982 Jul 23, 217(4557), 371 - 3
Lethal disruption of the yeast actin gene by integrative DNA transformation; Shortle D et al.; A mutant allele of the chromosomal locus corresponding to the cloned actin gene of the yeast Saccharomyces cerevisiae has been constructed by DNA transformation with a hybrid plasmid which integrates into, and thereby disrupts, the protein-encoding sequences of the gene . In a diploid strain of yeast, disruption of the actin gene on one chromosome results in a mutation that segregates as a recessive lethal tightly linked to a selectable genetic marker on the integrated plasmid . The actin gene, therefore, must encode an essential function for yeast cell growth.

Mol Cell Biochem, 1982 Jul 23, 46(2), 65 - 71
Low Km cyclic AMP phosphodiesterase of yeast may be bound to ribosomes associated with the nucleus; Londesborough J et al.; The sub-cellular distribution of low Km cyclic AMP phosphodiesterase (defined as the EDTA-sensitive activity at 1 microM cyclic AMP) was examined using spheroplast lysates and mechanical disintegrates of yeast . Close to 65% of the enzyme was particle-bound in each case . Most of the bound activity in mechanical disintegrates sedimented at 145 000 g in an RNA-rich fraction, and could be solubilised from this fraction by RNase treatment . With spheroplast lysates, however, 50% of the enzyme co-sedimented with DNA at 5 000 g, and the highest specific activity was in purified nuclei with a protein/DNA mass ratio of 16 . The results suggest that at least 50% of the enzyme is bound by ribosomes attached to the outer nuclear membrane.

Nature, 1982 Jul 22, 298(5872), 391 - 3
A bifunctional gene product involved in two phases of the yeast cell cycle; Piggott JR et al.; The cell cycle in Saccharomyces cerevisiae is divided into two distinct phases . Unbudded, mononucleate cells in the G1 phase can react to relevant environmental changes by mating, sporulating, or by entering stationary phase . DNA synthesis and bud initiation occur almost simultaneously and mark 'commitment' to the completion of mitosis . Temperature-sensitive mutations at the cdc28 locus are known to cause arrest in the G1 phase of the cell cycle at the restrictive temperature . Here we show that the cdc28 gene product is also active in post-G1 cell cycle functions, and that a different property of the gene product may be required for each phase of the cycle in which it acts.

Nature, 1982 Jul 22, 298(5872), 347 - 50
Synthesis and assembly of hepatitis B virus surface antigen particles in yeast; Valenzuela P et al.; The surface antigens of hepatitis B virus (HBsAg) has been synthesized in the yeast Saccharomyces cerevisiae by using an expression vector that employs the 5'-flanking region of yeast alcohol dehydrogenase I as a promotor to transcribe surface antigen coding sequences . The protein synthesized in yeast is assembled into particles having properties similar to the 22-nm particles secreted by human cells.

J Biol Chem, 1982 Jul 10, 257(13), 7879 - 86
Purification and polypeptide characterization of complex III from yeast mitochondria; Sidhu A et al.; Complex III was isolated and purified from bakers' yeast by ammonium sulfate fractionation and column chromatography on Ultrogel AcA 34 . The purified complex contained 7.03 nmol/mg of protein and 4.24 nmol/mg of protein of cytochromes b and c1, respectively . The specific activity of the complex was 17.1 mumol/min/mg of protein, using the decyl analog of coenzyme Q as substrate . Electrophoresis of the purified complex revealed the presence of seven polypeptides with molecular weights ranging from 15,500 to 50,000 . Polypeptides having molecular weights lower than 15,000 were not observed, except when the complex was dissociated in the absence of proteolytic inhibitors, suggesting that these low molecular weight species arise as a result of proteolytic digestion of the complex . The isoelectric points of the subunits of complex III and their stoichiometry wee determined . Trypsin and chymotrypsin digestion of the oxidized and reduced forms of the isolated complex suggested that the two high molecular weight core proteins are embedded within the complex and hence are inaccessible to the exogenous proteases, while cytochromes b and c1, the iron-sulfur protein, and the 17,500-dalton subunit are substantially exposed to the surface of the complex . The iron-sulfur protein appears to undergo a conformational change upon reduction of the complex, rendering it less susceptible to trypsin digestion . The core proteins and the iron-sulfur protein were purified, and antibodies against these proteins were raised . Immunoinhibition studies with these antibodies also indicated that the antigenic sites of the core proteins were embedded in the complex.

J Biol Chem, 1982 Jul 10, 257(13), 7756 - 61
The molecular characterization of three transcriptional mutations in the yeast iso-2-cytochrome c gene; Montgomery DL et al.; Three mutations, each of which causes overproduction of iso-2-cytochrome c, were characterized biochemically . Two, CYP3-4 and CYP3-15, were previously shown to be cis-dominant and map to the CYC7 locus which encodes the iso-2 protein, while the third, cyp1-16, maps to an unliked locus . All three mutations caused dramatically increased levels of transcription of the CYC7 gene, and the CYC7 mRNA in mutant cells was found to be the same size as that in wild type cells . The CYP3-4 mutation was found to be caused by the integration of a transposable element, Tyl, 269 base pairs 5' to the coding sequences . The CYP3-15 mutation was also found to alter the DNA, probably through a deletion or inversion with one endpoint 285 base pairs upstream from the coding sequence . The CYC7 gene in both wild type and mutant cells was not subject to catabolite repression.

Biochimie, 1982 Jul, 64(7), 477 - 86
{Comparative study on the chemical modification of sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenases from yeast and rabbit muscle . The relationship between structure and chemical reactivity}; Bodo JM et al.; Chemical modification of cystein 149 residues from yeast apo-glyceraldehyde-3-phosphate dehydrogenase either by iodoacetamidonaphtol or N-(4-dimethylamino-3,5-dinitrophenyl) maleimide results in the disappearance of free sulfhydryl groups according to "full sites reactivity", whereas loss of the dehydrogenase activity occurs following "half of the sites reactivity" . Chemical modification of the same cystein residues of the rabbit muscle apoenzyme by N-(4-dimethylamino-3,5-dinitrophenyl) maleimid shows that both loss of activity and disappearance of the sulphydryl groups may be described as "full sites reactivity" phenomena . After chemical modification by iodoacetamidonaphtol both processes follow "half of the sites reactivity".

J Bacteriol, 1982 Jul, 151(1), 177 - 85
13C nuclear magnetic resonance study of trehalose mobilization in yeast spores; Barton JK et al.; Using high-resolution 13C nuclear magnetic resonance, we examined the mobilization of endogenous trehalose in suspensions of yeast asci . Sporulation of yeast cells in {1-13C}acetate resulted in incorporation of label into the C-3 and C-4 positions of trehalose within the asci . During germination of these asci with {1-13C}glucose, the consumption of both endogenous trehalose and exogenous glucose were followed simultaneously by 13C nuclear magnetic resonance, as was the formation of glycerol and ethanol, their glycolytic and products . Time courses for carbohydrate consumption indicated that trehalose, although it decreased to 25% of its initial value upon germination, was not preferentially catabolized and did not provide the primary energy supply for germination with glucose . The ratio of trehalose to glucose catabolized was 0.09 . Exogenous glucose levels appeared to regulate trehalose mobilization since trehalose was only consumed when sufficiently high levels (more than 2 mM) of glucose were present . Upon glucose depletion newly synthesized {1-13C}trehalose was observed . Nuclear magnetic resonance spectra of extracts confirmed the trehalose peak assignments and showed products of {1-13C}glucose catabolism . In addition by quantitating trehalose consumption and 2-deoxyglucose incorporation in dormant yeast asci, we found that 3.8 +/- 0.l4 molecules of 2-deoxyglucose were incorporated for each trehalose molecule consumed . Trehalose can therefore function as a carbohydrate source for ATP formation during dormancy.

Fundam Appl Toxicol, 1982 Jul-Aug, 2(4), 168 - 72
The nature of salicylate inhibition of sugar transport in yeast; Scharff TG et al.; A study was made in yeast of the cellular site associated with inhibition of sugar transport by salicylate . Time-course and stereospecificity studies were done on the uptake of salicylic acid (sal), meta-hydroxybenzoic acid (m-OH), D-glucose (gluc) and 3-0-methyl-D-glucose (3-OMG) at 20 and 30 degrees C . Sal added simultaneously with 3-OMG had little effect on initial uptake rates of the latter . Inhibition of 3-OMG transport occurred only after appreciable uptake of sal . Rates of glucose utilization in cells preincubated with sal increased with time despite increased drug in the medium as sal decreased in the cells . Benzoic acid also inhibited 3-OMG transport . m-OH or para-hydroxybenzoic acid entered cells but neither substance inhibited 3-OMG transport nor affected the inhibition by sal of 3-OMG transport . Benzyl and salicyl alcohols had no effect on 3-OMG transport . The results indicate a stereospecific binding site or sites for sal located in the cell interior and associated with inhibition of sugar transport . The known inhibition of sugar transport by sal in susceptible mammalian cells, including human cells, may also occur through binding of sal to intracellular, stereospecific sites.

Biochimie, 1982 Jul, 64(7), 509 - 22
How the loss of several residues, at the level of one interglobule junction, modulates the lactate dehydrogenase activity of yeast flavocytochrome b2: a study of the nicked enzymes resulting from clostripain and trypsin action; Gervais M et al.; The native chain of flavocytochrome b2 is folded into three globules linked together by two protease-sensitive bridges "a" and "cd" . We show in this paper that zone "a" of H-flavocytochrome b2 is the first to be cleaved under clostripain action . The alpha c and beta c fragments thus formed are homologous to alpha T and beta'T trypsic fragments . The remaining activities of the resulting (alpha c beta c) and alpha T beta'T) forms are only 25 per cent and 4 per cent of the native flavocytochrome b2 one . The study of the catalytic properties of (alpha c beta'T) and (alpha T beta c) species resulting from the crossed reassociation of the isolated fragments show that the beta type fragment plays a critical role in the catalytic process . A dramatic activity decrease may be correlated with the loss of 6 amino acid residues at the N-terminal of beta c . Our best hypothesis is that these amino acids are involved in the active site, which may be located in the contact zone between alpha and beta . These results are in agreement with previous results obtained in this laboratory which showed the necessity of both alpha T and beta'T fragments for the correct conformation of the flavin binding site.

Eur J Biochem, 1982 Jul, 125(2), 445 - 51
Yeast mutant defective in synthesis of phosphatidylinositol . Isolation and characterization of a CDPdiacylglycerol--inositol 3-phosphatidyltransferase Km mutant; Nikawa J et al.; 1 . A Km mutant of Saccharomyces cerevisiae with a lesion in CDPdiacylglycerol-inositol 3-phosphatidyltransferase was isolated . The mutant required a high concentration of myo-inositol for growth . 2 . The CDPdiacylglycerol-inositol 3-phosphatidyltransferase in the mutant cells showed an apparent Km for myo-inositol over 200-times higher than that of the enzyme in wild-type cells . The maximum velocity of the mutant enzyme was comparable to that of the wild-type enzyme . 3 . In mutant cells, labelled myo-inositol, phosphate and acetate were incorporated into phosphatidylinositol at much slower rates than in wild-type cells . The phosphatidylinositol content of mutant cells was markedly lower than that observed in wild-type cells . 4 . Genetic analysis showed that the growth phenotype of the mutant arose from a single nuclear gene mutation in a gene coding for CDPdiacylglycerol-inositol 3-phosphatidyltransferase . 5 . The mutant showed a normal level of phosphatidylserine synthase activity . The phosphatidylserine synthase gene was located between ura3 and hom3 on chromosome V, whereas the CDPdiacylglycerol-inositol 3-phosphatidyltransferase gene showed no linkage with ura3 . 6 . Labelled acetate was incorporated into various lipids including triacylglycerols, diacylglycerols, sterol esters and phospholipids other than phosphatidylinositol at faster rates in mutant cells than in wild-type cells . Incorporation into both the fatty acid and the sterol moieties was facilitated in the mutant . 7 . A striking change in the cell-division process was observed when phosphatidylinositol synthesis was limited . The results showed that phosphatidylinositol synthesis is involved in the cell-division cycle of yeast.

Can J Biochem, 1982 Jul, 60(7), 757 - 62
Some properties of a mitochondrial endonuclease from yeast; Morosoli R et al.; A mitochondrial endonuclease from Saccharomyces cerevisiae was previously shown to cut both strands of native DNA at opposite or nearby sites . The present studied demonstrate that the endonuclease activity is dependent on the strength of the hydrogen bonds between the DNA strands; the activity was measured at different ionic strengths, with substrates of different base compositions and also with DNA in which the double helix has been locally destabilized by ultraviolet irradiation, by depurination, and by single-stranded nicks . The activity is 30% greater with mitochondrial DNA (mt-DNA) than with nuclear DNA . At 0.08 ionic strength, the relative activities with double-stranded polydeoxyribonucleotides are 2.4:1:0.6 for poly(dA).poly(dU) : poly(dA).poly(dT) : poly(dG) . poly(dC) . Increasing ionic strength decreases similarly the activity with poly(dA).poly(dU) and poly(dA).poly(dT), but has little effect with poly(dG).poly(dC) . The greater activity with poly(dA).poly(dU) than with poly(dA).poly(dT) was confirmed with nick-translated mt-DNA and with DNA synthesized in isolated mitochondria using {3H}TTP and {3H}dUTP in both cases . The endonuclease cuts modified DNA preferentially in the thymine dimer regions, at the apurinic sites, and at sites opposite to nicks . The possible involvement of this endonuclease in the degradation of mitochondrial DNA during "petite" induction is discussed.

Proc Natl Acad Sci U S A, 1982 Jul, 79(13), 4138 - 42
sigma, a repetitive element found adjacent to tRNA genes of yeast; del Rey FJ et al.; sigma is a DNA element of about 340 base pairs (bp) that is repeated many times in the yeast genome . The element has 8-bp inverted repeats at its ends and is flanked by 5-bp direct repeats . The 5-bp repeats are different for each sigma and have no homology with the ends of the sigma sequence . sigma is located 16 or 18 bp from the 5' end of several tRNA genes . Southern analysis of different yeast strains shows that the pattern of hybridization is different even for closely related strains.

Biochim Biophys Acta, 1982 Jun 30, 697(3), 363 - 70
Activation of a latent RNAase from yeast by nucleoside triphosphates; Belhadj O et al.; A latent RNAase activity stimulated by nucleoside triphosphates has been isolated from a yeast chromatin extract, by filtration on Sepharose 6B and hydroxyapatite chromatography . The RNAase was separated from a thermolabile proteic inhibitor on phosphocellulose . When separated from the inhibitor, the RNAase hydrolyses RNA to 5'-mononucleotides . Its activity is retained in the presence of EDTA, and 50% inhibited by 1 mM ATP or CTP . The RNAase is inhibited by the thermolabile component only in the presence of divalent cations . The activity is recovered upon addition of 0.01 mM ATP to the mixture . The Km for ATP is 10 microM . ATP can be replaced by other ribo- or deoxyribonucleoside triphosphates with varying efficiency but not by ADP, AMP or cAMP . These results suggest multiple interactions between the RNAase, a regulatory component, divalent cations and nucleoside triphosphates.

Nucleic Acids Res, 1982 Jun 25, 10(12), 3715 - 32
Enzymatic replacement in vitro of the first anticodon base of yeast tRNAAsp: application to the study of tRNA maturation in vivo, after microinjection into frog oocytes; Carbon P et al.; A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C) . This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position . Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions . Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte . Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U . In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte . The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.

Nucleic Acids Res, 1982 Jun 25, 10(12), 3617 - 26
Initiation of transcription of genes for mitochondrial ribosomal RNA in yeast: comparison of the nucleotide sequence around the 5'-ends of both genes reveals a homologous stretch of 17 nucleotides; Osinga KA et al.; The DNA sequence around the beginning of the genes coding for the large and small ribosomal RNAs in yeast mitochondria has been established . In order to determine the 5'-end points of the ribosomal RNAs, DNA fragments were labelled in vitro at a restriction site within each gene and hybridized with ribosomal RNA . The hybrids were then treated with S1 nuclease and the products analysed for size by gel electrophoresis . This enabled us to identify where in the determined DNA sequence the 21S ribosomal RNA and the precursor for 15S ribosomal RNA (15.5S rRNA) start, since both transcripts are initiated de novo (Levens et al . (1981) J.Biol.Chem., 256, 5226-5232) . Comparison of the DNA sequences around the start points of transcription reveals the existence of a homologous stretch of 17 nucleotides . This conserved sequence may be an essential element of a promoter in mtDNA.

J Biol Chem, 1982 Jun 25, 257(12), 7181 - 8
Targeted deletion of a yeast enolase structural gene . Identification and isolation of yeast enolase isozymes; McAlister L et al.; Yeast contain two nontandemly repeated enolase structural genes which have been isolated on bacterial plasmids designated peno46 and peno8 (Holland, M . J., Holland, J . P., Thill, G . P., and Jackson, K . A . (1981) J . Biol . Chem . 256, 1385-1395) . In order to study the expression of the enolase genes in vivo, the resident enolase gene in a wild type yeast strain corresponding to the gene isolated on peno46 was replaced with a deletion, constructed in vitro, which lacks 90% of the enolase coding sequences . Three catalytically active enolases are resolved differ DEAE-Sephadex chromatography of wild type cellular extracts . As expected, a single form of enolase was resolved from extracts of the mutant cell . Immunological and electrophoretic analyses of the multiple forms of enolase confirm that two enolase genes are expressed in wild type cells and that isozymes are formed in the cell by random assortment of the two polypeptides into three active enolase dimers . The yeast enolase loci have been designated ENO1 and ENO2 . The deletion mutant lacks the enolase 1 polypeptide confirming that this polypeptide is encoded by the gene isolated on peno46 . The intracellular steady state concentrations of the two polypeptides are dependent on the carbon source used to propagate the cells . Log phase cells grown on glucose contain 20-fold more enolase 2 polypeptide than enolase 1 polypeptide, whereas cells grown on ethanol or glycerol plus lactate contain similar amounts of the two polypeptides . The 20-fold higher than in cells grown on the nonfermentable carbon sources . In vitro translation of total cellular RNA suggests that the steady state concentrations of the two enolase mRNAs in cells grown on different carbon sources are proportional to the steady state concentrations of the respective enolase polypeptides.

Nucleic Acids Res, 1982 Jun 11, 10(11), 3341 - 52
Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes; Uhlenbeck OC et al.; Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase . The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe . Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all . Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding . However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.

J Biol Chem, 1982 Jun 10, 257(11), 6494 - 500
Transcriptional initiation and processing of the small ribosomal RNA of yeast mitochondria; Christianson T et al.; We have identified the nucleotide at which transcription initiates on the yeast mitochondrial small (14 S) rRNA gene by sequencing of RNA labeled at the 5' initiating triphosphate with vaccinia virus guanylyltransferase {alpha-32P}GTP (in vitro capping reaction) . Initiation occurs within the stem of a 12-base palindromic repeat . The initiation sequence has homology with the large (21 S) ribosomal RNA initiation sequence that has been previously determined . We have also sequenced the 5' and 3' ends of the mature 14 S rRNA after labeling with T4 polynucleotide kinase and RNA ligase, respectively . These sequences demonstrate that about 80 nucleotides are cleaved from the 5' end of a precursor to produce the mature 14 S rRNA . This cleavage is imprecise in that the processing occurs at one of five adjacent nucleotides 77 to 81 nucleotides downstream from the 5' initiation site . The 3' ends of this precursor and the mature 14 S rRNA are unique and identical.

Cell, 1982 Jun, 29(2), 585 - 94
Clonal lethality caused by the yeast plasmid 2 mu DNA; Holm C; Strains of Saccharomyces that carry the nib allele of a nuclear gene exhibit a "nibbled" colony morphology if they also harbor the plasmid 2 mu DNA . I have found that the expression of the nibbled phenotype is correlated with the presence of a subpopulation of abnormally large cells that give rise to mortal clones . Large cells apparently become large as a consequence of a defect in DNA replication or nuclear division . Large nib cells contain twice as much 2 mu DNA per microgram of total DNA as small nib cells do, and elevated 2 mu DNA copy number is the cause, not the effect, of increased cell size . It appears that the NIB allele can prevent an increase in 2 mu DNA copy number, but cannot produce a decrease once the copy number has exceeded the normal level . I propose, therefore, that the NIB gene product normally represses the amplification of 2 mu DNA copy number, and that the nib allele is partially defective in this function.

Cell, 1982 Jun, 29(2), 527 - 36
Expression of the split gene cob in yeast: evidence for a precursor of a "maturase" protein translated from intron 4 and preceding exons; Weiss-Brummer B et al.; Intron 4 (14) of the split gene cob in mitochondrial DNA contains a long open reading frame in phase with the preceding exon . Mutations in this intron block the excision of the 14 sequence from the cob precursor RNA and, at the same time, generate a series of new polypeptides, parts of which apparently result from translation of 14 sequences . We sequenced six mutations clustered in the upstream part of the open reading frame, about 340 bp from the exon-intron boundary (box9 cluster) . Four are base pair exchanges in the same triplet of this region; these form the polypeptides typical for 14 plus a trans-acting product encoded by 14, as shown by complementation studies . The other two mutations--a -2 bp deletion at the same site, causing frameshift with a chain-terminating codon within a few triplets, and a base pair exchange at a nearby site--affect both the formation of 14 typical translation products and the trans-acting function . These results on box9 mutants combined with results on box7 mutants suggest that an 14-encoded "maturase" protein (apparent molecular weight, 27,000) is cleaved off a precursor protein (apparent molecular weight, 55,000) encoded by exon sequences B1 to B4 and the intron open reading frame . We further discuss the role of the box9 nucleotide sequence in the maturation of cob-specific RNA.

Cell, 1982 Jun, 29(2), 347 - 55
Yeast use translational control to compensate for extra copies of a ribosomal protein gene; Pearson NJ et al.; The efficient assembly of ribosomes requires a balanced synthesis of ribosomal RNA and each ribosomal protein . In an attempt to establish the mechanisms responsible for such balanced synthesis we have altered the gene dosage for one of the components by introducing into yeast an autonomously replicating plasmid containing the gene tcm1, which codes for ribosomal protein L3 . The plasmid is maintained at 5-10 copies per cell by selection for expression of its URA3 gene . The plasmid-containing cells transcribe 7.5 times as much L3 mRNA as control cells, maintain 3.5 times as much L3 mRNA as control cells and synthesize no more than 1.2 times as much L3 protein as control cells . We conclude that the balanced synthesis of ribosomal proteins is maintained by modulating both the efficiency of translation and the lifetime of their mRNAs.

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