Yeasts

FEBS Lett, 1984 Apr 9, 169(1), 73 - 8
Nucleotide substitutions in a yeast mitochondria cis-acting mutant located in the last intron of the apocytochrome b gene; Bonjardim CA et al.; The region of mitochondrial DNA corresponding to the intron mutant M6-200 in Saccharomyces cerevisiae D273-10B has been isolated, and the nucleotide sequence of a 519 bp RsaI fragment has been determined . Three nucleotide substitutions were found at nucleotides +2650 (G----T), +2668 (G----A) and +2798 (A----G), all within the genetically defined location in the gene . Particular significance can be attributed to the first two changes (+2650 and +2668), that can be genetically isolated from the third substitution and, in addition, alter conserved sequence features detected in a study {(1982) Biochimie 64, 867-881} of fungal mitochondrial introns.

Eur J Biochem, 1984 Apr 2, 140(1), 143 - 6
Catalytical mechanism of the phenylalanyl-tRNA synthetase from yeast . Reactivity of ATP in the absence of phenylalanine; Thiebe R; Phenylalanyl-tRNA synthetase catalyses an AMP-ATP exchange under conditions where no aminoacylation of tRNA occurs . A plausible explanation for this reaction had not been given so far . The results of the present investigation provide evidence for the following interpretation . tRNAPhe induces a polarisation of the ATP in complex with the enzyme; this stimulates (a) the formation of phenylalanyl-adenylate in the presence of phenylalanine, (b) the hydrolysis of ATP in the absence of phenylalanine and AMP and (c) the transfer of diphosphoryl onto AMP in the presence of AMP, especially when phenylalanine is absent.

Eur J Biochem, 1984 Apr 2, 140(1), 157 - 61
Synthesis and properties of (4-13C)NAD+ . Observation of its binding to yeast alcohol dehydrogenase by 13C-NMR spectroscopy; Oberfrank M et al.; Starting from (13C)formic acid, acetone and cyanoacetamide samples of (4-13C)nicotinic acid and (4-13C)-nicotinamide were synthesised in an overall and additive yield of 11% . 1H-NMR and mass spectroscopy showed 90% enrichment of 13C in the expected position . NADase-catalysed exchange between thionicotinamide-adenine dinucleotide and (4-13C)nicotinamide furnished (4-13C)NAD+ which was purified, characterized and quantified by 1H-NMR and 13C-NMR spectroscopy and by enzymic assay . The 13C-NMR signal of (4-13C)beta-NAD+ (146.09 ppm) was broadened and shifted (147.83 ppm) upon binding to yeast alcohol dehydrogenase.

Mol Cell Biol, 1984 Apr, 4(4), 771 - 8
Transformation of protoplasted yeast cells is directly associated with cell fusion; Harashima S et al.; The frequency of cell fusion during transformation of yeast protoplasts with various yeast plasmids with a chromosome replicon (YRp or YCp) or 2 mu DNA (YEp) was estimated by two methods . In one method, a mixture of protoplasts of two haploid strains with identical mating type and complementary auxotrophic nuclear markers with or without cytoplasmic markers was transformed . When the number of various phenotypic classes of transformants for the nuclear markers was analyzed by equations derived from binominal distribution theory, the frequency of nuclear fusion among the transformants was 42 to 100% in transformations with the YRp or YCp plasmids and 28 to 39% with the YEp plasmids . In another method, a haploid bearing the sir mutation, which allows a diploid (or polyploid) homozygous for the MAT (mating type) locus to sporulate by the expression of the silent mating-type loci HML and HMR, was transformed with the plasmids . Sporulation ability was found in 43 to 95% of the transformants with the YRp or YCp plasmids, and 26 to 31% of the YEp transformants . When cytoplasmic mixing was included with the nuclear fusion, 96 to 100% of the transformants were found to be cell fusants . Based upon these observations, we concluded that transformation of yeast protoplasts is directly associated with cell fusion.

J Bacteriol, 1984 Apr, 158(1), 337 - 9
Activation of trehalase by membrane-depolarizing agents in yeast vegetative cells and ascospores; Thevelein JM; The membrane-depolarizing agents 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazone, and nystatin are known to cause a rapid increase in the cyclic AMP level in fungal cells . Addition of these proton ionophores to yeast stationary-phase cells or ascospores causes an immediate 10-fold increase in trehalase activity . This observation is in agreement with a role for cyclic AMP-induced phosphorylation in the activation process of trehalase . It also provides an explanation for previous results on the induction of trehalose breakdown by 2,4-dinitrophenol in resting yeast cells.

EMBO J, 1984 Apr, 3(4), 829 - 34
Processing of yeast mitochondrial messenger RNAs at a conserved dodecamer sequence; Osinga KA et al.; The yeast mitochondrial genes coding for cytochrome c oxidase subunit I ( COX1 ) and the ATPase subunits 8 and 6 are organized in one transcription unit . Precise mapping of RNA termini with S1 nuclease and primer extension analysis shows that the 3' end of the COX1 mRNA and the 5' end of the ATPase precursor RNA are juxtaposed within a conserved dodecamer sequence (5'- AAUAAUAUUCUU -3') . Sequence comparison reveals that this motif is present downstream of nearly all protein-encoding genes, including extragenic unassigned reading frames ( URFs ) and two URFs located within introns . Also the 3' terminus of an RNA species derived from the URF -containing intron of the large rRNA gene maps within such a dodecamer sequence . It is likely, therefore, that this motif serves as a processing point in the generation of mature mRNA . From a comparison of the various transcription units, we infer that RNAs that originate from an endonucleolytic cleavage at this sequence have stable 3' termini, while further processing of the 5' ends occurs . The efficiency of the initial cleavage varies between the different positions at which the motif is present.

Radiat Res, 1984 Apr, 98(1), 74 - 81
Comparisons of the effects of vacuum-uv and far-uv synchrotron radiation on dry yeast cells of different uv sensitivities; Hieda K et al.; Far-uv-sensitive (rad l/rad l) and wild-type cells of diploid Saccharomyces cerevisiae were irradiated in vacuum at 155, 170, 220, and 250 nm using synchrotron radiation (SR) . Inactivation, gene conversion at leu l, and membrane damage as judged by methylene blue penetration were measured . Radiations of all these wavelengths killed dry yeast cells . In the vacuum uv, radiation at 155 and 170 nm induced membrane damage but not gene conversion, whereas far-uv radiation at 220 and 250 nm induced gene conversion but not membrane damage . The far-uv-sensitive strain showed no enhanced sensitivity to vacuum-uv radiation . These results indicate that damage to the cell membrane is considerably more important than to nuclear DNA for yeast cell inactivation by vacuum-uv radiation.

Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 905 - 12
Sequence of tRNA Ile IAU from brewer's yeast; Pixa G et al.; The nucleotide sequence of tRNAIle from brewer's yeast Saccharomyces cerevisiae was determined . Its primary structure is pG-G-U-C-U-C-U-U-m1G-m2G- C-C-C-A-G-D-D-G-G-D-D-A-A-G-G-C-A-C-C-G-U-G-C-U-I-A-U-t6 A-A-C-G-C-G-G-G-GA-D-m5 C-A-G-C-G-G-T-psi-C-G-m1 A-U-C-C-C-G-C-U-A-G-A-G-A-C-C-A-C-C-A . Its anticodon is I-A-U . It should therefore recognize the three isoleucine codons and is for this reason probably the only isoacceptor tRNA for isoleucine in brewer's yeast . It presents a large homology with its counterpart from Torulopsis utilis (87%).

Nucleic Acids Res, 1984 Mar 26, 12(6), 2955 - 68
Structure and function of the nontranscribed spacer regions of yeast rDNA; Skryabin KG et al.; The sequences of the nontranscribed spacers (NTS) of cloned ribosomal DNA (rDNA) units from both Saccharomyces cerevisiae and Saccharomyces carlsbergensis were determined . The NTS sequences of both species were found to be 93% homologous . The major disparities comprise different frequencies of reiteration of short tracts of six to sixteen basepairs . Most of these reiterations are found within the 1100 basepairs long NTS between the 3'-ends of 26S and 5S rRNA (NTS1) . The NTS between the starts of 5S rRNA and 37S pre-rRNA (NTS2) comprises about 1250 basepairs . The first 800 basepairs of NTS NTS2 (adjacent to the 5S rRNA gene) are virtually identical in both strains whereas a variable region is present at about 250 basepairs upstream of the RNA polymerase A transcription start . In contrast to the situation in Drosophila and Xenopus no reiterations of the putative RNA polymerase A promoter are present within the yeast NTS . The strands of the yeast NTS reveal a remarkable bias of G and C-residues . Yeast rDNA was previously shown to contain a sequence capable of autonomous replication (ARS) (Szostak, J.W . and Wu, R (1979), Plasmid 2, 536-554) . This ARS, which may correspond to a chromosomal origin of replication, was located on a fragment of 570 basepairs within NTS2.

Nucleic Acids Res, 1984 Mar 26, 12(6), 2705 - 15
Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence; Haumont E et al.; We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs . For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U) . This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34 . Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme . Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all . This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.

J Mol Biol, 1984 Mar 25, 174(1), 163 - 73
Structure of the ribotrinucleoside diphosphate codon UpUpC bound to tRNAPhe from yeast . A time-dependent transferred nuclear Overhauser enhancement study; Clore GM et al.; The structure of the ribotrinucleoside diphosphate UpUpC, the codon for phenylalanine, bound to yeast tRNAPhe in solution is elucidated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons . The glycosidic bond and ribose conformations are low anti and 3'-endo, respectively, typical of an A-RNA type structure . The main chain torsion angles are all within the range of those expected for A-RNA but small differences from those in conventional A-RNA 11 result in a special structure with a larger rotation per residue (40 to 45 degrees compared to 32.7 degrees in R-RNA 11) and almost perfect stacking of the bases . These two structural features, which are similar to those found in the anticodon triplet of the monoclinic crystal form of tRNAPhe, can account for the known greater stability of the codon-anticodon complex relative to an equivalent double helical RNA trimer with a conventional A-RNA structure.

J Biol Chem, 1984 Mar 25, 259(6), 3714 - 9
Yeast LEU1 . Repression of mRNA levels by leucine and relationship of 5'-noncoding region to that of LEU2; Hsu YP et al.; Yeast LEU1 encodes the second enzyme in leucine biosynthesis . A 3.5-kilobase pair (kb) yeast genomic DNA fragment which complements a leu1 auxotroph was isolated by yeast transformation . After recloning into an integrating vector, a subfragment (of the 3.5-kb fragment) directs a URA3 marker to integrate at the LEU1 locus . About 1.9 kb was sequenced from the 5'-end of the 3.5-kb insert, and a long open reading frame and potential ATG start codon were located . S1 nuclease mapping showed a major start for LEU1 transcripts at 79 nucleotides upstream of the ATG codon . Northern blots with a LEU1-specific probe showed the size of the LEU1 transcript (about 2.9 kb) is consistent with the size of the enzyme and steady state levels of the transcript are sharply reduced in cells grown in the presence of an elevated leucine concentration . The latter observation correlates with the repression by leucine of LEU1 gene product levels . Other work has shown that the level of the LEU2 gene product is also repressed by leucine . Sequence comparisons between LEU1 and LEU2 show that the LEU2 5'-sequences which are cognate to leucine are not found in LEU1; and three blocks of nucleotide sequence homology between LEU1 and LEU2 occur in the 330 nucleotides upstream of the respective start codons.

Biochem Biophys Res Commun, 1984 Mar 15, 119(2), 447 - 51
Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance; Tijssen JP et al.; Non-penetrating cations, like UO2+(2) and Eu3+, are bound to the outside of yeast cells in a reversible fashion . Binding of these ions was attended with a decrease of the 31P NMR polyphosphate signal . Subsequent addition of EDTA to the suspension restored the original spectrum . These experiments confirm the localization of a polyphosphate fraction outside the plasma membrane of yeast.

FEBS Lett, 1984 Mar 12, 168(1), 61 - 4
Inhibition of ribonuclease activity during RNA synthesis in isolated yeast nuclei by cadmium; Schulz-Harder B et al.; We have developed an efficient transcription system in isolated yeast nuclei . If MnCl2 is substituted by CdCl2, degradation of newly synthesized RNA is markedly reduced . This effect is due to the inhibition of nuclear ribonuclease activity, since microsomal ribonuclease activity is less affected by the cation . The extent to which the addition of CdCl2 to the in vitro transcription assay inhibits ribonuclease activity is demonstrated by the measurements of the size of newly synthesized RNA . Efficient RNA synthesis in this system is not affected up to a concentration of 0.1 M CdCl2.

Nucleic Acids Res, 1984 Mar 12, 12(5), 2407 - 19
In vitro generation of specific deletions in DNA cloned in M13 vectors using synthetic oligodeoxyribonucleotides: mutants in the 5'-flanking region of the yeast alcohol dehydrogenase II gene; Chan VL et al.; Deletion mutants are particularly useful in defining the boundaries of noncoding genetic functions . Such mutants can be precisely generated using synthetic oligodeoxyribonucleotides as mutagens . In this paper we describe the application of this method to recombinant DNA cloned in a phage M13-derived vector . The mutagenic oligodeoxyribonucleotides, 20 and 21 nucleotides in length, were used to delete a tract of 20 dA-dT base-pairs and an adjacent 22 base-pair perfect dyad from the ADR3 locus, the 5'-flanking regulatory region of the ADR2 gene, of Saccharomyces cerevisiae with high efficiency.

J Biol Chem, 1984 Mar 10, 259(5), 3124 - 6
Solution X-ray scattering studies of the yeast phosphofructokinase allosteric transition . Characterization of an ATP-induced conformation distinct in quaternary structure from the R and T states of the enzyme; Laurent M et al.; The allosteric transition of yeast phosphofructokinase has been studied by solution x-ray scattering . The scattering curves corresponding to the native enzyme (T conformation) were found to be similar to the curves recorded in the presence of saturating concentrations of fructose 6-phosphate (R conformation) or AMP (R or R' conformation) . However, the curves obtained in the presence of ATP are clearly different: the radius of gyration increases and the secondary minima and maxima are systematically shifted to lower angles, suggesting a swelling of the enzyme in the presence of ATP . These results give the first direct evidence for the existence of an ATP-induced T' conformation, distinct in quaternary structure from the R and T states of the enzyme oligomer, in agreement with our previous modeling of yeast phosphofructokinase regulation . X-ray scattering data are discussed in relation to the distinct molecular mechanisms of the ATP and fructose 6-phosphate allosteric effects involving, respectively, sequential and concerted conformational changes of the enzyme oligomer.

J Biol Chem, 1984 Mar 10, 259(5), 2886 - 95
Investigations of the metal ion-binding sites of yeast inorganic pyrophosphatase; Knight WB et al.; Yeast inorganic pyrophosphatase was found to bind two Mn2+ per subunit in the absence of phosphate and three Mn2+ per subunit in the presence of phosphate . Kinetic studies of the pyrophosphatase-catalyzed hydrolysis of Cr(NH3)4PP and Cr(H2O)4PP were carried out with Mn2+ and with Mg2+ as activators . The results from these studies suggest that three divalent cations per pyrophosphatase active site are required for catalysis . NMR and EPR studies were conducted to evaluate the relative location of the metal ion binding sites on the enzyme . The two Mn2+ ions bound to the free enzyme are in close enough proximity to magnetically interact . Analysis of the NMR and EPR data in terms of a dipolar relaxation mechanism between Mn2+ ions provides an estimate of the distance between them of 10-14 A . When the diamagnetic substrate analog {Co(NH3)4PNP}- or intermediate analog {Co(NH3)4 (P)2}- are bound to pyrophosphatase, two Mn2+ ions still bind to the enzyme and their magnetic interaction increases . In the presence of these Co3+ complexes, the Mn2+--Mn2+ separation decreases to 7-9 A . Several NMR and EPR experiments were conducted at low Mn2+ to pyrophosphatase ratios (approximately 0.3), where only one Mn2+ ion binds per subunit, in the presence of Cr3+ or Co3+ complexes of PNP or PP . Analysis of the Mn2+--Cr3+ dipolar relaxation evident in proton NMR and EPR data provided for the calculation of Mn2+--Cr3+ distances . When the substrate analog CrPNP was present, the Mn2+--Cr3+ distance was congruent to 7 A whereas, when Cr(P)2 was bound to pyrophosphatase, the Mn2+--Cr3+ distance was congruent to 5 A . These results strongly support a model for the catalytic site of pyrophosphatase that involves three metal ion cofactors.

Cell, 1984 Mar, 36(3), 741 - 51
Sequence of the preprotoxin dsRNA gene of type I killer yeast: multiple processing events produce a two-component toxin; Bostian KA et al.; The preprotoxin gene of the 1.9 kb M1 dsRNA genome from type I killer yeast has been sequenced employing a partial-length cDNA derived from an in vivo transcript . A single open reading frame, commencing with AUG at M1 dsRNA bases 14-16, terminates with UAG at 963-965 and codes for a 316 amino acid protein, believed to be identical to the 34 kd preprotoxin species, M1-P1, synthesized by in vitro translation of denatured M1 dsRNA . N-terminal sequencing of M1-P1 confirms this prediction . Secreted toxin is shown to consist of two dissimilar, disulfide-bonded subunits, alpha and beta, of apparent size 9.5 and 9.0 kd, respectively, whose N-terminal sequences are also found in the predicted preprotoxin sequence . Its proposed domains consist of delta, a 44 amino acid N-terminal segment, followed by alpha and beta, which are separated by gamma, a large central glycosylated segment . Processing sites, domain functions, and the potential role of gamma in immunity are discussed.

Biophys Chem, 1984 Mar, 19(2), 171 - 81
A new theoretical index of biochemical reactivity combining steric and electrostatic factors . An application to yeast tRNAPhe; Lavery R et al.; A new theoretical index of the chemical reactivity of sites within macromolecules is developed, which combines both steric and electrostatic factors . It is applied to the study of yeast tRNAPhe and the results obtained are compared with known experimental reactivities . A comparison indicates the superiority of the new index over the sole use of the surface accessibility.

Biochem J, 1984 Mar 1, 218(2), 405 - 13
Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda; Rytka J et al.; The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins . Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation . Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type . The excreted porphyrins comprised mainly dehydroisocoproporphyrin . Analysis of uroporphyrinogen decarboxylase activity in the cell-free extract revealed a 70-80% decrease of activity in the mutant and showed that the relative rates of the different decarboxylation steps were modified with the mutant enzyme . A 2-3-fold increase in 5-aminolaevulinate synthase activity was measured in the mutant . The biochemical characteristics of the Sm1 mutant are very similar to those described for porphyria cutanea tarda.

Cell, 1984 Mar, 36(3), 645 - 53
Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast; Langford CJ et al.; Our previous deletion experiments have shown that a short region of yeast nuclear gene introns containing the conserved sequence 5'-TACTAACA-3' is essential for splicing . In this report we show that the chemically synthesized decanucleotide 5'-TGTACTAACA-3', when introduced into a hybrid gene forming unspliceable RNA molecules, results in the generation of spliceable transcripts . Single A----C transversions in the fourth or eighth position of this sequence eliminated its intron-generating capacity . The C----T transition in the fifth position, generated by sodium bisulphite mutagenesis, did not affect the efficiency and accuracy of splicing . These results clearly demonstrate the biological significance of this conserved intron sequence and shed further light on its possible functioning.

Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1475 - 9
Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA; Pluta AF et al.; The termini of macronuclear DNA molecules from the protozoan Oxytricha fallax share a common sequence and structure, both of which differ markedly from those deduced for yeast telomeres . Despite these differences, terminal restriction fragments from O . fallax macronuclear DNA can support telomere formation in yeasts . Two linear plasmids (LYX-1 and LYX-2) constructed by ligating BamHI-digested total Oxytricha macronuclear DNA to a yeast vector were analyzed . One end of LYX-1 and both ends of LYX-2 are derived from the Oxytricha DNA that encodes rRNA (rDNA) whereas the other end of LYX-1 is from an Oxytricha fragment other than rDNA . After propagation in yeast, both ends of LYX-1 and LYX-2 retain the C4A4 repeat characteristic of the O . fallax terminal sequence . In addition, both ends of both plasmids acquire 300-1000 base pairs of DNA containing the sequence (C-A)n, a sequence found near the termini of yeast chromosomes . Thus, at least two different Oxytricha termini display distinctive properties in yeast cells in that linear plasmids containing them are not degraded nor are they integrated into chromosomal DNA . These Oxytricha termini may act directly as telomeres in yeast; alternatively, the Oxytricha DNA may serve as a signal that results in the elaboration of a yeast telomere on the ciliate DNA.

Eur J Biochem, 1984 Mar 1, 139(2), 201 - 8
The duck alpha A globin but not the yeast actin gene is transcribed by a HeLa cell extract; Horcher R et al.; We have investigated the transcription in a HeLa whole-cell extract of two evolutionary widely separated structural genes coding for duck alpha A globin and yeast actin . Transcription of isolated DNA fragments of the duck alpha A globin gene increases linearly up to relatively high concentrations of DNA . Size analyses and S1 mapping of the transcripts synthesized in vitro on either linear DNA fragments or supercoiled templates reveal that the alpha A globin RNA is initiated at the in vivo cap site and remains unspliced . The same assay conditions were used to transcribe the yeast actin gene . In contrast to the duck gene, size analyses and S1 mapping of the RNA products synthesized on both linear DNA fragments and the supercoiled template containing the actin gene show that the transcripts found in vitro do not stem from the in vivo cap site . The promoter of the yeast actin gene is not recognized in this system in vitro.

J Hosp Infect, 1984 Mar, 5(1), 83 - 91
Oral ketoconazole and amphotericin B for the prevention of yeast colonization in patients with acute leukaemia; Donnelly JP et al.; Forty-eight neutropenic patients with acute leukaemia were randomly allocated to receive, as antifungal prophylaxis, either ketoconazole, 400 mg once daily (K), or amphotericin B tablets and lozenges (A), or both ketoconazole and amphotericin B together (K + A) . Antifungal prophylaxis was considered to have failed if (1) there was evidence of increasing colonization of the oropharynx or faeces with Candida spp . or other yeasts, or (2) if systemic antifungal therapy was begun empirically . Prophylaxis failed in nine of 17 patients given K, in four of 19 given A, and in four of 12 given K + A . The differences between the three regimens were not statistically significant, neither was there any significant difference in the mean duration of neutropenia before prophylaxis failed . The absorption of ketoconazole was impaired when patients were neutropenic . We conclude that ketoconazole was neither more nor less effective than amphotericin B in the prevention of yeast colonization in neutropenic patients.

Biochemistry, 1984 Feb 28, 23(5), 797 - 801
Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate; Gonzalez MA et al.; In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled {gamma-17O,18O}ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio{17O,18O}phosphate proceeds with inversion of configuration . Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb {Webb, M . R . (1982) Methods Enzymol . 87, 301-316} . We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O . These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.

Nucleic Acids Res, 1984 Feb 24, 12(4), 1889 - 900
Initiation of transcription in yeast mitochondria: analysis of origins of replication and of genes coding for a messenger RNA and a transfer RNA; Osinga KA et al.; The initiation of transcription of the yeast mitochondrial genes coding for subunit I of cytochrome c oxidase (COX1) and for tRNA1Thr has been examined . COX1 messenger RNA synthesis is initiated in a conserved nonanucleotide sequence (ATATAAGTA) which we have previously found immediately upstream of ribosomal RNA genes at positions at which RNA synthesis starts . The 5'-end of the precursor of tRNA1Thr is located in a variant nonanucleotide motif (TTATAAGTA), which may be characteristic for tRNA genes . Using a partially purified fraction of mtRNA polymerase, we demonstrate that RNA synthesis is precisely initiated in vitro in nonanucleotide sequences preceding both ribosomal RNA-, tRNA- and messenger RNA-encoding genes and origins of replication.

J Mol Biol, 1984 Feb 15, 173(1), 1 - 13
Location of DNAase I sensitive cleavage sites in the yeast 2 micron plasmid DNA chromosome; Fagrelius TJ et al.; We have studied the uniformity with which the yeast 2 micron plasmid DNA within its nucleoprotein complex is protected from digestion by DNAase I . To probe for relatively unprotected regions, plasmid nucleoprotein complexes were digested with DNAase I to yield a preparation in which approximately half of the circular DNA molecules had been converted to full-length linear molecules . The sites of the double-strand breaks were then mapped in relation to restriction endonuclease sites using end-label probes . The most prominent sensitive sites were found at positions very close to the beginning and end of a 122 base-pair sequence with dyad symmetry located within the 599 base-pair inverted repetition of the plasmid . The sequence is known to be necessary for plasmid site-specific recombination . Other sensitive sites were mapped to the 5'-side of known coding regions . A unique plasmid sequence located to one side of the replication origin was also sensitive to DNAase I digestion yet did not yield discrete cleavage sites . Cleavage of plasmid DNA stripped of proteins did not result in the appearance of distinct fragments as found after cleavage of the same DNA within the nucleoprotein complex . We conclude from these results that, when complexed with proteins, specific plasmid DNA sequences involved in transcription, replication and recombination are more accessible to nuclease digestion.

FEBS Lett, 1984 Feb 13, 167(1), 165 - 9
Nucleotide sequence of a yeast tRNAArg3A gene and its transcription in a homologous in vitro system; Villanueva J et al.; Twelve bacterial clones containing complementary sequences to yeast tRNAArg3 were isolated from a gene library . The size of the yeast BamHI inserts ranges from 5.4 to 10 MDa . There are at least 6 copies of this gene in different loci of the yeast genome . Insert from clone pYAT-3 was mapped, and the presence of a tRNAArg3A gene was confirmed by DNA sequence . The coding region is colinear with the transcriptional product . Unlike other reported tRNAArg3A genes, this one is not linked to a tRNAAsp gene . In vitro transcription using a yeast extract produces a transcript of 76 +/- 1 bases.

J Biol Chem, 1984 Feb 10, 259(3), 1661 - 6
Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation . II . Lanosterol metabolism by purified P-450(14)DM and by intact microsomes; Aoyama Y et al.; A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen . This conversion did not occur anaerobically or when either P-450(14)DM, the reductase, or NADPH was omitted from the system . In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns . Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol . Upon aerobic incubation of microsomes from semianaerobically grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol . This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-450(14)DM . It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.

J Biol Chem, 1984 Feb 10, 259(3), 1655 - 60
Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation . I . Purification and spectral properties; Yoshida Y et al.; A form of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation (tentatively called "P-450(14)DM") was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoretic homogeneity . An apparent monomeric Mr = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Both optical and EPR spectra of oxidized P-450(14)DM are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm . As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-450(14)DM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state . Oxidized P-450(14)DM was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350- and 414-nm troughs possessed larger and relatively smaller {theta} values, respectively, than those reported for other low spin ferric cytochromes P-450 . Lanosterol was the only compound which caused a Type I spectral change in oxidized P-450(14)DM . The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-450(14)DM adduct was in a spin state equilibrium.

Cell, 1984 Feb, 36(2), 309 - 18
Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway; Julius D et al.; Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway . In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro . At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates . In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed . The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus . Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5).

J Biochem (Tokyo), 1984 Feb, 95(2), 589 - 92
Nucleotide sequence of an essential region for autonomous replication of cloned yeast mitochondrial DNA; Mabuchi T et al.; A 341 bp sequence from yeast mtDNA was cloned, which consisted of an upstream 98 bp AT stretch and a downstream 206 bp AT stretch separated by a single 37 bp GC cluster . Cleavage of this GC cluster did not cause loss of the autonomously replicating function of this sequence . The recloned first 98 bp AT stretch was incapable of replication, while the recloned 206 bp AT stretch could replicate . We were able to confine an essential sequence for autonomous replication within a 186 bp AT stretch . Sequencing data revealed a sequence of ATATAAAT and stem and loop structures within the AT stretch.

Biochem J, 1984 Feb 1, 217(3), 641 - 7
The regulatory properties of yeast pyruvate kinase; Morris CN et al.; The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic . The findings were represented empirically by the exponential model for a regulatory enzyme . The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively {Ainsworth (1977) J . Theor . Biol . 68, 391-413} . The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account . The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme {Ainsworth, Kinderlerer & Gregory (1983) Biochem . J . 209, 401-411}, and it is suggested that they have a common origin in charge interactions within the active site.

Genetics, 1984 Feb, 106(2), 207 - 26
Healing of broken linear dicentric chromosomes in yeast; Haber JE et al.; In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome . Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores . The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle) . The Ura+ Mal+ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura+ Mal+, others Ura+ Mal- and others Ura- Mal+ . The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.--Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic breakage have demonstrated that these stable derivatives arose from mitotic breakage of the dicentric chromosome, followed by one of several different healing events . The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome . A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere . In two instances we recovered chromosome III partially duplicated with a novel right end . We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres.

Radiat Res, 1984 Feb, 97(2), 329 - 40
Interpretation of the dose and LET dependence of RBE values for lethal lesions in yeast cells; Frankenberg D; Survival data on yeast cells proficient or deficient in the repair of DNA double-strand breaks (dsb) and data on the induction of dsb are used to interpret the dose dependence of the RBE value for lethal lesions after irradiation at high dose rate followed by 72-hr liquid holding providing optimum conditions for repair of potentially lethal lesions (RBEDP, DP = delayed plating) . The radiations applied are conventional (150 kV), soft (50 kV), and ultrasoft (4 kV) X rays, 30-MeV electrons (or 60Co gamma rays), and 3.5-MeV alpha particles . Analysis shows that the dose dependence of the RBEDP value can be explained by the combination of two dose-independent RBE values, one for the single-particle traversal effect (RBEspt) and the other for the accumulation of dsb (RBEdsb) due to the traversal of more than one particle through the cell nucleus . Furthermore, it is shown that the LET dependence of RBEspt values describing the linear component of the lethal lesions must be considered separately for "electron" and "particle" radiations.

Biochimie, 1984 Feb, 66(2), 127 - 34
Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (L-lactate cytochrome c reductase) upon binding of pyruvate, the reaction product; Tegoni M et al.; Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b2 moiety were carried out at room temperature, in the presence of added pyruvate or in its absence . Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate, the reaction product in the oxidation of L-lactate . The proportion of flavin semiquinone nearly reaches then 100 per cent.

Infect Immun, 1984 Feb, 43(2), 467 - 71
Influence of yeast mannan on release of myeloperoxidase by human neutrophils: determination of structural features of mannan required for formation of myeloperoxidase-mannan-neutrophil complexes; Wright CD et al.; Structural features of mannan which participate in the formation of myeloperoxidase-mannan-neutrophil complexes have been studied by using a battery of structurally modified mannans . Mannan was isolated from Saccharomyces cerevisiae X2180 wild type and modified by strong alkaline degradation, selective (mild) alkaline degradation, and selective acetolysis . Mannose oligosaccharides bound to the peptide portion of mannan appeared to be required for binding of mannan to the neutrophil . The interaction of mannan with myeloperoxidase appeared to occur through phosphate groups of the mannan outer chain . The myeloperoxidase-mannan interaction was determined to be ionic in nature . The mannan-neutrophil interaction may involve cell membrane receptors for mannose.

FEBS Lett, 1984 Jan 30, 166(2), 321 - 5
Vacuoles are not the sole compartments of proteolytic enzymes in yeast; Emter O et al.; Localization in vacuoles, the lysosome-like organelle of yeast, was checked for several newly detected proteolytic enzymes . While aminopeptidase Co and carboxypeptidase S were found in vacuoles, proteinase D and proteinase E as well as a variety of other proteolytic activities detectable with the aid of chromogenic peptide substrates do not reside in this cell compartment.

J Biol Chem, 1984 Jan 25, 259(2), 1004 - 10
Yeast cells recover from mating pheromone alpha factor-induced division arrest by desensitization in the absence of alpha factor destruction; Moore SA; Saccharomyces cerevisiae MATa cells arrest cell division in response to the mating pheromone, alpha factor . After some interval of time the cells resume division . It is demonstrated here that cells recover from division arrest by becoming insensitive to the alpha factor under conditions of low cell density (10(2) cells/ml) where no alpha factor destruction occurs . The time of desensitization occurs later at higher alpha factor concentrations . Using the technique of perfusion photomicroscopy developed in this study, it was found that 95% of the desensitized cells remain insensitive to the alpha factor for greater than or equal to 3 generations at the alpha factor concentration where recovery occurred . Upon recovery, cells have generation times which are similar or identical to the calculated time from the cdc28 "start" step of cell division to cell separation . Therefore, the abnormally large recovered cells behave as if they have no growth time requirement for cell division for several generations . Desensitization was found to occur asymmetrically for parent and daughter cells . While parents (cells which have budded) are insensitive to alpha factor, their daughters (cells which have never budded and which were formed from desensitized parents during continuous perfusion with alpha factor) show 54% with a delayed generation time compared to control daughter cells.

FEBS Lett, 1984 Jan 23, 166(1), 131 - 5
Reaction of thionitrobenzoate-modified yeast cytochrome c with monomeric and dimeric forms of beef heart cytochrome c oxidase; Darley-Usmar VM et al.; Thionitrobenzoate-modified yeast cytochrome c was shown to react with both monomeric and dimeric forms of beef heart cytochrome c oxidase through subunit III . This cytochrome c derivative was found to inhibit electron transfer in the dimer but not in the monomer . These results are interpreted to show that the high affinity binding site for cytochrome c is a cleft at the interface between monomers in the cytochrome c oxidase dimer.

Nature, 1984 Jan 12-18, 307(5947), 178 - 80
Human hepatitis B vaccine from recombinant yeast; McAleer WJ et al.; The worldwide importance of human hepatitis B virus infection and the toll it takes in chronic liver disease, cirrhosis and hepatocarcinoma, make it imperative that a vaccine be developed for worldwide application . Human hepatitis B vaccines are presently prepared using hepatitis B surface antigen (HBsAg) that is purified from the plasma of human carriers of hepatitis B virus infection . The preparation of hepatitis B vaccine from a human source is restricted by the available supply of infected human plasma and by the need to apply stringent processes that purify the antigen and render it free of infectious hepatitis B virus and other possible living agents that might be present in the plasma . Joint efforts between our laboratories and those of Drs W . Rutter and B . Hall led to the preparation of vectors carrying the DNA sequence for HBsAg and antigen expression in the yeast Saccharomyces cerevisiae . Here we describe the development of hepatitis B vaccine of yeast cell origin . HBsAg of subtype adw was produced in recombinant yeast cell culture, and the purified antigen in alum formulation stimulated production of antibody in mice, grivet monkeys and chimpanzees . Vaccinated chimpanzees were totally protected when challenged intravenously with either homologous or heterologous subtype adr and ayw virus of human serum source . This is the first example of a vaccine produced from recombinant cells which is effective against a human viral infection.

J Biol Chem, 1984 Jan 10, 259(1), 412 - 7
Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway; Runge KW et al.; Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a {3H}mannose suicide enrichment . Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro . When UDP-{3H}Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose . When dolicholphospho{3H}glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides . These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide . We also present evidence that both mutants transfer Man9GlcNAc2 to protein.

FEBS Lett, 1984 Jan 9, 165(2), 251 - 3
Investigation of the role of the substrate metal ion in the yeast inorganic pyrophosphatase reaction; Ting SJ et al.; The substrate activities of a series of tripositive metal ion-pyrophosphate complexes with yeast inorganic pyrophosphatase were examined . While the Michaelis constants for these complexes were shown to be between one and two orders of magnitude greater than that of the natural substrate, {Mg(H2O)4PPi}2-, the turnover numbers were in general comparable to that of {Mg(H2O)4PPi}2- . These data suggest that the nature of the metal ion cofactor effects substrate binding but in most cases not catalysis . Thus, the role of the metal ion in catalysis is probably restricted to that of an electron sink.

Biochemistry, 1984 Jan 3, 23(1), 69 - 73
Stimulation of yeast RNA polymerase II transcription by critical values of supercoiling; Pedone F et al.; RNA chains of discrete length were obtained in vitro by yeast RNA polymerase II directed transcription of a supercoiled plasmid . On the basis of the amount and the molecular weight of the RNA chains synthesized in the absence of reinitiation events, the number of actively transcribing RNA polymerase molecules has been calculated . A stimulation of transcriptional activity was found to be related to the torsional strength of negative supercoiling of the template . The DNA unwinding angle measured in the complexes formed with the enzyme in the presence of three ribonucleoside triphosphates equals 485 +/- 30 degrees, marking a melting effect of 14 base pairs per bound enzyme molecule.

Eur J Biochem, 1984 Jan 2, 138(1), 77 - 81
Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys; Vacher J et al.; In this paper we describe the construction of a yeast tRNACys UGA suppressor . After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil . The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities . The level of aminoacylation was similar to that of the parent molecule . Readthrough of the UGA termination codon in beta-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions . We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes . This may be related to the low suppressor activity; however, it is also possible that other features of tRNACys structure may be unadapted to efficient UCA anticodon function.

Eur J Biochem, 1984 Jan 2, 138(1), 169 - 77
The biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast . DNA sequence analysis of the nuclear gene coding for the 14-kDa subunit; De Haan M et al.; The nuclear gene coding for the imported 14-kDa subunit of the ubiquinol-cytochrome c reductase of yeast mitochondria has been sequenced in an attempt to define regulatory and protein topogenic elements . The gene has a length of 381 base pairs and is potentially capable of encoding a polypeptide of 14561 Da . It is transcribed into a single low-abundance RNA of 680 nucleotides whose 5' and 3' termini map, respectively, 30-35 nucleotides upstream and 180-190 nucleotides downstream of the initiator and termination codons . Consistent with the estimated low level of the mRNA, codon usage in the gene is not strongly biased and other features, characteristic of highly expressed genes in yeast, are absent . The 14-kDa protein is predicted to be a predominantly hydrophilic protein, with only a single, short hydrophobic stretch located between positions 19-38 . Comparison with other imported mitochondrial proteins so far sequenced has failed to reveal unifying features that might serve as targeting elements . Steady-state levels of the 14-kDa and 11-kDa subunits are reduced in mit- mutants which synthesize truncated forms of apocytochrome b and in these, newly synthesized subunits exhibit a specifically increased turnover rate . We suggest that association of these two subunits with the complex may be mediated or enhanced by interaction with other subunits, in particular cytochrome b.

Eur J Cell Biol, 1984 Jan, 33(1), 19 - 23
Monitoring yeast spindles in the fluorescence microscope; Baumstark-Khan C et al.; Formation of the complete spindles during the budding process of Saccharomyces uvarum was investigated by fluorescence microscopy of protoplasted cells . Protoplasts were treated with anti-tubulin antibodies and DAPI, a fluorescent dye staining DNA . Thus, both chromatin and spindles could be visualized . Duplication as well as formation of separated spindle pole bodies during the different stages of budding are documented, demonstrating the occurrence and behaviour of microtubules during yeast cell cycle.

Antonie Van Leeuwenhoek, 1984, 50(4), 369 - 78
Trichosporon adeninovorans sp . nov., a yeast species utilizing adenine, xanthine, uric acid, putrescine and primary n-alkylamines as the sole source of carbon, nitrogen and energy; Middelhoven WJ et al.; A new yeast species, Trichosporon adeninovorans, was isolated from soil by the enrichment culture method . Apart from adenine, the strain utilized uric acid, guanine, xanthine, hypoxanthine, 6,8-dihydroxypurine, putrescine, propylamine, butylamine, pentylamine, hexylamine and octylamine as sole source of carbon, nitrogen and energy . The structure of the cell wall of Tr . adeninovorans was ascomycetous . On the subcellular level growth on adenine or uric acid was accompanied with the development of microbodies in the cell . These cell organelles probably were the site of urate oxidase, an enzyme that, after growth on purine substrates, together with allantoinase was present at high activities . Low activities of adenine amidohydrolase and xanthine dehydrogenase were also demonstrated.

Antonie Van Leeuwenhoek, 1984, 50(3), 249 - 60
The induction of mycelial development in Aureobasidium pullulans (IMI 45533) by yeast extract; Cooper LA et al.; Germ tube and subsequent mycelial development from yeast-like and swollen cells of Aureobasidium pullulans (IMI 45533) was induced by yeast extract in defined liquid medium . This morphogenetic transition was dependent on inoculum size; pH effects were not involved and once mycelial development was induced in the cells it continued even in the absence of yeast extract . The progeny of mycelium and future generations were unaffected by yeast extract . Cessation of germination was not due to any obvious medium changes but appeared to be partly due to the production of a germination inhibitor, which could also be produced by control cells grown in the absence of yeast extract.

Antonie Van Leeuwenhoek, 1984, 50(3), 219 - 25
Candida lignophila sp . nov., a new basidiomycetous yeast anamorph from rotting wood of Drimys winteri; Dill I et al.; Two strains of an undescribed Candida species were isolated from samples of a rotting trunk of Drimys winteri collected on the isle of Chiloe in South Chile . A description of the new species Candida lignophila is given and its relationship to other species is discussed with particular emphasis on its typical basidiomycetous properties as well as on its ecological habitat.

J Immunopharmacol, 1984, 6(4), 305 - 21
The immunomodulatory effect of yeast glucan on delayed hypersensitivity; Perez HA et al.; The effect of yeast beta-1, 3-glucan as an immunopotentiator of delayed type-hypersensitivity reactions (DHR) was studied . Delayed-type-hypersensitivity reactions in mice sensitized intraperitoneally (IP) with sheep red blood cells (SRBC) and pretreated three days previously with glucan given IP were significantly increased . However, mice sensitized IP with SRBC three days after the subcutaneous (SC) administration of glucan showed depressed DHR . Glucan given at the same site but not at distance strongly potentiated the DHR induced by SC sensitization with SRBC . Subcutaneous injection of glucan and SRBC given together also resulted in a sustained DHR which persisted twelve days after sensitization when DHR of control mice had waned.

Antonie Van Leeuwenhoek, 1984, 50(4), 379 - 81
The yeast Candida sequanensis sp . nov; Saez H et al.; A strain of an undescribed Candida species was isolated from animal fodder . A description of the new species is given and its distinction from the most closely resembling species of the genus is discussed.

Antonie Van Leeuwenhoek, 1984, 50(4), 305 - 20
Nematospora sinecauda sp . nov., a yeast pathogen of mustard seeds; Holley RA et al.; An undescribed yeast species was recovered from oriental (Brassica juncea) and yellow (B . hirta) mustard seeds . The new species most closely resembled Nematospora coryli but its asci were rarely cylindrical . The asci and ascospores of N . sinecauda were smaller and the spores did not possess a whip-like appendage . During germination a sprout cell formed first on the smooth anterior surface of the spore above the median ridge . The posterior region of the spore was decorated with interrupted concentric ridges . A description of the new species is given.

Mol Gen Genet, 1984, 197(3), 519 - 21
Mutagenesis in yeast-misreplication or misrepair?
Kilbey BJ.
Evidence from the phenotype of mutants which partially block mutagenesis and from experiments made to time induced mutagenesis relative to cell division in yeast is used to question whether mutagenesis in yeast should be regarded as an error-prone repair phenomenon.

Sabouraudia, 1984, 22(6), 487 - 91
Immunodiffusion studies on the antigens of Histoplasma capsulatum: comparison of mycelial histoplasmin with the yeast phase reagent Histolyn-CYL; Owens RD et al.; Immunodiffusion assays were performed to determine if H and M antigens associated with mycelial histoplasmin were present in the yeast phase reagent, Histolyn-CYL (H-CYL) . Neither H nor M antigens could be detected in H-CYL . However, when H-CYL was reacted against human histoplasmal sera a positive reaction was evidenced in all instances in which a response was elicited with a reference mycelial immunodiffusion reagent.

Enzyme, 1984, 31(4), 197 - 208
Studies on NADPH-cytochrome c reductase . II . Steady-state kinetic properties of the crystalline enzyme from ale yeast; Tryon E et al.; From a study of the steady-state kinetics (at pH 7.6, 30 degrees C) of the reduction of cytochrome c, a 'ping-pong' mechanism may be postulated for the crystalline NADPH-cytochrome c reductase from ale yeast, Saccharomyces cerevisiae {1}, a result derivable from a three-substrate ordered system with a rapid equilibrium random sequence in substrates, NADPH and FAD, followed by reactions of the third substrate, Cyt C3+ . On this basis, estimates for the kinetic parameters were made together with the inhibitor dissociation constants for NADP+ (competitive with respect to NADPH as variable substrate, but noncompetitive with respect to cytochrome c3+ as the variable substrate) . A noncompetitive type of inhibition was also found for cytochrome c2+ with NADPH as variable substrate, in confirmation of the proposed mechanism . With 2,6-dichloroindophenol as the acceptor, in place of cytochrome c3+, a value for KNADPH could be estimated which agreed with that estimated above, with cytochrome c3+ as the acceptor, again, in confirmation of the postulated mechanism . The reactions with molecular O2 catalyzed by the enzyme with NADPH as the reductant have been studied polarographically, and its Km for O2 estimated to be about 0.15 mmol/l at pH 7.6, 25 degrees C . The product of the reaction appears to be H2O2, which acts as a noncompetitive inhibitor for NADPH (Ki = 0.5 mmol/l), and tentatively an enzyme ternary complex containing oxygen and FADoh (semiquinone of FAD) may be assumed to be the kinetically important intermediate, which may be postulated to be in quasi-equilibrium with an enzyme ternary complex containing Oo2 (superoxide) and FAD.

Sabouraudia, 1984, 22(1), 1 - 5
Effects of divalent cations and functionally related substances on the yeast to mycelium transition in Sporothrix schenckii; Alsina A et al.; In a minimal basal medium with glucose at pH 4.0 and 25 degrees C, a lowering of the magnesium and zinc concentrations or increase in the calcium concentration of the medium favoured the yeast-mycelium transition in Sporothrix schenckii . Addition of zinc (1 and 10 mM) inhibited mycelial development and induced reversion to a yeast-like morphology . EDTA and EGTA also delayed germ tube formation, possibly by their calcium-chelating effects or by altering intracellular concentrations of this or other ions . Ionophore X537A also caused a delay in germ tube formation, possibly by interfering with magnesium metabolism in these cells.

J Cell Sci Suppl, 1984, 1, 43 - 58
Structural and functional analysis of a yeast centromere (CEN3); Carbon J et al.; Structure-function analysis of a yeast (Saccharomyces cerevisiae) centromere (CEN3) has been carried out by altering the nucleotide sequence of the DNA within and surrounding the centromere of yeast chromosome III, and observing the behaviour of the resulting altered chromosomes during mitotic and meiotic cell divisions . A centromere substitution vector (pJC3-13) was constructed, which contains in the proper orientation: the DNA sequences that normally flank the chromosome III centromere, a wild-type URA3 gene for selection, and a unique BamHI restriction site for insertion of various DNA sequences to be assayed for centromere activity . Cleavage of the plasmid DNA with EcoRI generates a linear DNA fragment whose ends are homologous with the regions flanking the centromere . Transformation of the appropriate homozygous ura3 diploid yeast strain with this linear DNA results in URA3+ transformants in which the CEN3 region on one copy of chromosome III has been replaced by the URA3 gene and the DNA sequence previously inserted into the vector . These studies identify a 289 base-pair (bp) DNA fragment from the CEN3 region that retains full centromere function when used to replace the normal CEN3 sequence . Centromeres function equally well in either orientation, and the chromosome XI centromere (CEN11) can be used to replace CEN3, with no observable effect on mitotic or meiotic chromosome segregation . Various DNA restriction fragments occurring within the CEN3 region were used alone or in combinations to replace the normal CEN3 sequence . Yeast centromeres contain a high A + T region about 82-89 bp in length (element II) flanked by a highly conserved 11 bp sequence (III) and a less-conserved 14 bp sequence (I) . The experiments demonstrate that both regions II and III are necessary for normal centromere function, although centromeres containing III plus truncated or rearranged portions of the high A + T region II retain partial activity . Chromosomes of the latter type often give abnormal segregation patterns through meiosis, including separation and random segregation of sister chromatids during the first meiotic division.

Antonie Van Leeuwenhoek, 1984, 50(5-6), 799 - 805
The adventures of the yeast genus Endomycopsis Dekker; von Arx JA et al.; The yeast genera Endomyces, Endomycopsella, Guilliermondella and Saccharomycopsis are delimited by the size, structure and pigmentation of the ascospores; they include mycelial yeasts formerly classified in the invalid genus Endomycopsis . The ultrastructure of the cell wall and the septa of yeasts is briefly discussed.

Mol Gen Genet, 1984, 197(3), 491 - 6
A hybrid DNA sequence containing the replication origin of the multicopy yeast plasmid 2 micron circle and an additional repeated sequence can convert maltose-negative into maltose-positive strains; Rodicio R et al.; Yeast DNA pools were prepared by ligating partial Sau3A genomic digests from strains carrying various MAL genes into the BamHI site of the yeast-Escherichia coli shuttle vector YRp7 . They were used to transform recipient yeast strains that could not utilize maltose since they lacked a classical MAL gene . Transformants were obtained that could use maltose and also formed normal levels of maltase . They were unstable . They would lose the selective marker TRP1 of YRp7 alone, together with the ability to utilize maltose or only the ability to utilize maltose . The insertion of one of the plasmids was used as a hybridization probe for the others and found to share homologous sequences with all . They were then shown to contain the replication origin of the yeast 2 micron circle plasmid and additional sequences . These additional sequences were used to probe genomic digests of total yeast DNA . They hybridized at various degrees of efficiency with several bands, indicating that they were part of a family of repeated sequences . Apparently, it was the combination of the replication origin of the 2 micron circles with the additional sequences that promoted maltose utilization.

Mol Gen Genet, 1984, 197(3), 351 - 7
Conformational alterations in the proximal portion of the yeast invertase signal peptide do not block secretion; Brown PA et al.; Various amino acid insertions have been introduced into the proximal portion of the signal sequence of secreted yeast invertase . The altered invertase genes have been reintroduced into yeast and monitored for their ability to direct synthesis of secreted invertase in vivo . The insertions should alter the signal polypeptide local secondary structure as predicted by the Chou and Fasman rules (1978) . Secretion of these altered invertase polypeptides is not blocked by the amino acid insertions.

Mol Gen Genet, 1984, 197(2), 345 - 6
A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance; Boeke JD et al.; Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection . Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3- mutants grow normally . This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells.

Microbiol Immunol, 1984, 28(9), 997 - 1007
Lethal effect of neutral mannan fraction of bakers' yeast in mice; Nagase T et al.; A simple polysaccharide, the neutral mannan from Saccharomyces cerevisiae wild type strain (WNM) was found to kill ddY strain mice by intravenous administration, showing a LD50 value of 12.2 mg/kg . On the other hand, the acidic mannan fraction from the same yeast containing phosphate (WAM025), and chemically phosphorylated WNM (WNM-P) were practically non-toxic . Concerning the relationship between chemical structure and lethal effect of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger lethality than those with less branched structures . Against C3H/HeJ strain mice with no responsiveness to lipopolysaccharide, the LD50 value of WNM was as high as 75 mg/kg . Pretreatment with 500 mg/kg of D-mannose, N-acetyl-D-glucosamine, D-galactose, and L-fucose prevented mice from the lethal effects of WNM . However, WNM (LD100) did not show any lethal effect in mice for 2 to 12 hr after treatment with dexamethasone, an anti-inflammatory steroid.

Radiat Environ Biophys, 1984, 23(4), 287 - 94
Release of P and K from yeast cells irradiated by vacuum UV below 170 nm; Matsumoto S et al.; Yeast cells were irradiated with monochromatic synchrotron radiation (SR) under wet conditions in the wavelength region from 160 to 185 nm at INS-SOR, Tokyo . By the particle-induced X-ray emission (PIXE) method applied to whole cells several elements were found to be released from the irradiated cells at the wavelengths shorter than 170 nm . The most drastic release occurred with phosphorus, followed potassium . Sulphur and calcium were not released over the whole wavelength region studied . It was also revealed that the release of these elements paralleled the cell inactivation . The cause of these element releases upon vaccuum-UV irradiation was inferred in relation to the dissociation of H2O molecules located in the vicinity of the cell surface region.

Mol Gen Genet, 1984, 196(2), 266 - 74
Organization and processing of the mitochondrial oxi3/oli2 multigenic transcript in yeast; Simon M et al.; In the present article, we confirm our previous proposal (Faye and Simon 1983a, b) that the oxi3 and oli2 genes belong to the same transcription unit . Furthermore, we have shown that a primary polycistronic transcript covers oxi3, aap1, oli2 and extends beyond URF2 . Transcriptional analysis of this region revealed several cleavage points . The examination of the DNA sequence at and surrounding these cleavage points disclosed that some of them take place at or near specific sequences found also in other known multigenic transcripts . Two of the major cleavages involve the stem-loop structure of GC rich clusters . We discuss the possibility that some of these cleavage sites serve as post-transcriptional processing signals and may be necessary for the maturation of the precursor RNA.

Mol Gen Genet, 1984, 195(1-2), 260 - 6
Replicon size of yeast ribosomal DNA; Walmsley RM et al.; The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9K bp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 microns long, on chromosome XII . Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined . We have used immobilised rRNA as a probe to examine the size of radioactively labelled rDNA replicons resolved on alkaline sucrose gradients . The replicons were found to be uniformly sized, about 5 repeat units in length, and groups of 4 adjacent replicons may be activated simultaneously . These observations suggest that replicon initiation events are not determined solely by the recognition of specific DNA sequences that function as origins of replication.

Prep Biochem, 1984, 14(2), 163 - 71
An evaluation of methods used to prepare yeast mitochondria for transcriptional studies; Rickwood D et al.; This report describes experiments which compare conditions necessary to isolate mitochondria uncontaminated with nuclear chromatin using a KDL grinding mill.

Microbiol Immunol, 1984, 28(6), 651 - 7
Pyrogenicity of yeast mannans in rabbits; Nagase T et al.; A few yeast mannans free from protein and phosphorus showed pyrogenic activity in rabbits although the extent of this activity was considerably lower than that of the bacterial lipopolysaccharides (LPS) . The pyrogenic activity was not abolished by treatment with sodium deoxycholate . This result showed that the mannans themselves participated in the pyrogenicity, excluding any possibility of LPS contamination in the mannans . Concerning the relationship between chemical structure and pyrogenicity of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger pyrogenicity than that of a less branched one.

Biochem Int, 1984 Jan, 8(1), 105 - 12
Yeast mitochondrial inner membrane 30 kd hydrophobic protein: properties and interaction with phospholipids; Alkoutayni M et al.; A 30 kd hydrophobic protein is extracted from yeast mitochondrial inner membrane . It is present in wild yeast strains but absent in mitochondrial DNA lacking mutants . The isoelectric point of the protein and its solubility in various organic solvents are determined . The fluorescence of a tryptophan residue near the surface of the 30 kd protein dissolved in butanol-1, can be quenched by phospholipids containing unsaturated fatty acids . Results are in accordance with the 30 kd protein being an integral protein of the yeast mitochondrial inner membrane.

Mol Gen Genet, 1984, 195(3), 487 - 90
UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast; Lawrence CW et al.; The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis . The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles . The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes . If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.

Mol Gen Genet, 1984, 194(3), 489 - 93
Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha; Roggenkamp R et al.; The biosynthesis of methanol oxidase, a peroxisomal enzyme in the methanol-utilizing yeast Hansenula polymorpha, was studied in vitro . Translation of Hansenula mRNA in a rabbit reticulocyte lysate yields methanol oxidase protein in high amounts . The apparent molecular mass of the protein was found to be identical to the subunit of the functional multimeric enzyme, which indicates the absence of an N-terminal extension typical of most transported proteins . The regulation of methanol oxidase by glucose repression and depression as well as by induction of methanol was shown to be controlled at the level of transcription . Two mutants of Hansenula polymorpha, unable to grow on methanol as a carbon and energy source were shown to be affected in methanol oxidase synthesis.

Mol Gen Genet, 1984, 194(1-2), 7 - 14
Genetic mode of action of cocarcinogens and tumor promoters in yeast and mice; Fahrig R; In experiments with yeast, cocarcinogens were found to be comutagenic and antirecombinogenic , tumor promoters to be corecombinogenic and antimutagenic . Substances that were cocarcinogens as well as tumor promoters had an intermediary effect . These results were confirmed in the mammalian spot test: By in vivo treatment of mice with the cocarcinogen catechol and the tumor promoter limonene carcinogen-induced recombination due to mitotic crossing over and gene mutations was reduced and enhanced, respectively . Our results support the hypothesis that mutagenesis is the mechanism by which chemicals induce malignancy, and that cocarcinogens modify the process by enhancement of mutagenicity whereas tumor promoters effect carcinogenesis by increase of the spontaneous frequency of recombination . In addition, induced mitotic recombination in mammals in vivo has been demonstrated for the first time.

Gene, 1984 Jan, 27(1), 23 - 33
Cloning and expression of a yeast copper metallothionein gene; Butt TR et al.; The induction of a copper-binding metallothionein (Cu-MT) was studied in yeast, Saccharomyces cerevisiae, and a relationship between copper resistance and intracellular levels of Cu-MT in these eukaryotes was established . Poly(A)-containing RNA from a copper-resistant (Cur) yeast strain, which synthesized abundant quantities of Cu-MT and in which Cu-MT gene transcription was enhanced 50-fold upon exposure to CuSO4, was used to screen yeast genomic DNA clones . Restriction analysis revealed common XbaI and KpnI sites in five genomic clones isolated . The transcription of these clones was regulated by copper . Transformation of a copper-sensitive (Cus) yeast strain by one of these clones confers copper resistance in yeast . The results suggest that the expression of the Cu-MT gene is, in part, responsible for mediating copper resistance in yeast.

Mikrobiologiia, 1984 Jan-Feb, 53(1), 5 - 9
{Intracellular pool of free amino acids in dehydrated yeast organisms}; Novichkova AT et al.; The dehydration of Saccharomyces cerevisiae was found to result in a noticeable decrease of the free amino acids content in the cells and in a considerable increase of cytoplasmic membrane permeability for these compounds . When the dehydrated organisms were reactivated, the normal permeability of the cytoplasmic membrane gradually restored and the pool of free amino acids increased in the cells.

J Cell Biol, 1984 Jan, 98(1), 44 - 53
Genes required for completion of import of proteins into the endoplasmic reticulum in yeast; Ferro-Novick S et al.; Yeast secretory mutants sec53 and sec59 define a posttranslational stage in the penetration of glycoprotein precursors into the endoplasmic reticulum (ER) . In the previous report we showed that at the restrictive temperature (37 degrees C) these mutants accumulate enzymatically inactive and incompletely glycosylated forms of the secretory enzyme invertase and the vacuolar enzyme carboxypeptidase Y . Cell fractionation experiments reveal that these precursor forms remain firmly bound to the ER membrane . However, upon return to the permissive temperature (24 degrees C), the invertase precursors are glycosylated, become partially active, and are secreted . Thermoreversible conversion does not require protein synthesis, but does require energy . In contrast to the effect of these mutations, inhibition of oligosaccharide synthesis with tunicamycin at 37 degrees C causes irreversible accumulation of unglycosylated invertase . The effect of the drug is exaggerated by high temperature since unglycosylated invertase synthesized in the presence of tunicamycin at 25 degrees C is secreted . A portion of the invertase polypeptide accumulated at 37 degrees C is preserved when membranes from sec53 and sec59 are treated with trypsin . In the presence of Triton X-100 or saponin, the invertase is degraded completely . The protected fragment appears to represent a portion of the invertase polypeptide that is embedded in or firmly associated with the ER membrane . This association may develop early during the synthesis of invertase, so that in the absence of translocation, some of the completed polypeptide chain remains exposed on the cytoplasmic surface of the ER.

J Cell Biol, 1984 Jan, 98(1), 35 - 43
Yeast secretory mutants that block the formation of active cell surface enzymes; Ferro-Novick S et al.; Yeast cells secrete a variety of glycosylated proteins . At least two of these proteins, invertase and acid phosphatase, fail to be secreted in a new class of mutants that are temperature-sensitive for growth . Unlike the yeast secretory mutants previously described (class A sec mutants; Novick, P., C . Field, and R . Schekman, 1980, Cell., 21:205-420), class B sec mutants (sec 53, sec 59) fail to produce active secretory enzymes at the restrictive temperature (37 degrees C) . sec 53 and sec 59 appear to be defective in reactions associated with the endoplasmic reticulum . Although protein synthesis continues at a nearly normal rate for 2 h at 37 degrees C, incorporation of {3H}mannose into glycoprotein is reduced . Immunoreactive polypeptide forms of invertase accumulate within the cell which have mobilities on SDS PAGE consistent with incomplete glycosylation: sec 53 produces little or no glycosylated invertase, and sec 59 accumulates forms containing 0-3 of the 9-10 N-linked oligosaccharide chains that are normally added to the protein . In addition to secreted enzymes, maturation of the vacuolar glycoprotein carboxypeptidase Y, incorporation of the plasma membrane sulfate permease activity, and secretion of the major cell wall proteins are blocked at 37 degrees C.

J Biochem (Tokyo), 1984 Jan, 95(1), 109 - 15
A kinetic study on the binding of monomeric and polymeric derivatives of NAD+ to yeast alcohol dehydrogenase; Yamazaki Y et al.; The binding to yeast alcohol dehydrogenase of NAD+ and its five derivatives (N6-{2-{N-{2-{N-(2-methacrylamidoethyl)carbamoyl}ethyl} carbamoyl}ethyl}-NAD (I), N6-{N-{2-{N-(2-methacrylamidoethyl) carbamoyl}ethyl}carbamoylmethyl}-NAD (II), copolymer of I with acrylamide (PA-I), copolymer of II with acrylamide (PA-II), and copolymer of I with N,N-dimethylacrylamide (PDMA-I} were studied statically and kinetically by the stopped-flow method by using the quenching of the enzyme fluorescence in the presence of pyrazole . Apparent dissociation constants and apparent rate constants were determined therefrom . It was concluded that (1) the N6-CH2CH2CO group (of I) is effective in making the derivative bind more strongly as well as faster than NAD+, while the N6-CH2CO group (of II) is not; and (2) the binding of the polymer derivatives of NAD+ to the enzyme is not essentially weaker and slower than that of native NAD+, but is even faster in some cases . The coenzymic activities of the above compounds were also determined with yeast alcohol dehydrogenase, pig heart malate dehydrogenase, and rabbit muscle lactate dehydrogenase.

Int J Biochem, 1984, 16(2), 177 - 82
The stimulation of yeast mitochondrial protein synthesis by low molecular weight cytoplasmic factors: requirement for intact mitochondria and lack of effect by folate derivatives; Finzi E et al.; Protein synthesis by GTP-supplemented yeast mitochondria is stimulated by a fraction of molecular weight less than 2,000 isolated from yeast high-speed supernatant (S-150) . The low molecular weight fraction works independently of the respiratory chain as the stimulation effect is not cyanide-sensitive . Stimulation of mitochondrial protein synthesis by cytoplasmic factors is dependent upon the method of mitochondrial isolation . The low molecular weight stimulatory factor(s) are not reduced folate derivatives which supply formyl groups required for initiation of mitochondrial protein synthesis.

EMBO J, 1984 Jan, 3(1), 107 - 11
Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA; Skipper N et al.; Complementary DNA (cDNA) copies of the M1-1, toxin-coding region of the yeast M1 double-stranded RNA (dsRNA) have been cloned and sequenced . These sequences, in combination with the known terminal sequence of M1-1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation . Potential glycosylation sites in the preprotoxin peptide are identified . Based on its methionine content the extracellular yeast toxin appears to be contained within the C-terminal region of the precursor.

Br J Cancer Suppl, 1984, 6, 157 - 61
Recovery from transcription inhibition in irradiated yeast cells; Weber KJ et al.; Repair process operating on radiation damaged DNA may be investigated by studying its functional integrity, e.g . its ability to serve as template for transcription . We measured the synthesis of ribosomal RNA and of the inducible enzyme arginase in yeast cells and followed their recovery after X-ray, alpha-particle and heavy ion exposure . Transcription inhibiting lesions formed upon X-irradiation in yeast cells are repaired during post-exposure incubation ("liquid-holding") . The inactivation curves are strictly exponential immediately after irradiation . After the "liquid holding" treatment the inactivation curves are still exponential but with a progressive decrease of the slopes as a function of incubation time . This indicates that a constant fraction of the lesions is repaired per time interval . But even after long incubation times (24 h) there is still a sizeable unrepaired fraction . Comparing different yeast strains and different irradiation temperatures recovery can also be demonstrated in cells exposed to alpha-particles . In addition, recovery is detected by the arginase assay after irradiation of yeast with very heavy charged particles, e.g . titanium ions.

Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 337 - 41
Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast; Karin M et al.; Copper resistance in yeast is controlled by the CUP1 locus . The level of resistance is proportional to the copy number of this locus, which can be found in up to 15 tandemly iterated copies . To elucidate the molecular mechanisms controlling the amplification and expression of the CUP1, locus, we determined its full nucleotide sequence . We have also identified and mapped two transcription units within the basic amplification unit of CUP1 in laboratory yeast strains . One of those transcription units is inducible by copper and encodes a low molecular weight copper binding protein--copper chelatin . The increased production of chelatin, due to both gene amplification and induction of transcription, leads to increased resistance of yeast cells to copper ions.

J Inorg Biochem, 1984 Jan, 20(1), 39 - 52
Activation of yeast enolase by Cd(II); Spencer SG et al.; Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+ . The activity is weakly stimulated, then inhibited by increasing ionic strength . The activity increases, then falls with increasing Cd2+ concentration . The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme . The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH . This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's . The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme . We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc . The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex . The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7 . There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor . The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen . The apparent reaction rate constant increases linearly with Cd2+ concentration . Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly . The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.

J Cell Biochem, 1984, 24(3), 229 - 42
Mitochondrial membrane biogenesis: characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase; McEwen JE et al.; A nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene (COX5) by complementation . This mutant, E4-238 {24}, and its revertant, JM110, produce variant forms of subunit V . In comparison to the wild-type polypeptide (Mr = 12,500), the polypeptides from E4-238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively . These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis-acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type . Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4-238, with DNA from a yeast nuclear DNA library in the vector YEp13 . One plasmid, YEp13-511, with a DNA insert of 4.8 kilobases, was characterized in detail . It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V . The availability of mutants altered in the structural gene for subunit V (COX5) and of the COX5 gene on a plasmid, together with the demonstration that plasmid-encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase.

Gene, 1984 Jan, 27(1), 13 - 21
Cloning of the vaccinia virus telomere in a yeast plasmid vector; DeLange AM et al.; The vaccinia virus DNA telomere, which contains a covalently closed hairpin structure, has been cloned in a yeast plasmid vector . Restriction mapping indicates that the cloned vaccinia telomere is maintained in yeast not in its native hairpin configuration but as an inverted repeat structure, within a circular plasmid, with the sequences of the viral hairpin now at the axis of symmetry of an imperfect palindrome . As such, the cloned telomere resembles the telomeric replicative intermediate observed during vaccinia virus DNA replication . Small deletions and duplications in the viral inverted repeats of different clones suggest a model in which the observed circular plasmids were generated in yeast by the replication of hybrid linear DNA molecules consisting of the linearized yeast vector flanked by two hairpin-containing vaccinia termini.

Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 367 - 70
Regulated expression of a human interferon gene in yeast: control by phosphate concentration or temperature; Kramer RA et al.; The promoter/regulator region from the yeast repressible acid phosphatase gene was used to construct a vector for the regulated expression of cloned genes in yeast . The gene for human leukocyte interferon was inserted into this vector . Yeast cells transformed with the resulting plasmid produced significant amounts of interferon only when grown in medium lacking inorganic phosphate . Mutants in two acid phosphatase regulatory genes (coding for a defective repressor and a temperature-sensitive positive regulator) were used to develop a yeast strain that grew well at a high temperature (35 degrees C) but produced interferon only at a low temperature (23 degrees C), independent of phosphate concentration.

Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 8 - 12
Steps in processing of the mitochondrial cytochrome oxidase subunit I pre-mRNA affected by a nuclear mutation in yeast; Simon M et al.; In Saccharomyces cerevisiae, the mitochondrial gene encoding the subunit I of cytochrome c oxidase (oxi-3 gene) is interrupted by intervening sequences . In this report, a nuclear mutation {referred to as mss51 in Faye, G . & Simon, M . (1983) Cell 32, 77-87} that specifically affects the processing of oxi-3 pre-mRNA was further characterized . DNA probes covering each oxi-3 exon-intron boundary were individually hybridized to wild-type and mutant mitochondrial RNA . By a technique relying on the S1 nuclease resistance or sensitivity of the RNA X DNA hybrids thereof, we have shown which site needs the MSS51 gene product to be cleaved . The mutation in the MSS51 gene gave rise to a complex pattern of splicing: the third intron was excised efficiently but the first two introns remained bracketed by their flanking exons . Further, the fourth and fifth introns were only partially split from their common exon and remained fused to their upstream and downstream flanking exon, respectively . Several plausible roles for the MSS51 gene product are discussed.

Nature, 1984 Jan 26-Feb 1, 307(5949), 386 - 8
Preferential integration of yeast transposable element Ty into a promoter region; Eibel H et al.; Mobile genetic elements have been identified in several eukaryotic organisms and some classes have been found to share common structural features with the proviral forms of animal retroviruses . The representatives of this class of mobile elements in the yeast Saccharomyces cerevisiae are called Ty elements, which could be a useful model system for studying the transposition of retrovirus-like elements . Here we have attempted to answer two questions often raised in discussions of the biological importance of transposition: what is the frequency of spontaneous Ty transposition, and are there certain chromosomal regions into which Ty elements preferentially integrate? We chose the LYS2 gene to investigate these questions because it allows direct selection of both mutants and revertants . We have found that 2% of spontaneous lys2 mutants are caused by Ty transposition with a preferential integration into the transcription initiation region.

Mol Gen Genet, 1984, 197(3), 515 - 6
Multiple genes control particulate phosphofructokinase of yeast; Parmar L et al.; Mutants of Saccharomyces cerevisiae lacking the particulate phosphofructokinase define at least four unlinked genes, PFK2, PFK3, PFK4 and PFK5 . A structural role of PFK2 is indicated . Mutations in the other three have pleiotropic effects.

Biomed Biochim Acta, 1984, 43(10), 1083 - 9
Influence of inorganic phosphate on the kinetic properties of yeast phosphofructokinase; Przybylski F et al.; Yeast phosphofructokinase is effectively activated by inorganic phosphate . In the absence of other allosteric stimulators, inorganic phosphate increases the maximum activity of the enzyme only . In the presence of the activators AMP and fructose 2,6-bisphosphate inorganic phosphate causes changes in the maximum activity and the enzyme affinity to fructose 6-phosphate . Inorganic phosphate augments the sensitivity of phosphofructokinase to the activators AMP and fructose 2,6-bisphosphate and increases the respective maximum activities . The extent of activation of the enzyme by inorganic phosphate prevails at low levels of fructose 6-phosphate and high ATP concentrations.

Biomed Biochim Acta, 1984, 43(4), 535 - 40
Cooperation of fructose-2,6-bisphosphate and AMP in the activation of yeast phosphofructokinase; Nissler K et al.; Yeast phosphofructokinase is effectively activated by AMP and fructose-2,6-bisphosphate . Both effectors influence the sensitivity of the enzyme with respect to fructose-6-phosphate and increase the respective maximum activities . The dependence of phosphofructokinase activity on the concentration of fructose-2,6-bisphosphate was measured at different AMP concentrations and vice versa . By AMP the half activation constant for fructose-2,6-bisphosphate is decreased by one order of magnitude . The affinity to AMP is significantly increased by fructose-2,6-bisphosphate . AMP increases the maximum activity of the enzyme with respect to fructose-2,6-bisphosphate only slightly, while the maximum activity with respect to AMP is drastically increased by fructose-2,6-bisphosphate . The interaction of the two activators is most pronounced at low levels of fructose-6-phosphate and at high concentrations of ATP.

Biomed Biochim Acta, 1984, 43(4), 413 - 8
Interaction of Cibacron blue F3G-A with yeast phosphofructokinase; Frenzel J et al.; The binding of Cibacron blue F3G-A to yeast phosphofructokinase was investigated by means of ultracentrifugation . Four moles of Cibacron blue are tightly bound per subunit of phosphofructokinase (dissociation constant = 0.26 microM) . This stoichiometry does not correspond to the stoichiometry of ATP binding to yeast phosphofructokinase (two moles of ATP per subunit) . Moreover, 32 moles of the dye are bound per subunit of phosphofructokinase to a second class of binding sites with low affinity (dissociation constant = = 53 microM) . The action of Cibacron blue on yeast phosphofructokinase cannot be explained completely in terms of its function as ATP analogue.

Biochimie, 1984 Jan, 66(1), 49 - 58
Studies on the structure of yeast phosphofructokinase; Chaffotte AF et al.; In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase . This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations . The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods . However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer . On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase . Obviously, some methods of molecular weight determination have led to erroneous results . In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.

Acta Microbiol Pol, 1984, 33(2), 119 - 30
Effect of some quaternary benzylammonium salts on physiology of yeast; Kolodynski J et al.; In order to determine the biological activity of eight compounds belonging to a group of quaternary ammonium salts, their influence on the active methionine transport, the integrity of cell membranes, respiration, and viability of Saccharomyces cerevisiae and some other yeast species has been investigated . The earliest effect observed during ammonium salts action on yeast cells is an immediate methionine transport abolishment followed by its fast leakage, which indicates increasing cell membrane degradation . Gradual decline of other biological functions such as respiration and viability is thus a result of disintegration and lack of tightness of the cell membranes . The studied compounds are characterized by a rather unspecific spectrum of action on yeast resulting in irreversible damage of cell walls and cell membranes, which in consequence leads to cell death.

J Biochem (Tokyo), 1984 Jan, 95(1), 131 - 6
Purification and properties of factors in yeast mitochondria stabilizing the F1F0-ATPase-inhibitor complex; Hashimoto T et al.; A previously found yeast-mitochondrial protein fraction stabilizing the inactivated complex between mitochondrial ATPase and intrinsic ATPase inhibitor (Hashimoto, T., et al . (1983) J . Biochem . 94, 715-720) was separated into two proteins by high performance liquid chromatography on a cation exchanger . The molecular weights of the factors were estimated to be 9,000 and 15,000 daltons by sodium dodecyl sulfate (SDS)-gel electrophoresis . Both factors were required to stabilize a complex of inhibitor and proton-translocating ATPase (F1F0-ATPase) either in its purified form or in mitochondrial membranes . On the other hand both factors together could not stabilize a complex of the inhibitor and F1-ATPase, suggesting that both factors act together with the F0-portion . The factors also facilitated very efficiently the binding of ATPase inhibitor to F1F0-ATPase in the presence of ATP and Mg2+ . Both the 15,000 and 9,000 dalton stabilizing factors were hardly distinguishable from delta- and epsilon-subunit, respectively, on an SDS-gel electrophoregram, but immuno-diffusion assay showed that neither factor was present in the purified F1-ATPase containing the delta- and epsilon-subunit.

Teratog Carcinog Mutagen, 1984, 4(4), 365 - 75
Comparative genetic activity of cis- and trans-1,2-dichloroethylene in yeast; Bronzetti G et al.; The cis and trans isomers of 1,2-dichloroethylene were tested for mutagenic effects in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system, an S9 mouse liver fraction, and by an in vivo intrasanguineous host mediated assay . The effects of the same agents on aminopyrine N-demethylase activity and cytochrome P-450 level in liver were studied in nonpretreated and in phenobarbital + beta-naphtoflavone-pretreated mice . In the suspension test, both isomers exhibited dose dependent toxicity, and survival was lower with metabolic activation than without . In this test also, both isomers exhibited genetic activity as measured by increases in recombinants at the ade 2 locus in experiments with metabolic activation . In the host-mediated assay, only the cis isomer showed evidence of mutagenic activity with significant increases in convertants at the trp locus and revertants at the ilv locus . Such mutagenic activity was found both after acute and chronic doses and in liver, kidney, and lung tissue . The two isomers exhibited different effects with respect to aminopyrine N-demethylase activity and cytochrome P-450 level . In general, the trans isomer appeared to emphasize induction of enzyme activity or level while the cis isomer more frequently tended to inhibit activity or destroy the enzyme.

Mol Gen Genet, 1984, 197(3), 420 - 4
A yeast with linear molecules of mitochondrial DNA; Kovac L et al.; Mitochondrial DNA from the yeast strain SR23, tentatively allocated to the species Candida rhagii, consists of linear molecules 30 kb long . This has been demonstrated by restriction analysis and selective radioactive labelling of terminal restriction fragments . Preliminary sequence analysis indicated that the two ends of the molecule are formed by inverted repeats . The arrangement of several genes in the mitochondrial genome of C . rhagii SR23 was established by specific hybridisation with probes prepared from mitochondrial DNA of Saccharomyces cerevisiae . The arrangement is unique, with genes coding for the two ribosomal RNAs placed widely apart . Intron(s) may be present in the gene coding for cytochrome b.

Nucleic Acids Symp Ser, 1984, (15), 177 - 80
Terminal structures of F2, a linear DNA plasmid from yeast: two different terminal structures in one linear DNA molecule; Kikuchi Y et al.; The terminal structure of F2, a linear double-stranded DNA plasmid of 3.9 kb from yeast, was analyzed . Results obtained by electrophoretic analyses of terminal restriction fragments of F2 showed that one end of F2 has terminal protein at the 5'-terminus, but another end has hairpin structure . This is a novel type of autonomously replicating DNA molecule.

Mol Gen Genet, 1984, 197(2), 219 - 24
Nuclear genes coding for four subunits of the yeast ubiquinol-cytochrome c reductase complex are present in single copies in the haploid genome and at least two of these are located on different chromosomes; Van Loon AP et al.; Genes coding for the 40 kilodaltons (kDa), 17-kDa, 14-kDa and 11-kDa subunits of the ubiquinol-cytochrome c reductase in yeast are present in single copies in the haploid genome . We have mapped each gene to a unique genomic environment and demonstrate that integration of cloned segments into nuclear DNA by homologous crossing-over with the endogenous gene results in the replacement of the corresponding chromosomal restriction fragment by fragments of predicted sizes . Chromosomal mapping, carried out by the procedure of Falco and Botstein 1983, indicates that the gene for the 17-kDa subunit lies on chromosome VI and that for the 11-kDa subunit on chromosome XII.

J Biol Chem, 1983 Dec 25, 258(24), 15037 - 45
The catalytic mechanism of yeast thiosulfate reductase; Chauncey TR et al.; Thiosulfate reductase catalyzes the desulfuration of thiosulfonates while oxidizing GSH to GSSG . Kinetic studies of the enzyme-catalyzed reaction between GSH and benzenethiosulfonate have been carried out, and direct evidence for the occurrence of glutathione persulfide as an immediate product of the reaction has been obtained . The formal mechanism of this enzymic reaction has been shown to be rapid equilibrium-ordered with GSH as the leading substrate.

J Mol Biol, 1983 Dec 15, 171(3), 345 - 52
Homologous proteins encoded by yeast mitochondrial introns and by a group of RNA viruses from plants; Zimmern D; Hensgens et al . (1983a) have demonstrated the existence of distant homology (averaging 19.6%) between the central sections of seven proteins encoded by introns (and one product of an apparently independent gene) in yeast mitochondrial DNA . The homologous regions are typically segments of about 115 amino acids within open reading frames of about 10(3) bases . Genetic studies indicate that at least two of these proteins are required for the splicing of mitochondrial transcripts . This paper reports that two distantly related proteins of Mr 30,000 that are encoded by different strains of tobacco mosaic virus both contain central sections whose amino acid sequences are 15% to 23% identical in a single alignment to those of one group of four intron-encoded proteins, and possess certain groups of conserved residues also characteristic of the mitochondrial proteins . Genetic studies implicate these proteins in the spreading of viral lesions . While this level of identity cannot establish conclusively that the proteins are related, it suggests the possibility of a functional and/or evolutionary connection that would, if borne out, have important implications.

J Biol Chem, 1983 Dec 10, 258(23), 14271 - 5
Dicyclohexylcarbodiimide blocks proton ejection and affects antimycin binding but not electron transport in complex III from yeast mitochondria; Clejan L et al.; Treatment of complex III with dicyclohexyldicarbodiimide (DCCD) either before or after incorporation into liposomes resulted in a loss of electrogenic proton movements; however, only minimal decreases in cytochrome c reductase activity were noted in the liposomes containing DCCD-treated complex III . Thus, DCCD appears to act by "uncoupling" proton translocation from electron transport . A decreased sensitivity of the ubiquinol:cytochrome c reductase activity to antimycin was also noted in the DCCD-treated complex III . This loss of sensitivity to antimycin was reflected in a decreased binding of antimycin to the complex after DCCD treatment from 9.5 nmol/mg of protein in the control to 3.8 nmol/mg of protein in the DCCD-treated complex . DCCD also affected the red shift observed after antimycin addition to dithionite-reduced complex III resulting in a broad peak with no sharp maximum . Similarly, DCCD treatment of yeast mitochondria resulted in a complete loss in the red shift after antimycin addition to mitochondria previously reduced with succinate . No loss in enzymatic activity was observed in the DCCD-treated mitochondria . These results suggest that DCCD concomitant with the inhibition of proton ejection in the cytochrome b-c1 region of the respiratory chain causes modifications in the properties of cytochrome b which alter the binding of antimycin without significantly affecting the electron transfer activity of this cytochrome.

J Biol Chem, 1983 Dec 10, 258(23), 14065 - 8
oli1 Transcripts in wild type and in a cytoplasmic "petite" mutant of yeast; Thalenfeld BE et al.; Subunit 9 of ATPase is known to be encoded in the oli1 gene of yeast mitochondrial DNA . The oli1 transcripts of wild type and of a cytoplasmic "petite" mutant have been analyzed by hybridization of mitochondrial RNA to various DNA fragments from the internal and flanking regions of the gene and by S1 nuclease mapping of the 5' and 3' ends . The results of such studies indicate that the ATPase gene is co-transcribed with the downstream serine tRNA gene . The oli1 message and tRNA are generated by post-transcriptional processing . Two of the nucleolytic processing steps are blocked in the cytoplasmic petite mutant, resulting in the accumulation of several different intermediate transcripts containing both genes . Processing of the 3' ends occurs near a common seven-nucleotide sequence (5'-ATTCTTA-3') also found in the 3' regions of other mitochondrial genes . This sequence is proposed to be part of a signal necessary for either termination of transcription or RNA processing.

Nucleic Acids Res, 1983 Dec 10, 11(23), 8269 - 82
Initiation of transcription of the yeast mitochondrial gene coding for ATPase subunit 9; Edwards JC et al.; We have determined transcriptional initiation sites for the ATPase subunit 9 gene on the yeast mitochondrial genome . Using S1 nuclease mapping, in vitro capping of primary transcripts with GTP and guanylyl transferase, and in vitro transcription analysis with purified mitochondrial RNA polymerase, we find the major site of transcriptional initiation to be at a point 630 nucleotides upstream of the coding region for the gene . In addition, we find much lower levels of initiation at a second site 78 nucleotides downstream of the first . Both initiation sites occur at the same position within a nonanucleotide sequence which we have previously found associated with initiation of rRNA synthesis . This work further supports the notion that this nonanucleotide sequence is an integral component of mitochondrial promoters and indicates that the same RNA polymerase is used for transcription of both mRNA and rRNA in yeast mitochondria.

Antonie Van Leeuwenhoek, 1983 Dec, 49(6), 571 - 8
Liquid nitrogen storage of yeast cultures . II . Stability of characteristics of stored strains; Kockova-Kratochvilova A et al.; Nineteen strains of yeasts possessing different characteristics were stored in liquid nitrogen and after 5 years phenotypic characters were evaluated and compared with equivalent strains preserved under paraffin oil . All qualitative characters tested remained stable, and quantitative characters varied only within the range of natural variability.

Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7466 - 70
Yeast mutants deficient in protein glycosylation; Huffaker TC et al.; The synthesis of asparagine-linked oligosaccharides involves the formation of a lipid-linked precursor oligosaccharide that has the composition Glc3Man9GlcNAc2 . We have used a {3H}mannose suicide selection to obtain mutants in yeast that are blocked in the synthesis of this precursor oligosaccharide . The alg1 mutant accumulated lipid-linked GlcNAc2, alg2 mutants accumulated Man1-2GlcNAc2, alg3 mutants accumulated Man5GlcNAc2, alg4 mutants accumulated Man1-8GlcNAc2, and alg5 and alg6 mutants accumulated Man9GlcNAc2 . Some of these mutants appeared to transfer oligosaccharides other than Glc3Man9GlcNAc2 from the lipid carrier to invertase . These aberrant protein-linked oligosaccharides were processed by the addition of outer chain residues in the alg3, alg5, and alg6 mutants . There was virtually no outer chain addition in the alg2 and alg4 mutants . alg4 was the only mutant that failed to secrete invertase.

Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7461 - 5
Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast; Urdea MS et al.; We have chemically synthesized and expressed in yeast a gene coding for human epidermal growth factor (urogastrone), a 53-amino-acid polypeptide that has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion . The synthetic gene, consisting of 170 base pairs, was designed with yeast-preferred codons and assembled by enzymatic ligation of synthetic fragments produced by phosphoramidite chemistry . The DNA synthesis protocol used allows for facile synthesis of oligonucleotides larger than 50 bases . Yeast cells were transformed with plasmids containing the synthetic gene under control of a yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter and were shown to synthesize a biologically active human epidermal growth factor.

J Biochem (Tokyo), 1983 Dec, 94(6), 1967 - 71
Magnesium cation induced conformational change of yeast tRNAPhe as studied by singlet-singlet energy transfer; Nagamatsu K et al.; The A76 or A73 nucleotide at the 3'end of tRNAPhe was modified with the fluorescent reagent of proflavine (PF) . The distance between the fluorophore of the 3'end and the Y base was measured by singlet-singlet energy transfer under the conditions of 10 mM and 0.01 mM Mg2+ . The distance obtained at 10 mM Mg2+ is very close to that obtained by the X-ray diffraction method, while the distance at 0.01 mM Mg2+ is significantly smaller . The difference in the distance is explained as a result of destabilization of the tertiary structure with reduction of the Mg2+ concentration . The calculated distance between A73 and A76 shows the stacked conformation of the CCA strand . Fluorescent quenching experiments showed that the degree of stabilization of 3'end A76 by stacking is lower than that of A73 . The removal of the CCA segment causes a difference not only in the thermal melting curves but also in the fluorescent wavelength of the Y base at 0.01 mM Mg2+ . The results suggest that the 3'end CCA strand has a helical structure and contributes to the stabilization of the whole structure of tRNAPhe.

Toxicol Lett, 1983 Dec, 19(3), 217 - 24
Comparative study of the effect of ochratoxin A analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells; Creppy EE et al.; Ochratoxin A (OTA), a naturally occurring mycotoxin of Aspergillus and Penicillium species, consists of a 5' chlorinated dihydromethyl isocoumarin linked to L,beta-phenylalanine by an alpha-amide bond . 8 analogues of OTA were prepared in which the phenylalanine was always substituted by another amino acid . The effects of these analogues on yeast tRNA amino acylation reaction and on growth and protein synthesis of hepatoma culture cells were compared with those of OTA . In addition, Ochratoxin B (OTB) and ochratoxin alpha (OT alpha) were examined . All the analogues of OTA had inhibitory effects in the 3 test systems, although to a lesser degree than OTA . The degree of inhibition depended on the kind of substituted amino acid, the tyrosine, valine, serine and alanine analogues being most effective, in contrast to the proline analogue . OTB and OT alpha were ineffective.

Cell, 1983 Dec, 35(3 Pt 2), 711 - 9
Yeast histone H2B containing large amino terminus deletions can function in vivo; Wallis JW et al.; The basic amino terminus of each histone is external to the nucleosome core particle . In vitro studies have shown that the amino termini are not required for nucleosome assembly . To address the significance of these results in vivo we constructed mutations in yeast histone H2B in genetic backgrounds lacking wild-type H2B protein . We found that the protein can function in vivo even with large deletions at its amino terminus . These mutations produce no obvious phenotype and there appears to be no selection against the mutant proteins in chromatin assembly . A deletion removing a large portion of the carboxyl terminus was lethal . We conclude that much of the amino terminus of histone H2B has no essential function in vivo.

Cell, 1983 Dec, 35(2 Pt 1), 521 - 9
Binding of alpha-factor pheromone to yeast a cells: chemical and genetic evidence for an alpha-factor receptor; Jenness DD et al.; The division cycle of yeast a cells is inhibited by alpha-factor . Haploid a cells were found to bind 35S-labeled alpha-factor, whereas haploid alpha cells and diploid a/alpha cells showed little binding . The association of alpha-factor with a cells was reversible upon dilution . Unlabeled alpha-factor competed for binding of 35S-alpha-factor; the concentration dependence for competition indicated 9 X 10(5) binding sites per cell with a dissociation constant (KD) of 3 X 10(-7) M . The rates of association (kon = 3 X 10(3) M-1 sec-1) and dissociation (koff = 9 X 10(-4) sec-1) were consistent with the equilibrium constant . The alpha-factor binding activity associated with five temperature-sensitive ste2 mutants was thermolabile, suggesting that the STE2 gene encodes the receptor for alpha-factor . In contrast, the binding activity of other temperature-sensitive mutants (ste4, ste5, ste7, ste11, and ste12) showed no thermolability.

Proc Natl Acad Sci U S A, 1983 Dec, 80(23), 7080 - 4
An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast; Emr SD et al.; The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae) . An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence . Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction . The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media . This extracellular invertase appears to contain no prepro-alpha-factor sequences . The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18) . Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase . Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants . Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration . Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.

J Bacteriol, 1983 Dec, 156(3), 1363 - 5
Yeast thermotolerance does not require protein synthesis; Hall BG; Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance . Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C . This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.

Genetics, 1983 Dec, 105(4), 857 - 72
A rapid chromosome-mapping method for cloned fragments of yeast DNA; Falco SC et al.; A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated . The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2 mu plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2 mu DNA was integrated (Falco et al . 1982) . After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4.

Gene, 1983 Dec, 26(2-3), 231 - 41
Direct selection for gene replacement events in yeast; Struhl K; A method that facilitates gene replacement at the HIS3 locus of Saccharomyces cerevisiae (yeast) has been developed . First, an internal region of the cloned HIS3 gene was replaced by a DNA segment containing the wild-type ribosomal protein gene, CYH2 . Second, by using standard yeast transformation methods, the wild-type HIS3 locus of a cycloheximide resistant strain (cyh2r) was replaced by this his3-CYH2 substitution . The resulting strain is sensitive to cycloheximide because CYH2 is dominant to cyh2r . Third, his3 mutations cloned into integrating or replicating vectors were introduced into this strain by selecting transformants via the vector-encoded marker . Selection for cycloheximide-resistant colonies resulted in the replacement of the his3-CYH2 allele by newly introduced his3 alleles . Thus, this scheme provides for the direct selection of gene replacement events at the HIS3 locus independently of the phenotype of the cloned his3 derivatives . In principle, it can be extended to any region of the yeast genome.

Eur J Biochem, 1983 Dec 1, 137(1-2), 179 - 83
The presence of the iron-sulfur protein (subunit V) of complex III in mitochondria of heme-deficient yeast cells lacking iron-sulfur clusters detectable by electron paramagnetic resonance; Lin CI et al.; The presence of subunit V, the iron-sulfur protein, of complex III has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae which lacks 5-aminolevulinic acid synthase and, hence, is devoid of heme . The mature form (24 K Da) of the iron-sulfur protein was observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against subunit V and either the sensitive immuno-transfer technique or immunoprecipitation from dodecylsulfate-solubilized mitochondria . In addition, a slight shoulder with a molecular mass 1.5 kDa larger than the mature form was present in mitochondria from the heme-deficient cells . Electron paramagnetic resonance spectroscopy revealed the absence of iron-sulfur signals due to clusters S-1, S-2 and S-3 of succinate dehydrogenase or to Rieske's iron-sulfur cluster of complex III in mitochondria from the heme-deficient cells . The lack of iron-sulfur centers in these cells may be a consequence of the absence of sulfite reductase in the cells without heme.

Cell, 1983 Dec, 35(2 Pt 1), 487 - 93
Yeast plasmid requires a cis-acting locus and two plasmid proteins for its stable maintenance; Kikuchi Y; Unstable ars1 (autonomously replicating sequence)-containing plasmids can be stabilized by connecting particular DNA segments of yeast 2 mu-plasmid . A cis-acting locus for the plasmid's stability (the STB locus) was found in the large unique region and separated from the replication origin of 2 mu-plasmid . Several direct repeats found in this region were essential for its function . Two proteins encoded by the plasmid (B and C) were required as trans-acting factors . The average copy number of the plasmid was not affected whether the plasmid carried the stability system or not, suggesting that the system was not directly associated with the replication function but was involved in partitioning at cell division.

Proc Natl Acad Sci U S A, 1983 Dec, 80(23), 7279 - 83
Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase; Letts VA et al.; The structural gene (CHO1) for phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was isolated by genetic complementation in Saccharomyces cerevisiae from a bank of yeast genomic DNA on a chimeric plasmid . The cloned DNA (4.0 kilobases long) was shown to represent a unique sequence in the yeast genome . The DNA sequence on an integrative plasmid was shown to recombine into the CHO1 locus, confirming its genetic identity . The cho1 yeast strain transformed with this gene on an autonomously replicating plasmid had significantly increased activity of the regulated membrane-associated enzyme phosphatidylserine synthase . Partial purification of phosphatidylserine synthase from microsomes of this transformed strain confirmed that the membrane-bound enzyme was overproduced 6- to 7-fold as compared with the wild-type strain . The strain also synthesized the product phospholipid, phosphatidylserine, at an increased rate . The transformed strain had altered proportions of a variety of other phospholipids, suggesting that their synthesis is affected by the rate of synthesis of phosphatidylserine in yeast.

Cell, 1983 Dec, 35(3 Pt 2), 753 - 62
A U4-like small nuclear RNA is dispensable in yeast; Tollervey D et al.; We have cloned a single copy gene that encodes a small nuclear RNA, designated snR3, from the yeast Saccharomyces cerevisiae . This RNA is highly conserved among fungi, and sequence and secondary structure analyses suggest that snR3 is analogous to mammalian U4 snRNA . To determine whether snR3 has an essential function in yeast, the gene (designated SNR3), was disrupted by replacing 35 nucleotides of coding sequences with 2.2 kb of yeast DNA containing the LEU2 gene . Since cells entirely lacking snR3 were expected to be inviable, the nonfunctional gene was used to replace one chromosomal copy in a diploid cell, and the diploid transformants were sporulated . Surprisingly, virtually all tetrads gave rise to four viable spores . Moreover, these haploid strains, which have been shown by DNA blot hybridization to lack an intact copy of the SNR3 gene, and which contain no detectable snR3 transcripts, are indistinguishable from their SNR3+ sister spores under a variety of growth conditions.

Cell, 1983 Dec, 35(3 Pt 2), 743 - 51
Yeast contains small nuclear RNAs encoded by single copy genes; Wise JA et al.; We have identified a group of RNA molecules in Saccharomyces cerevisiae that appears to be equivalent to the U class of small nuclear RNAs previously described in other eucaryotes, resembling them in size, metabolic stability, 5' cap structure, presence of modified bases, and nuclear localization . However, the yeast snRNAs differ from their counterparts in several potentially important ways . First, they are present in very low abundance, less than 200 copies per cell, as compared to 10(5)-10(6) for mammalian U1-U6 . Second, there appear to be more species in yeast than in any cell type previously examined . Finally, we have cloned five yeast snRNA genes, and find that each is present in a single copy per haploid genome, whereas all previously characterized snRNAs are encoded by multiple (5 to 100) gene copies . The presence of single copy genes in yeast will greatly facilitate the genetic analysis of snRNA function.

J Immunol Methods, 1983 Nov 25, 64(3), 303 - 11
A quantitative microassay for leukocyte chemotaxis, using a microscopic slide system with complement-activating yeast particles as gradient source; Coble BI et al.; A simple quantitative microassay was developed for studying polymorphonuclear leukocyte (PMNL) chemotaxis under conditions where the number of available cells is a limiting factor, e.g., pustules, neutropenia, small children and cerebrospinal fluid . PMNL suspensions are placed on glass slides to which fluorescein-labeled yeast particles have been fixed . After adherence, normal human serum is added to the slides . Owing to complement activation, a chemotactic gradient which attracts the adherent PMNL is formed around the yeast particles . The number of PMNL-associated yeast particles in the presence of normal serum is scored, and compared with cells migrating in the presence of inactivated serum or in the absence of serum . A locomotory index is calculated as the number of yeast particles associated with PMNL divided by the total number of yeast particles.

J Biol Chem, 1983 Nov 25, 258(22), 13849 - 56
Comparison of dose-response curves for alpha factor-induced cell division arrest, agglutination, and projection formation of yeast cells . Implication for the mechanism of alpha factor action; Moore SA; MAT alpha cells of the yeast Saccharomyces cerevisiae produce a polypeptide mating pheromone, alpha factor . MATa cells respond to the pheromone by undergoing several inducible responses: the arrest of cell division, the production of a cell surface agglutinin, and the formation of one or more projections on the cell surface commonly termed the "shmoo" morphology . Dose-response curves were determined for each of these inducible responses as a function of alpha factor concentration . It is shown that under conditions commonly employed in previous studies, the dose-response for cell division arrest is determined by the rate at which cells inactivate the alpha factor . In order to achieve conditions where inactivation would not be the dominant parameter, the cell division response to alpha factor was monitored at low cell densities . Under conditions of essentially no alpha factor destruction, the dose of alpha factor at which cells exhibit a half-maximal response for cell division arrest (2.5 X 10(-10) M) is nearly the same as that at which cells exhibit a half-maximal response for agglutination induction (1.0 X 10(-10) M) . On the contrary, the half-maximal response for projection formation was obtained at doses of alpha factor 2 orders of magnitude higher (1.4 X 10(-8) M) . These results are consistent with the same high affinity alpha factor receptor mediating both cell division arrest and agglutination induction . A different system of lower affinity must mediate projection formation . Alternatively, if the same system and receptor are used, then a much higher occupancy is required for the induction of projections compared to division arrest and agglutination induction.

J Biol Chem, 1983 Nov 25, 258(22), 14025 - 33
Identification of multiple transcriptional initiation sites on the yeast mitochondrial genome by in vitro capping with guanylyltransferase; Christianson T et al.; We have studied transcriptional initiation in the mitochondria of the yeast Saccharomyces cerevisiae by analyzing mitochondrial transcripts from grande and petite yeast after labeling in vitro with vaccinia virus guanylyltransferase and {alpha-32P}GTP . This procedure labels triphosphate-terminated RNA which arises from transcriptional initiation . Exploiting the extremely low GC content (18%) of yeast mitochondrial DNA, we digested the in vitro capped transcripts with the G-specific ribonuclease T1; this resulted in 27 oligonucleotides varying in size from 2 to 51 nucleotides . RNA from 14 overlapping petites was analyzed and 20 transcripts were localized by deletion mapping . Nineteen oligonucleotides were sequences and 13 were identified and precisely localized by comparison with known DNA sequences . In all cases, transcription is initiated at a consensus nonanucleotide sequence which can be considered part of the yeast mitochondrial promoter . We identified initiation sites for the 21 S and 14 S rRNAs; the phenylalanine, f-methionine, and glutamic tRNAs; two sites for the OLI-1 gene; and three for the ori (rep) regions . Most promoters appear to give rise to very long multigene primary transcripts . Examples are multigene transcripts for the glutamic tRNA and COB genes and for the OLI-1, serine tRNA, and Var genes . Since the consensus nonanucleotide sequences at the ori regions are similar to those at other transcriptional initiation sites, it is likely that the same RNA polymerase primes DNA replication and gene transcription.

J Biol Chem, 1983 Nov 25, 258(22), 13418 - 21
Nuclear genes for mitochondrial proteins . Identification and isolation of a structural gene for subunit V of yeast cytochrome c oxidase; Cumsky MG et al.; The gene for yeast cytochrome c oxidase subunit V, COX5, has been isolated from a Saccharomyces cerevisiae DNA library by complementation of a cytochrome c oxidase subunit V mutant, JM28 . One complementing plasmid, YEp13-511, with a DNA insert of 4.8 kilobase pairs, has been characterized in detail . This plasmid restores respiratory competency in JM28, results in increased cytochrome c oxidase activity and a new form of subunit V in JM28 mitochondria, and is capable of selecting mRNA for subunit V . These results indicate that YEp13-511 carries the COX5 gene and that the subunit V encoded by this plasmid gene is capable of entering the mitochondrion and assembling into a functional holocytochrome c oxidase.

Nucleic Acids Res, 1983 Nov 25, 11(22), 7759 - 68
Yeast ribosomal protein S33 is encoded by an unsplit gene; Leer RJ et al.; The structure of the gene coding for ribosomal protein S33, - a protein which escapes the coordinate control of ribosomal protein synthesis in rna 2 mutant cells -, was determined by sequence analysis . The gene comprises an uninterrupted coding region of 204 nucleotides encoding a protein of 8.9 kD . Like for other yeast ribosomal protein genes that have been sequenced so far, a relatively strong codon bias was observed . By S1 nuclease mapping the 5' end of the S33 mRNA was shown to be located at 11 to 15 nucleotides upstream from the initiation codon.

Science, 1983 Nov 18, 222(4625), 778 - 82
Yeast RNA polymerase II genes: isolation with antibody probes; Young RA et al.; Genes encoding yeast RNA polymerase II subunits were cloned . Efficient isolation of these genes was accomplished by probing a phage lambda gt11 recombinant DNA expression library with polyvalent antibodies directed against purified yeast RNA polymerase II . The identity of genes that specify the largest RNA polymerase II subunits, the 220,000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay . Both of these genes exist in single copy in the yeast Saccharomyces cerevisiae.

Biochim Biophys Acta, 1983 Nov 17, 741(2), 269 - 71
Extraction of histones H2A, H3 and H4 from yeast nuclei . Measurement of the extent of yeast histone acetylation following one-dimensional gel electrophoresis; Nelson DA et al.; Yeast histones H2A, H3 and H4 were specifically extracted from purified nuclei using a 2% NaCl/75% ethanol solution . The extraction resulted in the complete removal of H2A, H3 and H4 from the nuclear pellet, as monitored by SDS-polyacrylamide gel electrophoresis of the protein . The relative absence of nonhistone proteins from this histone subset simplifies the determination of the extent of histone modification in yeast . Levels of H4 acetylation were measured directly on Coomassie blue-stained Triton acid-urea gels and the levels verified by gel fluorography of the {3H}acetate-labeled histone.

Eur J Biochem, 1983 Nov 15, 136(3), 583 - 7
Reversibility characteristics of glucose-induced trehalase activation associated with the breaking of dormancy in yeast ascospores; Thevelein JM et al.; The breaking of dormancy in yeast ascospores by addition of glucose is associated with a sudden tenfold increase in the activity of trehalase . The rapid activation of trehalase is followed by a slower inactivation process which is greatly retarded in the presence of nitrogen sources and cycloheximide . When glucose is washed away from the spores after some time and the spores resuspended in glucose-free medium, the trehalase activity decreases sharply . Subsequent addition of new glucose partially reactivates the enzyme . The extent of reactivation decreases further with each subsequent activation/inactivation step . Changing the duration of the inactivation periods has no effect on this diminution of the reversibility . However, prolonging the duration of the activation step speeds up the loss of reversibility . On the other hand, addition of a nitrogen source or cycloheximide completely prevents the loss of reversibility . The results of the reversibility studies are in agreement with the phosphorylation mechanism which has been proposed for the underlying molecular process of trehalase activation . Apparently, they are also in agreement with proteolytic breakdown being responsible for the inactivation of trehalase after its initial activation . However, the effect of cycloheximide and nitrogen sources, at least in ascospores, does not appear to be due to inhibition or repression of protease synthesis, respectively, since the addition in the presence of glucose of a nitrogen source after trehalase inactivation immediately reactivates the enzyme completely.

Biochem Biophys Res Commun, 1983 Nov 15, 116(3), 822 - 9
Yeast pheromone alpha-factor is synthesized as a high molecular weight precursor; Emter O et al.; The sex pheromone alpha-factor of Saccharomyces cerevisiae, a tridecapeptide of approx . 1,700 molecular weight, was found to be synthesized in vivo as a high molecular weight precursor of Mr = 28,000 . Inhibition of N-linked glycosylation by tunicamycin leads to three precursor species of lower molecular weight indicating three carbohydrate residues linked to the alpha-factor precursor molecule . A molecular weight of 18,000 was determined for the unglycosylated molecule.

J Mol Biol, 1983 Nov 15, 170(4), 883 - 904
Structure and function of the yeast URA3 gene . Differentially regulated expression of hybrid beta-galactosidase from overlapping coding sequences in yeast; Rose M et al.; Expression of the URA3 gene of Saccharomyces cerevisiae was studied by analysis of URA3-lacZ gene fusions constructed in vitro . Synthesis of hybrid beta-galactosidase by fusions in frame with the coding sequence for orotidine-5'-phosphate decarboxylase (OMPdecarboxylase) was found to be normally regulated even when only 11 nucleotides of URA3 coding sequence remained, indicating that all transcription initiation and regulatory sites are present at the beginning of the URA3 gene . An upstream initiator codon that begins a short overlapping coding sequence in another reading frame was also found to be active in producing hybrid beta-galactosidase . However this beta-galactosidase synthesis showed little or no regulation . Nuclease protection experiments revealed numerous species of URA3 mRNA . The regulation of these is consistent with the idea that the URA3 protein and the overlapping peptide are translated from differentially regulated mRNAs of different lengths.

FEBS Lett, 1983 Nov 14, 163(2), 335 - 8
Activation of yeast mannan synthetase by alpha factor pheromone; Ruiz T et al.; The Saccharomyces cerevisiae mating pheromone alpha factor induces the activation of yeast mannan synthetase both in vivo and in vitro . In vivo the activating effect of the pheromone on the synthetase is specific for cells of the alpha mating type, while in vitro alpha factor is able to exert its action on the synthetase of either cell type (MAT a, MAT alpha and MAT a/MAT alpha).

Antonie Van Leeuwenhoek, 1983 Nov, 49(4-5), 369 - 85
Significance of yeast peroxisomes in the metabolism of choline and ethanolamine; Zwart KB et al.; The yeasts Candida utilis and Hansenula polymorpha were able to grow in media containing choline or ethanolamine as the sole nitrogen source . During growth in the presence of these substrates, large peroxisomes developed in the cells, and extracts of choline-grown C . utilis cells contained increased levels of amine oxidase and catalase . Incubation of whole cells with choline in the presence of the amine oxidase inhibitor aminoacetonitrile led to excretion of dimethylamine and methylamine . Cytochemical experiments in which spheroplasts prepared from choline-grown cells were incubated with CeCl3 and choline, trimethylamine, dimethylamine or methylamine revealed positively stained peroxisomes, whereas in the presence of 1 mM aminoacetonitrile staining was not observed . This indicated that choline was degraded via methylated amines and that peroxisomes played a role in its metabolism . A similar involvement of peroxisomes in choline degradation was observed in H . polymorpha . Cell-free extracts of ethanolamine-grown C . utilis and H . polymorpha also contained increased levels of amine oxidase and catalase . Ethanolamine was oxidized by cell-free extracts of both organisms after growth in the presence of ethanolamine or choline . Incubation of spheroplasts of ethanolamine- or choline-grown C . utilis with CeCl3 and ethanolamine resulted in positively stained peroxisomes . In this organism peroxisomes were therefore also involved in ethanolamine degradation.

Mol Biol (Mosk), 1983 Nov-Dec, 17(6), 1117 - 25
{Two-dimensional polyacrylamide gel electrophoresis in the study of yeast mitochondrial transfer RNA}; Martin RP et al.; An improved system of tRNA fractionation by two-dimensional polyacrylamide electrophoresis with high resolution power and also methods of visualization and identification of fractioned tRNAs are given . The system was used for studying mitochondrial tRNAs and their precursors.

Chem Biol Interact, 1983 Nov, 47(2), 239 - 47
Effects of ochratoxin A metabolites on yeast phenylalanyl-tRNA synthetase and on the growth and in vivo protein synthesis of hepatoma cells; Creppy EE et al.; The ochratoxin A (OTA) metabolite (4R)-4-hydroxyochratoxin A {4R)-OTA) inhibits the aminoacylation of phenylalanine tRNA catalyzed by phenylalanyl-tRNA synthetase (PheRS) with a Ki-value of 0.9 mM as compared to 1.3 mM for OTA . It also inhibits protein synthesis and cell growth in the same manner as OTA . Ochratoxin alpha (OT alpha) does not affect either protein synthesis or cell growth.

Biochimie, 1983 Nov-Dec, 65(11-12), 661 - 72
Primary structure of three tRNAs from brewer's yeast: tRNAPro2, tRNAHis1 and tRNAHis2; Keith G et al.; The primary structures of three brewer's yeast tRNAs: tRNAPro2 and tRNAHis1 and 2 have been determined (Formula:see text) The U* in the anticodon U*-G-G of tRNAPro2 is probably a derivative of U; tRNAPro2 has 80 per cent homology with mammalian tRNAsPro . tRNAHis1 and tRNAHis2 differ by only 5 nucleotides; they have identical anticodons and may therefore recognize both codons for histidine; they have an additional nucleotide at the 5' end . As in all other sequenced tRNAsHis this nucleotide is not paired with the fourth nucleotide from acceptor adenosine . All three sequenced tRNAs have a low degree of homology with their counterparts from yeast mitochondria.

Mol Biol (Mosk), 1983 Nov-Dec, 17(6), 1126 - 46
{Yeast mitochondrial transfer RNA . Structure, coding properties and genome organization}; Martin RP et al.; The up-to-date data on mitochondrial tRNAs of yeast, their structures and peculiarities of these structures, anomalies of the mitochondrial genetic code and anticodons of tRNAs, the structure and number of tRNA genes are reviewed in the present paper . New information concerning 17 types of yeast mitochondrial tRNAs, deciphered by the authors of the paper are given; among them 8 types are first published . The likeness and differences of yeast mitochondrial tRNAs from their cytoplasmic counterparts are discussed by comparison with other organisms.

J Inorg Biochem, 1983 Nov, 19(3), 255 - 67
Studies of activating and nonactivating metal ion binding to yeast enolase; Brewer JM et al.; Measurements of binding of certain divalent cations to yeast apoenolase were made using a pH-meter, chromatography, a divalent cation electrode, and ultrafiltration . The binding of the activating metal ions Mg2+ and Co2+ and the nonactivator Ca2+ were studied as functions of the presence or absence of substrate/product, phosphate, and fluoride or level of Tb3+ . The data suggest phosphate and fluoride increase Mg2+ binding but not Ca2+ binding . Substrate/product appears to increase Ca2+ binding as well as that of Mg2+ and Co2+ . In the presence of substrate, Co2+ binding was 5-6 mol/mol dimer . In the absence of substrate/product, Tb3+ reduced Co2+ binding from 4 mol/mol to 2 . These data are interpreted in terms of binding to "conformational," "catalytic" (substrate/product dependent), and "inhibitory" sites . Measurements of Tb3+ fluorescence quenching by Co2+ suggested that the distance between "conformational" sites on the two subunits was large, while the distance between "conformational" and "inhibitory" sites was ca . 17 +/- 4 A . Potentiometric titrations of apoenzyme with Ca2+ and Mg2+ showed that the metal ions produced the same proton release in the presence or absence of substrate/product . If phosphate and fluoride were present, then more protons were released if Ca2+ was the titrant rather than Mg2+, suggesting a difference in ionization state in the complex with the activating metal . Electron paramagnetic resonance studies of Co2+ binding to the various sites in the enzyme are presented . The Co2+ bound to all three sites appears to be high spin, consistent with a preponderance of oxyligands in an octahedral environment . Substrate, citrate, and a strongly binding substrate analogue strongly enhance the hyperfine structure of conformational Co2+ . This is interpreted as the result of a change in interaction of an axial ligand to conformational Co2+ produced by carbon-3 of substrate or analogue.

Eur J Cancer Clin Oncol, 1983 Nov, 19(11), 1575 - 83
Induction of petite mutants in yeast by non-intercalative DNA-binding antitumour agents; Ferguson LR et al.; A series of 17 bis-charged non-intercalative DNA-binding antitumour agents and 7 related inactive compounds have been tested for the induction of respiratory deficient (petite) mutants in Saccharomyces cerevisiae D5 . Many compounds were strong inducers of petite mutants at concentrations which were not toxic to the growth of the yeast cells . Mutagenicity is only weakly correlated with in vitro inhibition of L1210 cell growth; however, mutagenicity, yeast toxicity and in vitro and in vivo antitumour activity are all correlated with selective binding to polydeoxy(adenylic-thymidylic) acid rather than polydeoxy(guanylic-cytidylic) acid, as measured by an ethidium competition assay . It is concluded that A-T-rich DNA may be a target for all the biological effects measured in this study . Furthermore, the possibility that the target for antitumour action may be tumour cell mitochondrial DNA is supported by these results.

Proc Natl Acad Sci U S A, 1983 Nov, 80(21), 6465 - 9
Isolation of yeast DNA replication mutants in permeabilized cells; Kuo C et al.; A random population of temperature-sensitive mutants was screened by assaying for defects in DNA synthesis in a permeabilized yeast DNA replication system . Twenty mutants defective in in vitro DNA synthesis have been isolated . In this paper we describe eight of these mutants . Seven of them fall into three complementation groups--cdc2, cdc8, and cdc16--involved in the control of the cell-division cycle . Because synthesis in vitro represents propagation of replication forks active in vivo at the time of permeabilization, our finding that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23 degrees C but not at 37 degrees C supports the conclusion that these two mutations directly affect DNA synthesis at replication forks . Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16 . Finally, the usefulness of our screening procedure is demonstrated by the isolation of replication mutants in previously undescribed complementation groups . One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.

Cell Biol Int Rep, 1983 Nov, 7(11), 947 - 54
cAMP levels and in situ measurement of adenylate cyclase and cAMP phosphodiesterase activities during yeast-to-hyphae transition in the dimorphic fungus Mucor rouxii; Cantore ML et al.; Intracellular levels of cAMP and specific activities of adenylate cyclase and cAMP phosphodiesterase were measured during yeast-to-hyphae transition in the dimorphic fungus M . rouxii . Enzymatic activities were measured in permeabilized cells under conditions preventing protein dephosphorylation and proteolysis . A two-fold decrease in intracellular cAMP levels occurred shortly after exposure of the yeast to air quite before morphological changes became evident . Morphogenesis to hyphae after exposure to air was inhibited by the addition of 10 mM dibutyryl cAMP to the culture medium, and the yeast morphology was maintained for at least 24 hours . The decrease in cAMP levels that occurs shortly after exposure of yeast culture to air was mainly accounted for by variations in the state of activation of cAMP phosphodiesterase while the specific activity of adenylate cyclase did not vary significantly during yeast-to-hyphae transition.

Arch Biochem Biophys, 1983 Nov, 227(1), 106 - 10
Presence and stoichiometry of two forms of subunit 6 of the mitochondrial ATPase complex of yeast; Todd RD et al.; One of the mitochondrically coded components of the yeast mitochondrial ATPase complex (subunit 6) can be resolved into two components on certain polyacrylamide gels in the presence of sodium dodecyl sulfate . Purification of the ATPase complex from commercially processed yeast as well as immunoprecipitation of the holo-enzyme from cells labeled in vivo with 14C-labeled amino acids demonstrate that both forms of subunit 6 are physically associated with the assembled enzyme and present in two copies each per complex . One-dimensional papain-generated peptide maps of the two components are identical except for the mobility of a single fragment . It is concluded that the two components of subunit 6 are different forms of a single protein and are present on an average of two copies each per complex.

Cell, 1983 Nov, 35(1), 117 - 25
tRNA gene transcription in yeast: effects of specified base substitutions in the intragenic promoter; Newman AJ et al.; The yeast SUP53 gene encodes a leucine-inserting amber suppressor tRNA . We have introduced specific base substitutions into both 5' and 3' elements of the intragenic promoter of this tRNA gene . The influence of these sequence changes on promoter function has been investigated by transcription of the mutant genes in a homologous cell-free system . Our results do not support the idea that tertiary intragenic structure is important in tRNA gene transcription . For one of the SUP53 mutants we are able to suggest a plausible molecular basis for defective transcription: a single base substitution in the 3' element of the intragenic promoter prevents the interaction of this element with a putative transcription factor.

Cell, 1983 Nov, 35(1), 109 - 15
Role of RNA structure in splicing: excision of the intervening sequence in yeast tRNA3leu is dependent on the formation of a D stem; Baldi MI et al.; A substitution mutant of the yeast tRNA3leu gene results in the sequence change of GCC to AAA at positions 10, 11, and 12 in the noncoding strand . The ability to form a D stem is lost . Transcription in the heterologous Xenopus germinal vesicle system is not drastically affected, but splicing of the tRNA precursor does not occur . To determine whether this effect is caused by the change in sequence or the change in conformation we constructed two new mutants . In one, mutation results in the sequence change of GGC to TTT at positions 24, 25, and 26 . The ability to form a D stem is lost; transcription is unaffected, but excision of the intron does not occur . The other, a double mutant, is characterized by both substitutions described above, and the ability to form a D stem is retained . The precursor derived from the double mutant is accurately spliced in X . laevis germinal vesicle extracts, therefore excision of the intervening sequence appears to depend on the formation of a D stem.

J Pharmacol Methods, 1983 Nov, 10(3), 223 - 9
Determination of analgesic drug efficacies by modification of the Randall and Selitto rat yeast paw test; Chipkin RE et al.; This report describes a modified Randall and Selitto (1957) rat yeast paw test that can evaluate differences in efficacy of different analgesics . The modifications consist of a decrease in the rate of acceleration of the noxious stimulus (mechanical pressure) on the inflamed paw from 20 to 12.5 mmHg/sec and an extension of the cut-off time from 15 to 60 sec . All the narcoticlike drugs tested (morphine, codeine, and pentazocine) increased the response latencies of the inflamed paws to the cut-off time . The nonsteroidal antiinflammatory-like drugs tested (acetylsalicylic acid, acetaminophen, indomethacin, phenylbutazone, and proquazone) showed plateaus in their analgesic effects (i.e., increasing the dose failed to produce significantly greater increases in the response latencies compared to the next lower dose) . Zomepirac (80-240 mg/kg p.o.) did not show this plateau effect, but was unable to increase response latencies to greater than 30 sec because of the toxicity of higher doses (320 mg/kg p.o.) . Flunixin NMG (the meglumine salt of flunixin), a nonnarcotic analgesic, did not display a plateau effect and increased response latencies to maximum values . The methodology was therefore able to discriminate analgesics active against mild to severe clinical pain (narcoticlike) from those only useful against mild to moderate pain (nonnarcotic-like).

Arch Biochem Biophys, 1983 Nov, 227(1), 302 - 9
Yeast inorganic pyrophosphatase substrate recognition; Knight WB et al.; Monodentate Co(NH3)5PPi was determined not to be a substrate for yeast inorganic pyrophosphatase while P1,P2-bidentate Co(NH3)4PPi was turned over by the enzyme at a rate of 7.5 min-1 . A kinetic analysis of the substrate activities of the P1,P2-bidentate complexes, Co(en)2PPi, Cr(NH3)4PPi, Cr(H2O)(NH3)3PPi, and Cr(H2O)2(NH3)2PPi, and Cr(H2O)4PPi was carried out in order to access the potential role of the metal-water ligands in productive binding . While substitution of the H2O ligands with NH3 ligands had a minimal affect on the Km for Mg2+, the binding affinity of the complexes decreased with an increasing NH3/H2O ligand ratio as did the turnover number of the corresponding central complexes . The Co(en)2PPi complex was hydrolyzed at a rate approximately 0.6% of that for the Co(NH3)4PPi complex . The substrate activities of beta, gamma-bidentate Co(NH3)4PPPi and alpha, beta, gamma-tridentate Co(NH3)3PPP with pyrophosphatase were also tested . While both complexes were shown to bind tightly to the Mg2+-activated enzyme neither was hydrolyzed . On the other hand, in the presence of the Zn2+-activated enzyme the tridentate complex was turned over at a rate of 0.17 min-1 while the bidentate complex remained inert to hydrolysis.

FEBS Lett, 1983 Oct 31, 163(1), 33 - 6
Regulation of yeast phosphorylase by phosphorylase kinase and cAMP-dependent protein kinase; Wingender-Drissen R et al.; Yeast phosphorylase is phosphorylated and activated by a cyclic AMP-independent protein kinase (called phosphorylase kinase) and a cyclic AMP-dependent protein kinase . Only in the presence of both kinases is phosphorylase fully activated and phosphorylated . No evidence was found for the presence of two phosphorylation sites as an identical phosphopeptide pattern of phosphorylase is obtained after phosphorylation by either one or both kinases . The kinases probably phosphorylate identical sites but recognize different subunits of phosphorylase . Phosphorylase kinase phosphorylates the high-Mr subunit while cAMP-dependent protein kinase phosphorylates the low-Mr subunit.

FEBS Lett, 1983 Oct 31, 163(1), 28 - 32
Yeast cyclic AMP-dependent protein kinase; Wingender-Drissen R; We have purified cyclic AMP-dependent protein kinase from the yeast Saccharomyces cerevisiae . The purified enzyme was inactive in the absence of cyclic AMP and displayed two protein bands on SDS gel electrophoresis . One was identified as the cAMP-binding protein by chromatography on cAMP-agarose . Mr of the latter was 50 000 while the catalytic subunit had an Mr of 59 000 . The enzyme accepted yeast phosphorylase, glycogen synthase and fructose 1,6-bisphosphatase as substrates . No inhibition by the mammalian protein kinase inhibitor was observed.

Biochim Biophys Acta, 1983 Oct 28, 748(2), 184 - 93
pH Dependence of the inhibition of yeast glyoxalase I by porphyrins; Douglas KT et al.; A number of porphyrin derivatives have been found to inhibit yeast glyoxalase I (EC 4.4.1.5) at 25 degrees C, including haemin, protoporphyrin IX, coproporphyrin III, haematoporphyrin, deuteroporphyrin as well as meso-(tetrasubstituted) porphines . Bilirubin and chlorophyllin were also inhibitory, but not cobalamin, adipic, pimelic or suberic acids . Whilst the Ki value for linear competitive inhibition by meso-tetra(4-methylpyridyl)porphine was pH-dependent, analogous Ki values for meso-tetra(4-carboxyphenyl)- and meso-tetra(4-sulphonatophenyl)porphines followed the Henderson-Hasselbalch equation with pKapp values of 7.10 and 6.50, respectively . Protoporphyrin showed similar behaviour (pKapp 7.06) with a deviation at lower pH . The haemin pH profile for Ki showed a maximum at approx . pH 6.5 . The redox reaction between haemin and glutathione did not interfere in the inhibition studies . The Ki value for S-(p-bromobenzyl)glutathione was pH-independent . A detailed analysis of porphyrin binding modes was undertaken.

Biochim Biophys Acta, 1983 Oct 28, 748(2), 263 - 70
Steady-state kinetics of yeast cytochrome c peroxidase catalyzed oxidation of inorganic reductants by hydrogen peroxide; Yandell JK et al.; Rates of yeast cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) catalyzed oxidation of bis(tripyridine)cobalt(II) ion, penta(amine)pyridineruthenium(II) ion and ferrocyanide ion by hydrogen peroxide have been found to obey the empirical equation: (formula; see text) in the pH range 5 to 8, and at saturating H2O2 concentrations . {( S} and {CcP} are the concentrations of the reductant and the enzyme, respectively.) Values of k2 were found to be independent of the reductant . The term k0{S} is only significant with the cobalt and ruthenium complexes at high pH . The mechanism proposed to account for this rate equation differs significantly from previous mechanistic proposals . In particular, the rate data require the assignment of the rate-limiting step at high substrate concentrations to a slow electron-transfer within the enzyme, and not, as previously suggested, to saturation of substrate binding to the enzyme . Also, the term k0{S} implies that the reactive substrates, including the natural substrate (yeast cytochrome c), react with the hydrogen peroxide-heme complex and not with the radical species formed by reaction with hydrogen peroxide in the absence of reductants.

Biochemistry, 1983 Oct 25, 22(22), 5090 - 6
Activation of yeast pyruvate carboxylase: interactions between acyl coenzyme A compounds, aspartate, and substrates of the reaction; Myers DE et al.; Chicken liver pyruvate carboxylase has an absolute requirement for short-chain acyl coenzyme A (CoA), whereas the same enzyme from yeast has less stringent requirements . The yeast enzyme has now been studied in an effort to elucidate the mechanism by which acyl-CoA stimulates pyruvate carboxylase activity . Yeast pyruvate carboxylase has an apparent basal level of activity above which CoA and acyl-CoAs of 2-20 carbons activate; the concentration of acyl-CoA required for half-maximum activation (K0.5) decreases as the chain length of the acyl moiety increases to 16 carbons . Activation of yeast pyruvate carboxylase by acyl-CoA is brought about in part by increasing the affinity of pyruvate carboxylase for two substrates, bicarbonate and pyruvate . The affinity of pyruvate carboxylase for bicarbonate is also increased by potassium ions . The observation of only low levels of activity in the absence of acyl-CoA or potassium ion leads to the conclusion that the basal activity so frequently referred to is probably due to the presence of activating monovalent cations . Pyruvate carboxylase from yeast probably has an absolute requirement for monovalent cations or acyl-CoA with a combination of the two being required for optimum conditions for maximal activity . Stimulation by acyl-CoA and inhibition by aspartate are mutually antagonistic with each affecting the activation or inhibition constant and the degree of cooperativity brought about by the other . The enzyme from liver is unaffected by aspartate.

J Biol Chem, 1983 Oct 25, 258(20), 12308 - 14
Topographical orientation of complex III in the yeast mitochondrial membrane; Sidhu A et al.; The orientation of the different subunits of complex III in the yeast inner mitochondrial membrane has been investigated by several different approaches . Immunoinhibition studies of cytochrome c reductase activity in intact mitoplasts and submitochondrial particles using IgG obtained from specific antisera against complex III, the iron-sulfur protein, core protein I, and core protein II suggested a transmembranous orientation of the complex with the antigenic sites of the iron-sulfur protein exposed on the cytoplasmic surface of the membrane . A lack of immunoinhibition was observed with the IgG against either core protein suggesting that these proteins may not be involved in catalysis . Digestion of mitoplasts with chymotrypsin indicated that the protein mass of cytochromes b and c1 protrudes from the cytoplasmic surface of the membrane; however, the hemes of cytochrome b appear to be buried within the membrane while the heme of cytochrome c1 is partially exposed on the chymotrypsin-sensitive portion of the polypeptide . By contrast, the iron-sulfur protein does not protrude from the membrane as it is completely resistant to chymotrypsin digestion . Labeling with the hydrophilic membrane-impermeant probe diazobenzenesulfonate suggests that core protein II is exposed on both sides of the membrane but protrudes into the matrix; while core protein I is within the membrane . Immunoprecipitation studies of sodium dodecyl sulfate and Triton X-100-solubilized mitochondria with subunit-specific antisera suggest that cytochromes b and c1 and core protein I are tightly associated in complex III . By contrast, the iron-sulfur protein and core protein II are loosely associated with the other subunits of the complex such that they are dissociated by low concentrations of detergent.

Biochem Biophys Res Commun, 1983 Oct 14, 116(1), 162 - 6
Two species of cytochrome P-450 involved in ergosterol biosynthesis of yeast; Hata S et al.; Discrimination of cytochrome P-450 involved in delta 22-desaturation of ergosta-5,7-dien-3 beta-o1 (P-450(22)-DS) from that involved in lanosterol 14 alpha-demethylation (P-450(14)-DM) in ergosterol biosynthesis was investigated with microsomes of several strains of Saccharomyces cerevisiae . In mutant N22 which is partially defective in the delta 22-desaturation, the 14 alpha-demethylation was not blocked . In contrast, mutant SG1 which is known to lack the 14 alpha-demethylation showed a significant activity of the delta 22-desaturation . The delta 22-desaturation activity was markedly increased upon aerobic adaptation of yeast cells but the 14 alpha-demethylation was not affected . Buthiobate, a specific inhibitor of P-450(14)-DM, and rabbit antibodies against P-450(14)-DM did not inhibit the delta 22-desaturation activity at all . It is evident from the obtained observations that these phenomena are not explainable in terms of NADPH-cytochrome P-450 reductase . These results indicate that P-450(22)-DS is different from P-450(14)-DM in molecular species.

Biochim Biophys Acta, 1983 Oct 13, 741(1), 128 - 35
Synthesis and maturation of the yeast vacuolar enzymes carboxypeptidase Y and aminopeptidase I; Distel B et al.; We have studied the two vacuolar enzymes carboxypeptidase Y and aminopeptidase I from Saccharomyces cerevisiae with respect to biosynthesis, maturation and transfer from their site of synthesis into the organelle . The levels of translatable mRNA for these two proteins increase more than 10-fold at the end of the exponential growth period on glucose as carbon source and decrease again in the stationary phase . Two precursors of carboxypeptidase Y have been identified by in vivo pulse-labelling with {35S}methionine . These differ in their amount of carbohydrate as shown by inhibition of N-linked glycosylation with tunicamycin . The first is a protein with an apparent molecular weight of 67 kDa, which can be converted into the mature 60-kDa protein via an intermediate of 69 kDa . In the pep4-3 mutant, which is disturbed in the maturation of several vacuolar enzymes (Hemmings, B.A., Zubenko, G.S., Hasilik, A . and Jones, E.W . (1981) Proc . Natl . Acad . Sci . U.S.A . 78, 435-439), the 69-kDa precursor accumulates in the vacuole . This suggests that the final proteolytic cleavage of carboxypeptidase Y can occur in the vacuole.

Nucleic Acids Res, 1983 Oct 11, 11(19), 6913 - 21
Long-range conformational transition in yeast tRNAPhe, induced by the Y-base removal and detected by chloroacetaldehyde modification; Krzyzosiak WJ et al.; Chemical modification was used to study the conformational changes occurring in yeast tRNAPhe after the Y-base excision . The chemical probe was the adenine- and cytosine-specific reagent chloroacetaldehyde . Comparison of the modification patterns in tRNAPhe and tRNAPhe-Y shows that seven bases, adenines 35, 36 and 38 in the anticodon loop and adenines 73, 76 and cytosines 74, 75 in the 3'-terminus were modified in both tRNAs with a quantitative difference in the modification level of the anticodon loop bases . The most interesting, however, is the qualitative difference consisting in modification of cytosine-60 in the T psi C loop of tRNAPhe-Y . Some aspects of the mechanism of this long-distance conformational transition are briefly discussed.

J Biol Chem, 1983 Oct 10, 258(19), 11968 - 73
Analysis of yeast RNA polymerases with subunit-specific antibodies; Breant B et al.; Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection . The majority of enzyme A or B subunits were specifically recognized by their respective antiserum . A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane . Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C . Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding.

Nature, 1983 Oct 6-12, 305(5934), 543 - 5
Induction of yeast mating pheromone a-factor by alpha cells; Strazdis JR et al.; Saccharomyces cerevisiae cells of a and alpha mating types constitutively secrete cell-specific peptide mating pheromones . a-Factor is secreted by a cells and acts on alpha cells, while alpha-factor is secreted by alpha cells and acts on a cells . Confirming preliminary studies, we demonstrate here that cultures of a cells contain higher than constitutive levels of a-factor activity when grown with alpha cells or alpha-factor . This induction of a-factor may result from increased synthesis or increased secretion of a-factor, as opposed to modification or stabilization of preexisting a-factor, as part of the a cell response to alpha-factor, as an a ste2 mutant (which cannot respond to alpha-factor) is not induced by alpha-factor . In mixed cultures inoculated with equal numbers of a cells and alpha cells, a cells predominate by stationary phase . Thus, a series of sequential interactions between a and alpha cells may be involved in establishing optimal hormone concentrations and cell ratios for conjugation.

Biochim Biophys Acta, 1983 Oct 4, 760(1), 143 - 8
Isolation and characterization of polyphosphates from the yeast cell surface; Tijssen JP et al.; When cells of Saccharomyces fragilis are subjected to osmotic shock, they release a limited amount of inorganic polyphosphate into the medium, which represents about 10% of the total cellular content . The osmotic shock procedure causes no substantial membrane damage, as judged from the unimpaired cell viability, limited K+ leakage and low percentage of stained cells . It is therefore suggested that this polyphosphate fraction is localized outside the plasma membrane . The released polyphosphate fraction differs from the remaining cellular polyphosphates in two respects: the mean chain length of the shock-sensitive fraction is significantly higher than that of the total cellular polyphosphates and its metabolic turnover rate, subsequent to pulsing with {32P}orthophosphate is much lower compared to the rest of the cellular polyphosphate . Incubation of intact cells with the anion exchange resin Dowex AG 1-X4 results in the release of high molecular weight polyphosphates . These results suggest that the osmotic shock-sensitive polyphosphate fraction has specific characteristics in both its cellular localization and metabolism.

Eur J Biochem, 1983 Oct 3, 135(3), 577 - 81
Study of the Hansenula anomala yeast flavocytochrome-b2-cytochrome-c complex 2 . Localization of the main association area; Thomas MA et al.; The reversible association of the Zn2+-substituted Hansenula anomala cytochrome c dimer (Thomas et al., preceding paper in this issue) to flavocytochrome b2 in oxidized or lactate-reduced state has been investigated by fluorimetry . The same method has been used for the determination of Zn-cytochrome c complexing to defined proteolytic fragments of flavocytochrome b2, either heme-b2-containing monomers or a flavin-linked tetramer . All these fragments but the isolated cytochrome b2 core showed binding stoichiometries, Kd values and ionic strength dependences quite similar to those found for native flavocytochrome b2 . These data allowed localization of the single high-affinity binding site of cytochrome c on a particular globule in the dehydrogenase domain of the flavocytochrome b2 protomers . Quenching of the Zn-porphyrin c fluorescence in the various complexes occurred with only minor changes of the fluorescence lifetime and did not show any direct relationship to the presence or the redox state of the heme b2 group.

Eur J Biochem, 1983 Oct 3, 135(3), 569 - 76
Study of the Hansenula anomala yeast flavocytochrome-b2--cytochrome-c complex 1 . Characterization of fluorescent Zn(II)-substituted cytochrome c; Thomas MA et al.; Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H . anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized . H . anomala Zn-cytochrome c appears to be in the form of a stable though non-covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods . By contrast, metal-free porphyrin-cytochrome c, the precursor of Zn-cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration . Significant conformational perturbations of H . anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety . Titrations at low ionic strength with native, tetrameric H . anomala flavocytochrome b2 in the lactate-reduced state showed a simple binding equilibrium (Kd = 0.1 microM at I = 0.03 M, 10 degrees C) with a stoichiometry of one Zn-cytochrome c dimer per protomer of flavocytochrome b2 . Quenching of the Zn-porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.

Eur J Biochem, 1983 Oct 3, 135(3), 457 - 63
Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast . Characterization of precursor forms of the 44-kDa, 40-kDa and 17-kDa subunits and identification of individual messenger RNAs for these and other imported subunits of the complex; Van Loon AP et al.; The mitochondrial ubiquinol--cytochrome c reductase complex (complex III or cytochrome bc1 complex) is thought to consist of eight subunits, seven of which are specified by nuclear genes and synthesized in the cytoplasm . We have studied the synthesis of five of the nuclear-encoded subunits both in vivo and in vitro and show that of these the 44-kDa, 40-kDa and 17-kDa subunits are synthesized with cleavable extensions, while the 14-kDa and 11-kDa proteins are synthesized without detectable extra sequences . The sizes of the pre-sequences, as determined by the relative mobility of the precursor proteins in sodium dodecyl sulphate/polyacrylamide gels, range from 0.5-kDa for the 44-kDa and 40-kDa subunits to 9-kDa for the 17-kDa subunit . The existence in vivo of precursor forms to the 44-kDa, 40-kDa and 17-kDa subunits implies that import is at least partially a post-translational process . The precursor of the 44-kDa subunit can be processed post-translationally in vitro by isolated mitochondria . The messenger RNAs for subunits of the complex have been studied . Those coding for the 44-kDa, 40-kDa, 14-kDa and 11-kDa proteins and cytochrome c1 are of different sizes, indicating that each of these subunits is synthesized as a separate protein, rather than as part of a polyprotein precursor.

Arch Biochem Biophys, 1983 Oct 1, 226(1), 224 - 30
Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate; Shoukry S et al.; Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme . Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited . NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site . The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm . Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme . Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm . These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site . Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme . Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain . These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.

J Biomol Struct Dyn, 1983 Oct, 1(1), 209 - 23
Yeast tRNAAsp-aspartyl-tRNA synthetase: the crystalline complex; Moras D et al.; Aspartyl-tRNA synthetase from yeast, a dimer of molecular weight 125,000 and its cognate tRNA (Mr = 24,160) were co-crystallized using ammonium sulfate as precipitant agent . The presence in the crystals of both components in the two-to-one stoichiometric ratio was demonstrated by electrophoresis, biological activity assays and crystallographic data . Crystals belong to the cubic space group I432 with cell parameter of 354 A and one complex particle per asymmetric unit . The solvent content of about 78% is favorable for a low resolution structural investigation . By exchanging H2O for D2O in mother liquors, advantage can be taken from contrast variation techniques with neutron radiations . Diffraction data to 20 A resolution were measured at five different contrasts, two of them being close to the theoretical matching point of RNA and protein in the presence of ammonium sulfate . The experimental extinction of the diffracted signal was observed to be close to 36% D2O, significantly different from the predicted value of 41% . The phenomenon can be explained by the existence of a large interface region between the two tRNAs and the enzyme . These parts of the molecules are hidden from the solvent and their protons are less easily exchangeable . Accessibility studies toward chemicals of tRNAAsp in solution and in the presence of synthetase are in agreement with such a model.

J Biomol Struct Dyn, 1983 Oct, 1(1), 183 - 207
The solution structure of yeast tRNAPhe as studied by nuclear Overhauser effects in NMR; Hilbers CW et al.; Recently, the imino proton spectrum of yeast tRNAPhe has been assigned by means of the application of the nuclear Overhauser effect (NOE) . In the present paper it will be shown that even for tRNA (MW 28000) connectivities between the imino proton spins can be observed using two-dimensional NOE spectroscopy . In this way the imino proton resonances of the D-stem region are assigned . The results are discussed in relation to those obtained by the classical one-dimensional nuclear Overhauser effect . It turns out that in 2D-NOE experiments connectivities from overlapping resonances can be observed which cannot be determined by one-dimensional Overhauser experiments . Moreover, the total assignment of the imino proton spectrum of yeast tRNAPhe is used to relate the three-dimensional crystal structure of the tRNA to its solution structure . It is shown that the principle elements of the X-ray structure, i.e . the hydrogen bonding network and the stacking of the stems upon one another, are also found in solution . This is true for the presence as well as for the absence of magnesium ions . However, in absence of magnesium ions the tRNA structure appears to differ in details from that in the presence of magnesium ions . Finally, the influence of the elongation factor Tu from B.stearothermophilus on the tRNA structure is discussed.

Can J Genet Cytol, 1983 Oct, 25(5), 415 - 9
Translocation of zinc from vacuole to nucleus during yeast meiosis; Bilinski CA et al.; A novel staining procedure employing the UV fluorochrome DAPI (4',6-diamidino-2-phenylindole X 2HCl) and dithizone (diphenylthiocarbazone) was developed for microcytochemical determination of sites of zinc localization in Saccharomyces cerevisiae Hansen . In vegetative cells vacuolar polyphosphate bodies stained with dithizone, whereas in sporulating cells nucleoli and centriolar plaques were dithizone-positive . Hence, dithizone not only permitted localization of zinc but also indicated zinc translocation from vacuolar to nuclear compartments during differentiation from the vegetative to sporulated state.

Biosci Rep, 1983 Oct, 3(10), 963 - 71
Location in the yeast hexokinase structure of residues related to the enzyme activity; Gray AJ et al.; Seven residues implicated as acting directly in substrate binding in yeast hexokinase B have been identified in the crystallographic structure by chemical sequencing . The cysteine which is regarded as a residue critically maintaining the active conformation of yeast hexokinase has been selectively labelled and likewise located in the structure . In some parts of the amino acid sequence predicted from the high-resolution electron density map it is found that alignments of chemically sequenced peptides can be made unambiguously; however, the extent of matching to the predicted sequence varies considerably along the chain.

Gene, 1983 Oct, 24(2-3), 289 - 97
Synthesis of a human insulin gene . VI . Expression of the synthetic proinsulin gene in yeast; Stepien PP et al.; The construction of plasmid vectors for the controlled expression of a synthetic human proinsulin gene in the yeast Saccharomyces cerevisiae is described . Attempts to express the proinsulin gene using the yeast ADH1 promoter alone did not yield detectable levels of proinsulin . Successful expression was achieved when the proinsulin gene was fused with the promoter and protein leader sequence of the GAL1 gene (coding for yeast galactokinase) in the yeast-Escherichia coli plasmid vector pYT7810 . Two different-length leader sequences were employed; the longer leader (about 280 amino acids, fusion plasmid pPS13) gave about five times greater expression than the shorter leader fusion (30 amino acids, plasmid pPS5) . Both fusions gave soluble protein products, and the proinsulin could be cleaved by cyanogen bromide treatment from the leader polypeptide . Proinsulin was detected by radioimmunoassay for human C-peptide only in cells induced with galactose, and was not detected in the gene fusions that were out of phase with the GAL1 leader sequence . Methods of improving the level of expression of the proinsulin gene in yeast using this system are discussed.

Arch Biochem Biophys, 1983 Oct 1, 226(1), 292 - 305
Proteolysis in eucaryotic cells: aminopeptidases and dipeptidyl aminopeptidases of yeast revisited; Achstetter T et al.; Using nine different L-aminoacyl-4-nitroanilides and four different dipeptidyl-4-nitroanilides, aminopeptidases and dipeptidyl aminopeptidases active at pH 7.5 and (or) pH 5.5 in logarithmically growing and stationary-phase cells of Saccharomyces cerevisiae were searched for . Ion-exchange chromatography was used to separate the proteins of the soluble cell extract . Besides the three already-characterized aminopeptidases--aminopeptidase I (P . Matile, A . Wiemken, and W . Guyer (1971) Planta (Berlin) 96, 43-53; J . Frey and K . H . Rohm (1978) Biochim . Biophys . Acta 527, 31-41), aminopeptidase II (J . Frey and K . H . Rohm (1978) Biochim . Biophys . Acta 527, 31-41; J . Knuver (1982) Thesis, Fachbereich Chemie, Marburg, FRG), and aminopeptidase Co (T . Achstetter, C . Ehmann, and D . H . Wolf (1982) Biochem . Biophys . Res . Commun . 109, 341-347)--12 additional aminopeptidase activities are found in soluble cell extracts eluting from the ion-exchange column . These activities differ from the characterized aminopeptidases in one or more of the parameters such as charge, size, substrate specificity, inhibition pattern, pH optimum for activity and regulation . Also, a particulate aminopeptidase, called aminopeptidase P, is found in the nonsoluble fraction of disintegrated cells . Besides the described particulate X-prolyl-dipeptidyl aminopeptidase (M . P . Suarez Rendueles, J . Schwencke, N . Garcia-Alvarez and S . Gascon (1981) FEBS Lett . 131, 296-300), three additional dipeptidyl aminopeptidase activities of different substrate specificities are found in the soluble extract.

Cell, 1983 Oct, 34(3), 961 - 70
Pedigree analysis of plasmid segregation in yeast; Murray AW et al.; We have used pedigree analysis to investigate the mitotic segregation of circular and linear DNA plasmids in Saccharomyces cerevisae . Circular ARS plasmids, which bear putative chromosomal replication origins, have a high segregation frequency and a strong bias to segregate to the mother cell at mitosis . The segregation bias explains how the fraction of plasmid-bearing cells can be small despite the high average copy number of circular ARS plasmids . Linear ARS plasmids do not show strong segregation bias, nor does the 2 mu ori-containing plasmid YEp 13, when it is present in strains containing intact 2 mu circles . In the absence of endogenous 2 mu circles, YEp 13 behaves like an ARS plasmid, showing a strong maternal segregation bias . The presence of a centromere on circular ARS plasmids eliminates segregation bias . We discuss a model for plasmid segregation, which explains these findings and the possible biological significance of mother-daughter segregation bias.

Biokhimiia, 1983 Oct, 48(10), 1611 - 6
{Purification and several properties of dihydroxyacetone kinase from the methylotrophic yeast Candida boidinii}; Bystrykh LV et al.; A procedure for isolation of a homogeneous dihydroxyacetone kinase including fractionation by polyethylene glycol and ion-exchange chromatography on polyethylenimine-Biogel has been developed . The enzyme is a dimer with Mr = 139 000 (2.71 000 according to SDS disc electrophoresis) and has a pI of 4.64 and pH optimum of 7.8-8.2 . The enzyme phosphorylates dihydroxyacetone and, in a lesser degree, glyceraldehyde . ATP is the most efficient phosphate group donor for the enzyme . When ITP, GTP, CTP and UTP are used, the dihydroxyacetone kinase activity is about 30%.

Cell, 1983 Oct, 34(3), 911 - 7
Characterization of the yeast mitochondrial locus necessary for tRNA biosynthesis: DNA sequence analysis and identification of a new transcript; Miller DL et al.; Most components necessary for the biosynthesis of mitochondrial tRNAs are coded by nuclear genes, but one mitochondrial locus other than the tRNA genes themselves is required to make functional tRNAs in the yeast Saccharomyces cerevisiae . DNA sequence analysis of this yeast mitochondrial tRNA synthesis locus is reported here . This region of mitochondrial DNA is almost exclusively A+T-rich DNA with one G+C-rich element . Despite the unusual structure of the DNA in this region, we have demonstrated that it codes for a heretofore unidentified mitochondrial transcript about 450 bases in length . Since this RNA is the only RNA encoded by the tRNA synthesis locus, it must be the active agent of the locus . This RNA could either act autonomously through RNA-RNA interactions or as part of an RNA-protein complex to effect tRNA biosynthesis.

Biochemistry, 1983 Sep 27, 22(20), 4642 - 6
Inactivation of yeast hexokinase B by triethyltin bromide and reactivation by dithiothreitol and glucose; Siebenlist KR et al.; Binding of triethyltin bromide to yeast hexokinase B results in a rapid change in the reactivity of the sulfhydryl groups of the molecule . The change was characterized by an increased rate as well as extent of reaction of the -SH groups, and it preceded the onset of inhibition of the enzyme . Rapid gel filtration of the enzyme-triethyltin complex reversed this change in sulfhydryl reactivity, and when the eluted enzyme was subjected to short incubation periods, the slow inhibition that occurs with the unfiltered enzyme-triethyltin complex was no longer manifested . With prolonged incubation, however, the gel-filtered sample demonstrated increased rate of loss of enzyme activity, indicating that the gel filtration step did not completely reverse the effects of triethyltin on the enzyme . Active enzyme was recovered, following the inactivation of yeast hexokinase with triethyltin, by incubation of the inactivated enzyme with a large excess of glucose and dithiothreitol . Near total recovery of enzyme activity with reversion to native enzyme conformation was achieved following incubation at 35 degrees C of the enzyme with glucose and dithiothreitol each at 0.1 M . The possible involvement of either cysteine or histidine in the binding of triethyltin to the enzyme was probed, and it was concluded that neither of these amino acids are donor ligands for tin.

J Mol Biol, 1983 Sep 25, 169(3), 663 - 90
DNA sequences of yeast H3 and H4 histone genes from two non-allelic gene sets encode identical H3 and H4 proteins; Smith MM et al.; The complete DNA sequences of two loci encoding H3 and H4 histones in Saccharomyces cerevisiae have been determined . Each locus contains one H3 and one H4 gene . The genes at each locus are divergently transcribed and the coding sequences are separated by 646 base-pairs at one locus and 676 base-pairs at the other . The H3 genes code for identical histone H3 proteins and the H4 genes code for identical histone H4 proteins . The yeast proteins differ from histones H3 and H4 of calf by 15 and 8 amino acid substitutions, respectively, and these differences are largely confined to the carboxy-terminal halves of the proteins . The genes demonstrate a bias in synonymous codon usage similar to that noted for other yeast genes . This bias is confined to the coding sequences of the genes and is specific for the reading frame encoding the proteins . The coding sequence of each gene is flanked on both sides by DNA with an A + T content of 70 to 80% . Possible regulatory sequences are located relative to the 5' and 3'-termini of the histone H3 and H4 RNA transcripts.

J Biol Chem, 1983 Sep 25, 258(18), 10867 - 72
Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast; Uno I et al.; In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP . The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP . Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP . The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase . Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells . Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts . The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.

Biochem Biophys Res Commun, 1983 Sep 15, 115(2), 642 - 7
Buthiobate: a potent inhibitor for yeast cytochrome P-450 catalyzing 14 alpha-demethylation of lanosterol; Aoyama Y et al.; Buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbon-imidate), a fungicide, inhibited 14 alpha-demethylation of lanosterol catalyzed by a reconstituted enzyme system consisting of cytochrome P-450 (P-450(14)-DM) and NADPH-cytochrome P-450 reductase both purified from Saccharomyces cerevisiae . Concentration of buthiobate necessary for the 50% inhibition was 0.3 microM and this value was markedly lower than those of metyrapone and SKF-525A . Buthiobate bound stoichiometrically to P-450(14)-DM and induced Type II spectral change of the cytochrome . Buthiobate inhibited lanosterol-dependent enzymatic reduction of the cytochrome . These facts indicate that buthiobate binds to P-450(14)-DM with high affinity and acts as a potent inhibitor on the cytochrome.

Nature, 1983 Sep 15-21, 305(5931), 189 - 93
Construction of artificial chromosomes in yeast; Murray AW et al.; Fifty-five-kilobase long artificial chromosomes containing cloned genes, replicators, centromeres and telomeres have been constructed in yeast . These molecules have many of the properties of natural yeast chromosomes . Centromere function is impaired on short (less than 20 kilobases) artificial chromosomes.

Biochim Biophys Acta, 1983 Sep 14, 747(1-2), 71 - 7
Sedimentation behaviour of aminoacyl-tRNA synthetases from mixed lysates of yeast and rabbit liver; Mirande M et al.; The subcellular distribution of five aminoacyl-tRNA synthetases from yeast, including lysyl-, arginyl- and methionyl-tRNA synthetases known to exist as high-molecular-weight complexes in lysates from higher eukaryotes, was investigated . To minimize the risks of proteolysis, spheroplasts prepared from exponentially grown yeast cells were lysed in the presence of several proteinase inhibitors, under conditions which preserved the integrity of the proteinase-rich vacuoles . The vacuole-free supernatant was subjected to sucrose density gradient centrifugation . No evidence for multimolecular associations of these enzymes was found . In particular, phenylalanyl-tRNA synthetase activity was not associated with the ribosomes, whereas purified phenylalanyl-tRNA synthetase from sheep liver, added to the yeast lysate prior to centrifugation, was entirely recovered in the ribosomal fraction . A mixture of lysates from yeast and rabbit liver was also subjected to sucrose gradient centrifugation and assayed for methionyl- and arginyl-tRNA synthetase activities, under conditions which allowed discrimination between the enzymes originating from yeast and rabbit . The two enzymes from rabbit liver were found to sediment exclusively as high-molecular-weight complexes, in contrast to the corresponding enzymes from yeast, which displayed sedimentation properties characteristic of free enzymes . The preservation of the complexed forms of mammalian aminoacyl-tRNA synthetases upon mixing of yeast and rabbit liver extracts argues against the possibility that failure to observe complexed forms of these enzymes in yeast was due to uncontrolled proteolysis . Furthermore, this result denies the presence, in the crude extract from liver, of components capable of inducing artefactual aggregation of the yeast aminoacyl-tRNA synthetases, and thus indirectly argues against an artefactual origin of the multienzyme complexes encountered in lysates from mammalian cells.

Nucleic Acids Res, 1983 Sep 10, 11(17), 5969 - 88
The role of non-coding DNA sequences in transcription and processing of a yeast tRNA; Raymond GJ et al.; We have tested the hypothesis that conserved sequences in the intervening sequence (IVS) and 5'-flanking region of a yeast tRNALeu3 gene serve some function . Genes with deletions of 8, 10, 13 and 20 bp in the IVS are all active as templates in vitro . Yeast extracts produce mature tRNALeu3 from delta 8, delta 10 and delta 13 genes . Xenopus extracts do not detectably ligate the 5' and 3' half-molecules resulting from IVS excision . Neither extract is able to excise the IVS from delta 20 precursors . Genes with introns enlarged by 10, 21 or 30 bp of DNA produce mature tRNA . Insertion of 103 bp results in reduced levels of transcription, little if any end maturation, and no detectable mature product . A conserved 15 bp sequence is present at the 5'-end of the tRNA sequence . Replacement of yeast DNA up to position -22 leaves the tRNA gene transcriptionally active . With replacement extended to -2 the gene is active in Xenopus extracts but nearly inert in yeast extracts . We conclude that tRNA transcription in yeast is insensitive to IVS structure but can be positively influenced by 5'-flanking sequence.

J Biol Chem, 1983 Sep 10, 258(17), 10649 - 56
Kinetics of assembly of complex III into the yeast mitochondrial membrane . Evidence for a precursor to the iron-sulfur protein; Sidhu A et al.; Complex III immunoprecipitated from yeast cells labeled in vivo with {35S}sulfate or {3H}leucine contained seven subunits with molecular weights ranging from 15,000 to 47,000 when analyzed by electrophoresis on polyacrylamide gels . The subunit composition of the immunoprecipitates was identical with that of the purified complex III isolated from bakers' yeast suggesting that the antiserum recognizes the holoenzyme assembled properly in the membrane (Sidhu, A., and Beattie, D.S . (1982) J . Biol . Chem . 257, 7879-7886) . Kinetic studies using double-labeled yeast cells followed by immunoprecipitation of complex III indicated that the subunits of the complex are assembled into the holoenzyme at very different rates . Cytochromes b and c1 and the 15,000-dalton subunit were the first polypeptides to be assembled into the complex with a half-time of labeling of 2.0-2.4 min . Core protein I and the iron-sulfur protein were inserted more slowly into the complex with a half-time of labeling of 4.6 and 5.3 min, respectively . Calculations of precursor pool sizes of the subunits indicated that for both core protein I and the iron-sulfur protein, there are large pools of precursors . The iron-sulfur protein was synthesized in vivo as a larger precursor polypeptide of molecular mass 28,000 Da . The precursor was subsequently cleaved, in a process requiring an energized mitochondrial inner membrane, into an intermediate form 1,500 Da larger than the mature subunit . The conversion of the intermediate to the mature form occurred in the inner mitochondrial membrane.

Biochim Biophys Acta, 1983 Sep 9, 740(4), 460 - 5
The influence of spermine on the structural dynamics of yeast tRNAPhe; Nilsson L et al.; A tRNAPhe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA . As previously reported (Ehrenberg, M., Rigler, R . and Wintermeyer, W . (1979) Biochemistry 18, 4588-4599) in the tRNA derivative the ethidium is present in three states (T1-T3) characterized by different fluorescence decay rates . T-jump experiments show two transitions between the states, a fast one (relaxation time 10-100 ms) between T1 and T2, and a slow one (100-1000 ms) between T2 and T3 . In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy . Spermine shifts the distribution of states towards T3, as does Mg2+, but the final ratio {T2}/{T1} obtained with spermine is higher than with Mg2+, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.

Biochim Biophys Acta, 1983 Sep 7, 733(2), 234 - 41
Ammonia accumulation in acetate-growing yeast; Bogonez E et al.; During growth on acetate, the pH of yeast cultures rises from 5.8 to around 7-8 in the stationary phase . This was found to result from acetic acid uptake and accompanying H+ loss . In addition, acetate-growing yeast were found to accumulate ammonia . The influence of pH on ammonia transport and accumulation was studied with the analogue {14C}methylamine with the following results . (a) Methylamine uptake kinetics from 0.1-50 mM were consistent with a single-component uptake system (NH+4 permease) at pH values more acidic than 6.5, and with a two-component system (NH+4 permease and NH3 diffusion) above pH 7.5 . (b) Equilibrium accumulation of methylamine was found to increase with increasing pH . (c) Methylamine efflux from methylamine-loaded cells increased as the external pH decreased . It was concluded from measurements of the internal pH under various culture conditions that the accumulation of ammonia in acetate-growing alkaline cultures resulted from the sum of two processes: (1) an energy-driven NH+4 transport; and (2) NH3 diffusion dependent on the delta pH.

Nature, 1983 Sep 29-Oct 5, 305(5933), 391 - 7
The new yeast genetics; Struhl K; Gene cloning and yeast DNA transformation techniques have greatly enhanced the power of classical yeast genetics . It is now possible to isolate any classically defined gene, to alter the yeast genome at will by replacing normal chromosomal sequences with mutated derivatives produced in vitro, and to create DNA molecules that behave as autonomous replicons or minichromosomes . These unique features of the new yeast genetics have been used to study many problems in eukaryotic molecular biology.

Lipids, 1983 Sep, 18(9), 623 - 9
A comparison of the oleaginous yeast, Candida curvata, grown on different carbon sources in continuous and batch culture; Evans CT et al.; The oleaginous yeast, Candida curvata D, was grown in both batch and continuous culture on 5 different carbon sources to compare the efficiency of fat production from the various substrates . Maximum lipid accumulation occurred in batch culture with xylose as the carbon source on nitrogen-limited medium reaching a level of 49% (w/w) of the biomass, but this was reduced to 37% at the optimum dilution rate (D = 0.05/hr) in a chemostat . Both the highest biomass and lipid yields were attained in continuous culture with lactose as the sole carbon source at a dilution rate of D = 0.04/hr, giving an efficiency of substrate conversion of 60 g of biomass and 18.6 g lipid per 100 g lactose utilized . The relative proportions of the major fatty acids (16:0, 18:0, 18:1, 18:2) in the lipid were found to vary considerably in batch culture and in continuous culture under carbon-limited conditions . However, on nitrogen-limited media in the chemostat, the fatty acid composition remained relatively constant over the whole range of dilution rates employed . Lipid from xylose-grown cells contained the greatest percentage of stearic acid (18:0) 15% and the lowest linoleic acid (18:2) 4%, whereas lipid from ethanol-grown cells contained elevated levels of oleic acid (18:1) 51% and decreased palmitic acid (16:0) 25%.

Cell, 1983 Sep, 34(2), 395 - 403
Evidence for the biochemical role of an internal sequence in yeast nuclear mRNA introns: implications for U1 RNA and metazoan mRNA splicing; Pikielny CW et al.; Sequence comparison of the introns of two yeast genes (rp51A and rp51B) coding for the same ribosomal protein shows homology only in the last 50 bases of the intron . This region of the intron contains an internal conserved sequence (ICS) present near the 3' end of all sequenced yeast nuclear mRNA introns . Removal of a 29 bp sequence containing the ICS prevents splicing of an intron-containing hybrid gene . In cells containing the wild-type gene, we have detected RNA molecules that we suggest are normal splicing intermediates, generated by an endonucleolytic cut in the primary transcript at the ICS . The homology of the ICS with a sequence near the 5' end of U1 snRNA suggests a model in which an interaction in cis between the ICS and the 5' splice junction in yeast is the counterpart of the interaction in trans between U1 and 5' splice junctions in higher eucaryotes.

Gene, 1983 Sep, 24(1), 73 - 81
The orir to ori+ mutation in spontaneous yeast petites is accompanied by a drastic change in mitochondrial genome replication; Mangin M et al.; The orir petite mutants of Saccharomyces cerevisiae show a very low level of suppressivity (5-12%; suppressivity is the percentage of diploid petites issued from a cross of the parental haploid petite with a wild-type cell), indicating a poor replication efficiency of their mitochondrial genome . The latter is made up of repeat units containing two inverted ori sequences and arranged as tandem pairs in inverted orientation relative to their nearest neighbors . After subcloning orir petites or crossing with wild-type cells a large number of ori+ petites are found in the progeny . In contrast to the orir petites, from which they are derived, these ori+ petites are characterized by high suppressivity levels (approx . 90%) and contain mitochondrial genomes made up of tandem repeat units containing single ori sequences . The structural changes underlying the orir to ori+ mutation are therefore accompanied by a dramatic increase in suppressivity, indicating that the elimination of inverted ori sequences causes a drastic change from very poor to very good replicative efficiency in the mitochondrial genome . Finally, crosses of ori0 petites with wild-type cells were also studied; the results obtained have clarified the reasons for the high frequency of petites having genomes similar to those of orir petites after mutagenesis with ethidium bromide.

Gene, 1983 Sep, 24(1), 61 - 71
The mitochondrial genomes of spontaneous orir petite mutants of yeast have rearranged repeat units organized as inverted tandem dimers; Faugeron-Fonty G et al.; We have investigated the structure and organization of the mitochondrial genomes of two related orir (ori-rearranged) spontaneous petite mutants of Saccharomyces cerevisiae . In these mutant genomes every repeat unit contains an inverted terminal duplication harboring a second (inverted) ori sequence, and tandem pairs of repeat units alternate with tandem pairs in inverted orientation . We have shown that orir genomes are organized as the genomes with inverted repeat units of ethidium bromide (EtBr)-induced petites, and we have clarified the mechanism by which such mutant mitochondrial genomes arise.

Arch Biochem Biophys, 1983 Sep, 225(2), 861 - 71
Structural and physiological features of sterols necessary to satisfy bulk membrane and sparking requirements in yeast sterol auxotrophs; Rodriguez RJ et al.; A variety of sterols and stanols have been analyzed for their ability to satisfy bulk membrane and high-specificity (sparking) functions in three yeast sterol auxotrophs . While many sterols and stanols satisfied bulk membrane requirements, only those possessing a C-5,6 unsaturation or capable of being desaturated at C-5 fulfilled the high-specificity sparking requirement . Unsaturation of the A-ring or beta-saturation of a C-5,6 double bond rendered both sterol and stanol unsuitable for either function . The C-28 methyl group of ergosterol, while not required for growth, allowed for greater ease of desaturation at C-5 in vivo . As a result some sterols and stanols lacking the C-28 methyl were incapable of satisfying the sparking requirement while identical compounds possessing the C-28 methyl were able to fulfill the sparking function(s) . These data are extended to hypothesize a role for the C-28 methyl group of ergosterol in yeast.

Mutat Res, 1983 Sep, 121(3-4), 195 - 8
A nuclear mutation decreasing rho- production modifies the unstable nitrous-acid sensitivity of yeast; Koltovaya NA et al.; The nuclear mmgl mutation, which reduces rho- mutability in Saccharomyces cerevisiae, renders the rho+ cells less sensitive to inactivation by nitrous acid (NA) but has little or no effect on the NA sensitivity of the rho0 cells devoid of mitochondrial (mt) DNA . Therefore the cells' NA sensitivity seems to be influenced by an interaction of the mmgl mutation and the mt genome rather than the mmgl mutation itself . The clonal variation of NA sensitivity is high in MMG+ yeast and significantly reduced in rho0 mutants and mmgl cells . The results presented suggest that frequent spontaneous heritable changes of the mt genome occur in MMG+ cells, which, (i) unlike rho- mutations, do not damage the respiratory capacity, and (ii) manifest themselves in a high clonal variation of NA sensitivity.

Cell, 1983 Sep, 34(2), 655 - 64
The promoter sequence of a yeast tRNAtyr gene; Allison DS et al.; Thirty-one base substitution mutations within the yeast SUP4 tRNAtyr gene were used to probe the effects of different intragenic sequences on promoter activity . The various mutant plasmids were tested quantitatively for their in vitro template activity and for their ability to block competitively the transcription of a reference gene . Five mutations within the coding sequence of SUP4 decreased template activity for pre-tRNAtyr synthesis . The competition assays revealed 11 mutant genes that behaved differently than SUP4-o . Six were weaker competitors and five were stronger . The 12 mutations affecting template activity or competition are clustered in three regions: those encoding the dihydrouracil (D) arm, the extra loop, and the T psi arm of the tRNA . All of the mutations that reduce competition involve base changes that decrease homology to a eucaryotic tRNA consensus sequence in the highly conserved D and T psi regions . Three of the five up mutations increased homology to the tRNA consensus sequence.

J Pharmacobiodyn, 1983 Sep, 6(9), 668 - 76
Antitumor activity of acidic mannan fraction from bakers' yeast; Hashimoto K et al.; The acidic fraction of bakers' yeast mannan containing mannose (93.6%), nitrogen (1.0%), and phosphorus (0.6%), designated as WAM025, showed a marked antitumor activity against the ascites forms of Ehrlich, sarcoma 180 and Meth A tumors in vivo . Namely, a 100% survival of mice transplanted with Ehrlich or sarcoma 180 ascites tumor, 1 X 10(5) cells/ddY strain mouse, was obtained by the intraperitoneal administration of WAM025, 150 mg/kg/d for 10 d . This effect evoked by WAM025 seems to be attributed to the activation of peritoneal adherent cells in mice, because a significant elevation of lysosomal enzyme level and active oxygen generation was observed on the peritoneal adherent cells obtained from the mannan-administered mice.

J Biochem (Tokyo), 1983 Sep, 94(3), 715 - 20
Binding properties of an intrinsic ATPase inhibitor and occurrence in yeast mitochondria of a protein factor which stabilizes and facilitates the binding of the inhibitor to F1F0-ATPase; Hashimoto T et al.; The content of an intrinsic ATPase inhibitor in mitochondria was determined by a radioimmunoassay procedure which showed the molar ratio of the inhibitor to ATPase to be 1:1 . The ratio in submitochondrial particles, where half of the enzyme was activated, was the same as that of mitochondria, indicating that the inhibitor protein has affinity for the mitochondrial membrane as well as for F1-ATPase . The inhibitor protein could be removed from the mitochondrial membrane by incubation with 0.5 M Na2SO4 and concomitantly the enzyme was fully activated . The enzyme fully activated by the salt treatment was inactivated again by the externally added ATPase inhibitor in the presence of ATP and Mg2+ . The enzyme-inhibitor complex (inactive) on the mitochondrial membrane was more stable than the solubilized enzyme-inhibitor complex but gradually dissociated in the absence of ATP and Mg2+ . However, in mitochondria, the enzyme activity was inhibited even in the absence of the cofactors . A protein factor stabilizing the enzyme-inhibitor complex on the mitochondrial membrane was isolated from yeast mitochondria . This factor stabilized the inhibitor complex of membrane-bound ATPase while having no effect on that of purified F1-ATPase . It also efficiently facilitated the binding of the inhibitor to membrane-bound ATPase to form the complex, which reversibly dissociated at slightly alkaline pH.

Arch Biochem Biophys, 1983 Sep, 225(2), 704 - 12
An acyl-thioesterase from yeast mitochondria; Stack R et al.; A previously unstudied acyl-coenzyme A thioesterase activity has been demonstrated in submitochondrial particles from Saccharomyces cerevisiae . The preferred substrate for the enzyme activity is oleoyl-coenzyme A . Tests with inhibitors of the thioesterase showed that, in addition to common thiol inhibitors, the oxidative phosphorylation inhibitors oligomycin and venturicidin also blocked thioesterase activity . Purification of the enzyme catalyzing this activity revealed that thioesterase copurified with mitochondrial ATPase . When thioesterase was isolated from oxidative phosphorylation mutants selected for resistance to these two inhibitors, thioesterase activity was also resistant . The results suggest that thioester hydrolysis may be catalyzed by components associated with the isolated ATPase complex . Further attempts to link this activity to in vivo function of ATPase were not successful.

Proc Natl Acad Sci U S A, 1983 Sep, 80(18), 5564 - 8
Identification of a single transcriptional initiation site for the glutamic tRNA and COB genes in yeast mitochondria; Christianson T et al.; We have identified a single transcriptional initiation site for the glutamic tRNA and COB (cytochrome b) genes by using the complementary techniques of in vitro capping of RNA and in vitro transcription . In the capping reaction, mitochondrial RNA is labeled with {alpha-32P}GTP by vaccinia virus guanylyltransferase . This reaction is specific for the 5' ends of RNA retaining the terminal triphosphate of transcriptional initiation . Exploiting the extremely low G+C content (18%) of yeast mitochondrial DNA, we digested in vitro capped transcripts from various petite deletion mutants with the G-specific RNase T1 . By petite deletion mapping, a capped transcript giving rise to a 51-base RNase T1-generated oligonucleotide was localized near the glutamic tRNA gene . When the sequence of this oligonucleotide was determined, it perfectly matched the DNA sequence 391 base upstream of the glutamic tRNA . Purified yeast mitochondrial RNA polymerase initiated transcription in vitro at the same site as shown by the sequence of the 33-base oligonucleotide product of the reaction performed in the absence of CTP . Initiation starts at a nonanucleotide sequence previously implicated in yeast mitochondrial transcriptional initiation . Because there is no evidence of an initiation site in the 1,050 bases between the glutamic tRNA and COB genes, the two genes are likely to be transcribed together . Further evidence of a long common transcript was provided by RNA blot hybridization.

Biochemistry, 1983 Aug 30, 22(18), 4223 - 9
Determination of base pairing in ribonucleic acid by Fourier-transform infrared spectrometry: yeast ribosomal 5S ribonucleic acid; Burkey KO et al.; Fourier-transform infrared (FT-IR) spectra of yeast ribosomal 5S RNA have been acquired at several temperatures between 30 and 90 degrees C . The difference spectrum between 90 (bases unstacked) and 30 degrees C (bases stacked) provides a measure of base stacking in the RNA . Calibration difference spectra corresponding to stacking of G-C or A-U pairs are obtained from "reference" FT-IR spectra of poly(rG) X poly(rC) minus 5'-GMP and 5'-CMP or poly(rA) X poly(rU) minus 5'-AMP and 5'-UMP . The best fit linear combination of the calibration G-C and A-U difference spectra to the 5S RNA (90-30 degrees C) difference spectrum leads to a total of 25 +/- 3 base pairs (17 G-C pairs + 8 A-U pairs) for the native yeast 5S RNA in the absence of Mg2+ . In the presence of Mg2+, an additional six base pairs are detected by FT-IR (one G-C and five A-U) . FT-IR melting curve midpoints show that A-U and G-C pairs melt together (65 and 63 degrees C) in the presence of Mg2+ but A-U pairs melt before G-C pairs (47 vs . 54 degrees C) in the absence of Mg2+.

Biochem Biophys Res Commun, 1983 Aug 30, 115(1), 317 - 24
Cyclic AMP and fructose-2,6-bisphosphate stimulated in vitro phosphorylation of yeast fructose-1,6-bisphosphatase; Pohlig G et al.; Phosphorylation of purified yeast fructose-1,6-bisphosphatase was studied using purified preparations from yeast of two different cyclic AMP-independent protein kinases and a cyclic AMP-dependent protein kinase . Incorporation of 32P into fructose-1,6-bisphosphatase could be demonstrated only with the cyclic AMP-dependent protein kinase . Phosphorylation of fructose-1,6-bisphosphatase was stimulated by 3 microM fructose-2,6-bisphosphate and inhibited by 1 mM 5'-AMP.

Biochemistry, 1983 Aug 30, 22(18), 4229 - 34
Inactivation of yeast hexokinase B by triethyltin bromide; Siebenlist KR et al.; Triethyltin bromide was found to demonstrate temperature-dependent inactivation of yeast hexokinase B . At temperatures of 20 degrees C or lower, little or no inactivation of the enzyme was detected after 2 h of reaction with 50-300 microM concentrations of the reagent . However, incubation at 25 degrees C or higher resulted in an increased rate and extent of loss of the enzyme activity with increasing incubation temperatures . The Arrhenius plot for the inactivation process showed a sharp break at approximately 30 degrees C, with a heat of activation (delta H*) above this temperature of 55.2 kcal, indicating that a triethyltin-induced conformational change occurred at the elevated temperatures . Sugar substrates provided protection against the inactivating effect by reducing the binding of triethyltin to the enzyme . In the absence of glucose, two sites of different affinity for triethyltin exist in the hexokinase monomer . Binding of triethyltin to the enzyme shifted its monomer-dimer equilibrium toward the monomeric form in an early stage of the interaction . Inactivation of the enzyme was associated with a slower subsequent event . Comparative effects of various organotin compounds on the activity of the enzyme indicated that inhibitory potency was associated with increasing hydrophobicity of the alkyl groups attached to the tin.

J Chromatogr, 1983 Aug 26, 266, 265 - 71
Rapid chromatographic method for the isolation of glucose-6-phosphate dehydrogenase from yeast enzyme concentrate; Lindblom H; A simple method for the isolation of glucose-6-phosphate dehydrogenase (G6PDH) from yeast enzyme concentrate is described . The method is based on high-performance anion-exchange chromatography and was developed in three steps: (1) optimization of chromatographic conditions on Polyanion SI-8 microns, (2) transfer of the optimized conditions to a preparative column containing Polyanion SI-17 microns and (3) final purification on an analytical column . As the enzyme is a minor component of the crude mixture, its presence is detected by a simple enzymatic method . The purity of the final fraction is checked by polyacrylamide gel electrophoresis.

J Chromatogr, 1983 Aug 26, 266, 585 - 98
Reversed-phase high-performance liquid chromatographic purification of subunits of oligomeric membrane proteins . The nuclear coded subunits of yeast cytochrome c oxidase; Power SD et al.; Reversed-phase chromatography of the subunits of an oligomeric membrane protein such as yeast cytochrome c oxidase requires additional sample handling techniques which are not necessary for soluble proteins . This paper considers these and discusses (1) methods for the removal of ballast material by preliminary batchwise extraction with solvent mixtures similar to those used for reversed-phase elution; (2) the chromatographic heterogeneity induced by partial cysteine oxidation; (3) the removal of tightly bound proteins from the stationary phase; and (4) the generation of an elution system with continuously variable selectivity based on acetonitrile-1-propanol ratios (0.05% triethylamine, 0.05% trifluoroacetic acid) . These methods are designed to simplify complex mixtures of hydrophobic proteins prior to chromatography and to purify them chromatographically in high yield.

Nature, 1983 Aug 25-31, 304(5928), 747 - 9
Stimulation of transcription of the yeast tRNATyr gene in cell-free extracts by tyrosyl-tRNA synthetase; Smagowicz W et al.; Eukaryotic transfer RNA genes have two internal discontinuous control regions, the A and B blocks, which correspond approximately to the D-stem and the pseudo-U arm of tRNA . In reconstituted transcription systems at least two components are required to direct accurate initiation by RNA polymerase (refs 4,5) . However, little is known about the mechanism of interaction of the internal promoter sequences with factors and RNA polymerase C within the transcription complex, although tRNA-like conformation of the B block sequence was surmised to be critical for DNA recognition . By analogy with the 5S RNA system, where a transcription factor required for 5S DNA expression was shown to interact both with 5S RNA and with the noncoding strand of the 5S gene, we explored the possibility that a protein which normally binds to tRNA could also interact with the tRNA gene and regulate its transcription . Here we show that in vitro transcription of the yeast SUP4 tRNATyr gene in crude yeast extracts is strongly stimulated by tyrosyl-tRNA synthetase (TyrRS) but not by two other non-cognate synthetases . Substrates of the synthetase, tRNATyr and tyrosine, interfere with stimulation of tRNA synthesis.

Eur J Biochem, 1983 Aug 15, 134(3), 571 - 4
Solvent isotope effects on the reaction catalyzed by yeast hexokinase; Taylor KB et al.; The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O . In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7 . In 2H2O, complex behavior is observed for these parameters as a function of pD . The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters . When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7 . Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.

J Biol Chem, 1983 Aug 10, 258(15), 9040 - 2
Altered cytochrome P-450 in a yeast mutant blocked in demethylating C-32 of lanosterol; Aoyama Y et al.; Spectroscopic and enzymatic analysis of a Saccharomyces cerevisiae mutant in sterol biosynthesis (SG1 (erg 11); Trocha, P . J., Jasne, S . J., and Sprinson, D . B . (1977) Biochemistry 16, 4721-4726) that was blocked in demethylating C-32 of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) showed that it contained low levels of cytochromes P-450 and b5 compared to those present in the parent strain D-587, while NADPH-cytochrome c reductase activity was elevated . The fungicide buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate), which bound specifically to yeast cytochrome P450 responsible for demethylating C-32 of lanosterol and effected a Type II spectral change, did not react with SG1 cytochrome P-450 . On the other hand, cholate-solubilized microsomes from SG1 formed a single precipitin line on Ouchterlony plates with antibodies raised against purified (Yoshida, Y., Aoyama, Y., Kumaoka, H., and Kubota, S . (1977) Biochem . Biophys . Res . Commun . 78, 1005-1010) yeast cytochrome P-450 specific for demethylating C-32 of lanosterol . Hence, mutant SG1 contained an altered protein which retained the antigenicity of cytochrome P-450 responsible for demethylating C-32 of lanosterol but lost its catalytic activity.

J Biol Chem, 1983 Aug 10, 258(15), 9459 - 68
Assembly of the mitochondrial membrane system . Characterization of a yeast nuclear gene involved in the processing of the cytochrome b pre-mRNA; McGraw P et al.; The cytochrome b gene of Saccharomyces cerevisiae D273-10B was previously shown to be composed of three exons and two introns (Nobrega, F.G., and Tzagoloff, A . (1980) J . Biol . Chem . 255, 9828-9837) . In the present study nuclear respiratory deficient mutants of this strain have been screened for defects in processing of the cytochrome b pre-mRNA . Fifteen independently isolated mutants lacking cytochrome b have been assigned to a single genetic complementation group (G36) . Members of this complementation group are blocked in the excision of the second intervening sequence of cytochrome b and consequently are unable to produce the mature mRNA . The wild type gene defined by this class of mutants has been named CBP2 . A recombinant plasmid with the CBP2 gene has been selected from a library of wild type nuclear DNA and further subcloned by transformation of a cbp2 mutant to respiratory competency . The smallest plasmid (pG36/T5) capable of complementing cbp2 mutants and of restoring their ability to complete processing of the cytochrome b pre-mRNA has a nuclear DNA fragment of 2.6 kilobase pairs inserted at the BamHI site of the yeast vector YEp13 . The sequence of the cloned DNA fragment has revealed an 1890-nucleotide-long reading frame encoding a basic protein with a molecular weight of 74,000 . Deletion analysis confirms that the entire reading frame is required for complementation of cbp2 mutants . This reading frame is proposed to code for the CBP2 gene product.

Biochemistry, 1983 Aug 2, 22(16), 3735 - 40
Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate; Kuo DJ et al.; (E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate . An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding . The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively . The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB . Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB . The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers . {1-14C}CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme . Therefore, CPB was being turned over by PDC, in addition to inactivating it . CPB can be labeled a suicide-type inactivator for PDC.

Cell, 1983 Aug, 34(1), 95 - 104
The yeast plasmid 2mu circle encodes components required for its high copy propagation; Jayaram M et al.; The yeast plasmid 2mu and certain hybrid plasmids constructed from it are maintained stably and at high copy number in yeast cells . By examining various mutant hybrid 2mu plasmids, we show that these properties require the integrity of four plasmid loci . Two of these, designated REP1 and REP2, are active in trans and correspond to two open coding regions of 2mu . The other two loci are active only in cis and correspond to the origin of replication and to a region, designated REP3, located several hundred bp away from the origin and consisting of direct repeats of a 62 bp sequence . We propose that the REP loci constitute a copy control system that overrides normal cellular restriction on plasmid replication and amplifies the plasmid when copy number is low.

Food Chem Toxicol, 1983 Aug, 21(4), 499 - 501
A study of the induction of gene conversion in yeast by sodium bisulphite under radical reaction conditions; Murthy MS et al.; Sodium bisulphite (NaHSO3) at low concentrations is known to undergo radical reactions in the presence of Mn2+ ions, generating sulphite anion radicals (SO-3.) . The kinetics of pH variation in aqueous solutions of NaHSO3 in the presence of 0.1 mM-MnSO4 suggests that the anion radicals are generated rapidly at low bisulphite concentration (10 mM) and quenched at a higher concentration (100 mM) . The ability of NaHSO3 to induce gene conversion in the diploid yeast Saccharomyces cerevisiae BZ34 under conditions suitable for radical reactions has been investigated . The results show that NaHSO3 does not induce gene conversion under these conditions.

Gene, 1983 Aug, 23(2), 221 - 32
Copy number control by a yeast centromere; Tschumper G et al.; Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome . In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that normally drives the 2 mu plasmids to high copy number . The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern . Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number . Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.

Genetics, 1983 Aug, 104(4), 603 - 18
Genetic effects of UV irradiation on excision-proficient and -deficient yeast during meiosis; Resnick MA et al.; The lethal and recombinational responses to ultraviolet light irradiation (UV) by excision-proficient (RAD+) and deficient strains (rad1) of Saccharomyces cerevisiae has been examined in cells undergoing meiosis . Cells that exhibit high levels of meiotic synchrony were irradiated either at the beginning or at various times during meiosis and allowed to proceed through meiosis . Based on survival responses, the only excision repair mechanism for UV damage available during meiosis is that controlled by the RAD1 pathway . The presence of pyrimidine dimers at the beginning of meiosis does not prevent cells from undergoing meiosis; however, the spore products exhibit much lower survival than cells from earlier stages of meiosis . The reduced survival is probably due to effects of UV on recombination . Meiotic levels of gene conversion are reduced only two to three times in these experiments; however, intergenic recombination is nearly abolished after a dose of 4 J/m2 to the rad1 strain . Exposure to 25 J/m2 had little effect on the wild-type strain . Since normal meiotic reciprocal recombination is generally considered to involve gene conversion-type intermediates, it appears that unrepaired UV damage dissociates the two processes . These results complement those obtained with the mei-9 mutants of Drosophila which also demonstrate a dissociation between gene conversion and reciprocal recombination . These results are consistent with molecular observations on the UV-irradiated rad1 strain in that there is no excision of pyrimidine dimers or exchange of dimers during meiosis.

Genetics, 1983 Aug, 104(4), 583 - 601
Meiotic DNA metabolism in wild-type and excision-deficient yeast following UV exposure; Resnick MA et al.; The effects of UV irradiation on DNA metabolism during meiosis have been examined in wild-type (RAD+) and mitotically defined excision-defective (rad1-1) strains of Saccharomyces cerevisiae that exhibit high levels of sporulation . The rad1-1 gene product is not required for normal meiosis: DNA synthesis, RNA synthesis, size of parental and newly synthesized DNA and sporulation are comparable in RAD+ and rad1-1 strains . Cells were UV irradiated at the beginning of meiosis, and the fate of UV-induced pyrimidine dimers as well as changes in DNA and DNA synthesis were followed during meiosis . Excision repair of pyrimidine dimers can occur during meiosis and the RAD1 gene product is required; alternate excision pathways do not exist . Although the rate of elongation is decreased, the presence of pyrimidine dimers during meiosis in the rad1-1 strain does not block meiotic DNA synthesis suggesting a bypass mechanism . The final size of DNA is about five times the distance between pyrimidine dimers after exposure to 4 J/m2 . Since pyrimidine dimers induced in parental strands of rad1-1 prior to premeiotic DNA synthesis do not become associated with newly synthesized DNA, the mechanism for replicational bypass does not appear to involve a recombinational process . The absence of such association indicates that normal meiotic recombination is also suppressed by UV-induced damage in DNA; this result at the molecular level is supported by observations at the genetic level.

Mutat Res, 1983 Aug, 112(4), 201 - 14
Fate of photo-induced 8-methoxypsoralen mono-adducts in yeast . Evidence for bypass of these lesions in the absence of excision repair; Chanet R et al.; A fraction of UVA-induced 8-methoxypsoralen (8-MOP) mono-adducts can be transformed by a second UVA (365 nm) irradiation procedure into lethal cross-links in Saccharomyces cerevisiae . To follow the fate of cross-linkable mono-adducts, cells were incubated in complete medium between the two UVA doses and survival was measured . The killing effect of the second UVA dose decreases rapidly in haploid wild-type as well as in strains blocked in mutagenic (RAD6+ type) or in recombinogenic (RAD52+ type) repair pathways . This is also true in the pso1-1 and pso2-1 strains selected for sensitivity to 8-MOP plus UVA treatment . In contrast, persistence of mono-adducts is observed in strains blocked in the excision-resynthesis repair pathway . In other words, cross-linkable mono-adducts are repaired by the excision process . The use of the cell-cycle conditional mutant strain (cdc14-1) permitted us to apply the second dose at a specific cell-cycle stage (post-G2 phase) after a 'priming' UVA treatment on stationary (G1) phase cells . Such experiments showed a bypass of mono-adducts in an excision-deficient context for at least one round of DNA replication.

Cancer Res, 1983 Aug, 43(8), 3700 - 6
Likelihood of the new antitumoral drug 10-{gamma-diethylaminopropylamino}-6-methyl-5H-pyrido{3',4':4,5}pyrrolo {2,3-g}isoquinoline (BD-40), a pyridopyrroloisoquinoline derivative, to induce DNA strand breaks in vivo and its nonmutagenicity in yeast; Moustacchi E et al.; BD-40, a pyridopyrroloisoquinoline analogue of ellipticines, has dose-dependent cytostatic and cytotoxic effects on cultures of Saccharomyces cerevisiae . These inhibitory effects take place only in growing cells and are enhanced in the presence of oxygen . Among the different repair-deficient mutants examined, a mutant defective in DNA strand break repair (rad52-1) was found to be the most sensitive to such a toxic effect . A triple mutant blocked in the excision (rad2), the mutagenic (rad6), and the recombinogenic (rad52) repair pathways demonstrated the same sensitivity as the single rad52 mutant . Nuclear reversion and forward mutations as well as mitochondrial "petite" mutation were not induced by BD-40 . These results indicate that: (a) the lesions induced in vivo by BD-40 are likely to be DNA strand breaks; (b) such damage is repairable in the wild type and is not of the mutagenic type; and (c) the excision pathway is not involved in such a repair of BD-40-induced lesions, and the mutagenic pathway plays a minor role . Since DNA strand breaks were not detected in vitro whether exposure of DNA to BD-40 was achieved in the presence or the absence of microsomal S-9 mix, it is suggested that an oxygen-dependent enzymatic processing, not linked to the microsomal monooxygenase complex, is required for the development of the cytotoxic activity of BD-40.

Lipids, 1983 Aug, 18(8), 545 - 52
The effect of AY-9944 on yeast sterol and sterol ester metabolism; Pereira R et al.; The effects of the hypocholesterolemic drug AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride) at two concentrations (10(-4) M and 5 X 10(-4) M) on the synthesis of sterols and sterol esters by Saccharomyces cerevisiae were investigated . Although growth was not markedly affected by the drug, there was a decrease in the free sterol to sterol ester ratio with increased drug concentration . A concomitant increase in the saturated fatty acids esterified to sterol relative to the unsaturated fatty acids was also noted in response to increased drug concentration . Ergosterol accounted for 94.7% of the free sterol in the control culture and for 87.8% of the 5 X 10(-4) M drug-treated culture, respectively . However, in the sterol ester fraction, the ergosterol content decreased from a value of 45.1% in the control culture to 2.4% in the 5 X 10(-4) M AY-9944 treated culture . The sterol ester fraction simultaneously showed increased levels of the delta 8 sterol, fecosterol, in response to increased drug concentration from a 7.4% control value to 57.4% in the 5 X 10(-4) M drug-treated culture . The accumulation of the delta 8 sterol suggests that the site of action of the drug is probably at the delta 8 to delta 7 isomerase step in the biosynthesis of ergosterol . The fact that ergosterol is retained as the major free sterol suggests a biological advantage to the retention of this particular sterol . In addition, the near normal growth in the presence of the drug, in spite of the occurrence of an altered sterol ester profile, indicates that the composition of the sterol ester fraction is not as critical as the free sterol fraction.

Biokhimiia, 1983 Aug, 48(8), 1241 - 8
{Compartmentalization of metabolism of spatially-delimiting amino acid pools of yeast cells}; Davidova EG et al.; The spatially different amino acid pools (i.e . cytoplasmic, vacuolar and mitochondrial) of yeast cells are metabolically compartmentalized . The accumulation of amino acids in these pools occurs at different rates; the highest rates are observed for glutamate and alanine . The former is predominantly accumulated in the cytoplasm, the latter--in the vacuoles . The renewal rates of the amino acid pools are also different . Each of them contains at least two subpools, readily convertible and relatively stable ones . The readily convertible subpools of the cytoplasmic and mitochondrial pools predominantly contain glutamate, aspartate, valine and alanine; that of the vacuolar pool--alanine . The bulk of the readily convertible alanine subpool (67%) is localized in the vacuoles, that of glutamate and aspartate (85 and 68%, respectively)--in the cytoplasm.

Biochem Biophys Res Commun, 1983 Jul 29, 114(2), 518 - 25
Synthesis of a double-stranded cDNA transcript of the killer toxin-coding region of the yeast M1 double-stranded RNA; Skipper N; Reverse transcription of methylmercuryhydroxide-treated M1 double-stranded RNA of yeast produces several discrete single-stranded and double-stranded cDNA's from oligo (dT)12-18 primer . S1 nuclease analysis shows that the longest transcript of the 1.8 kb template, a 2.2 kb molecule, is a 1.1 kb duplex terminated at one end by a hairpin-like structure . The 1.1 kb ds cDNA contains a complete copy of the M1-1, toxin-coding, sequence of M1 double-stranded RNA, and its synthesis is primed from oligo (dT)12-18 that anneals to a sequence within the internal, AU-rich, region of the template.

Nucleic Acids Res, 1983 Jul 25, 11(14), 4879 - 90
The complete nucleotide sequence of the rat 18S ribosomal RNA gene and comparison with the respective yeast and frog genes; Torczynski R et al.; The complete nucleotide sequence of the rat 18S ribosomal RNA gene has been determined . A comparison of the rat 18S ribosomal RNA gene sequence with the known sequences of yeast and frog revealed three conserved (stable) regions, two unstable regions, and three large inserts . (A,T) leads to (G,C) changes were more frequent than (G,C) leads to (A,T) changes for three comparisons (yeast leads to frog, frog leads to rat, and yeast leads to rat) . GC pairs were inserted preferentially over AT pairs for the same three comparisons . These two factors contribute to the progressively higher GC content of 18S ribosomal RNA of yeast, frog, and rat.

Biochem Biophys Res Commun, 1983 Jul 18, 114(1), 81 - 7
Partial melting of the segment around pseudouridine in yeast 5S RNA; Nagamatsu K et al.; Pseudouridine in yeast 5S RNA was modified with 4-bromomethyl-7-methoxy-coumarin(BMC) . Temperature dependence of fluorescence intensity was measured at various concentrations of Mg2+ and K+ cations . Hyperchromicity was also measured . At 100mM KCl and 10mM Mg2+, fluorescence intensity decreased with temperature as free BMC except a plateau at 45 degrees C . Withdrawal of Mg2+ from the buffer resulted in a large quenching at 20 degrees C and showed a gradual increase of fluorescence intensity with temperature, indicating a partial melting of the segment around pseudouridine . The temperature range agrees with the low melting temperature shown by hyperchromicity . In 10 mM KCl solution, the effects are more exaggerated.

Biochem Biophys Res Commun, 1983 Jul 18, 114(1), 331 - 8
Characterization of phosphorylase kinase activities in yeast; Pohlig G et al.; Two phosphorylase kinase activities were resolved by DEAE-cellulose chromatography . The main activity peak was enriched 2800-fold, the minor appeared to be an aggregate of the enzyme . Phosphorylase kinase also phosphorylated histone and casein with no changes in phosphorylation ratios throughout the preparation steps but was most active on yeast phosphorylase . The molecular weight was 29000 +/- 2000 . ATP, UTP, GTP served as substrates while CTP was inactive . Mg-ions activated the kinase without inhibition at high concentrations (30 mM) . In addition to this cAMP-independent kinase, cAMP-dependent protein kinase also phosphorylated phosphorylase . The catalytic subunit and phosphorylase kinase were not identical since the latter was not inhibited by yeast cAMP binding protein.

J Am Vet Med Assoc, 1983 Jul 15, 183(2), 212 - 4
Failure of brewer's yeast as a repellent to fleas on dogs; Baker NF et al.; Active and inactive brewer's yeast, when given as a dietary supplement to dogs at the rate of 14 g/day, failed to repel or kill fleas . Twenty dogs in each of 3 groups were inoculated weekly for 7 weeks with 100 unfed cat fleas (Ctenocephalides felis) . One group served as a control while one group received inactive and the other active yeast during the last 5 weeks of the trial . There were no significant differences in flea counts among the 3 groups during the first 4 weeks of yeast supplemental feedings . Total flea counts during the 5th week of yeast supplementation did not differ significantly from the counts on control dogs, although there was a significant difference between counts from dogs receiving active and those receiving inactive yeast.

Arch Biochem Biophys, 1983 Jul 15, 224(2), 579 - 86
Hydroperoxide anion, HO-2, is an affinity reagent for the inactivation of yeast Cu,Zn superoxide dismutase: modification of one histidine per subunit; Blech DM et al.; Yeast Cu,Zn superoxide dismutase is inactivated by H2O2 at alkaline pH, and complete inactivation correlates with the modification of 1.0 histidine per subunit . At elevated concentrations of H2O2, a saturation process is evident and is characterized by kmax, the maximum pseudo-first-order rate constant for inactivation, and Kinact, the total hydrogen peroxide concentration at which the enzyme is half-saturated . In the pH range from 9.0 to 11.5 at 25 degrees C, kmax remains constant at 0.54 +/- 0.03 min-1, but Kinact decreases progressively with increasing pH, from 15.5 mM at pH 9.0 to 1.11 mM at pH 11.5 . It is proposed that the reason for the observed increased affinity with increasing pH is that the reactive species is not H2O2 per se, but rather the HO-2 anion (the pKa for H2O2 is 11.6) . An increase in pH would thus lead to an increased concentration of HO-2 at a fixed total peroxide concentration, and saturation would occur at a lower total peroxide concentration . By analogy with other anions, it is proposed that HO-2 coordinates directly to the Cu ion to form the reactive complex . Once the enzyme-peroxide complex is formed, however, the rate-determining step leading to modification of histidine and loss of activity is independent of pH between 9.0 and 11.5.

Eur J Biochem, 1983 Jul 15, 134(1), 27 - 32
The interaction of mammalian medium-chain hydrolase with yeast fatty acid synthetase; Slabas AR et al.; The interaction of rat mammary gland medium-chain thioesterase with yeast fatty acid synthetase has been investigated . Medium-chain thioesterase interacts with yeast fatty acid synthetase causing premature chain termination of the fatty acids synthesized from acetyl-CoA and malonyl-CoA . This effect is most marked under conditions of rate-limiting malonyl-CoA availability . Immobilized yeast fatty acid synthetase specifically binds rat mammary gland medium-chain thioesterase . This interaction has been used to purify medium-chain thioesterase to near homogeneity from samples of rat mammary gland cytosol . The stoichiometry of binding of medium-chain thioesterase to yeast fatty acid synthetase has been investigated . Yeast fatty acid synthetase binds 5.7 +/- 1 mol medium-chain thioesterase/mol yeast fatty acid synthetase . It is concluded that yeast fatty acid synthetase has a medium-chain thioesterase binding site.

Nucleic Acids Res, 1983 Jul 11, 11(13), 4501 - 20
Nuclear magnetic resonance studies on yeast tRNAPhe . III . Assignments of the iminoproton resonances of the tertiary structure by means of nuclear Overhauser effect experiments at 500 MHz; Heerschap A et al.; Resonances of the water exchangeable iminoprotons of the tertiary structure of yeast tRNAPhe were studied by experiments involving Nuclear Overhauser Effects (NOE's) . Direct NOE evidence is presented for the assignment of all resonances of iminoprotons participating in tertiary basepairing (except that of G19C56 which was assigned by an elimination procedure) . The present results in conjunction with our previous assignment of secondary iminoprotons constitute for the first time a complete spectral assignment of all iminoprotons participating in basepairing in yeast tRNAPhe . In addition we have been able to assign the non(internally) hydrogen bonded N1 proton of psi 55 as well as the N3 proton of this residue, which is one of the two iminoprotons hydrogen bonded to a phosphate group according to X-ray results . No evidence could be obtained for the existence in solution of the other iminoproton-phosphate interaction: that between U33 N3H and P36 located in the anticodon loop . Remarkable is the assignment of a resonance at 12.4 - 12.5 ppm to the iminoproton of the tertiary basepair T54m1A58 . The resonance positions obtained for the iminoprotons of G18 (9.8 ppm) and m2(2)G26 (10.4 ppm) are surprisingly far upfield considering that these protons are involved in hydrogen bonds according to X-ray diffraction results . As far as reported by changes in chemical shifts of iminoproton resonances the main structural event induced by Mg++ ions takes place near the tertiary interactions U8A14 and G22m7G46.

Nucleic Acids Res, 1983 Jul 11, 11(13), 4483 - 99
Nuclear magnetic resonance studies on yeast tRNAPhe . II . Assignment of the iminoproton resonances of the anticodon and T stem by means of nuclear Overhauser effect experiments at 500 MHz; Heerschap A et al.; Resonances of the water exchangeable iminoprotons of the T and anticodon stem of yeast tRNAPhe were assigned by means of Nuclear Overhauser Effects (NOE's) . Together with our previous assignments of iminoproton resonances from the acceptor and D stem (A . Heerschap, C.A.G . Haasnoot and C.W . Hilbers (1982) Nucleic Acids Res . 10, 6981-7000) the present results constitute a complete assignment of all resonances of iminoprotons involved in the secondary structure of yeast tRNAPhe with a reliability and spectral resolution not reached heretofore . Separate identification of the methylprotons in m5C40 and m5C49 was also possible due to specific NOE patterns in the lowfield part of the spectrum . Our experiments indicate that in solution the psi 39 residue in the anticodon stem is orientated in a syn conformation in contrast to the normally observed anti orientation of the uracil base in AU basepairs . Evidence is presented that in solution the acceptor stem is stacked upon the T stem . Furthermore, it turns out that in a similar way the anticodon stem forms a continuous stack with the D stem, but here the m2(2)G26 residue is located between the latter two stems (as is found in the X-ray crystal structure) . The stacking of these stems is not strictly dependent on the presence of magnesium ions . NOE experiments show that these structural features are preserved when proceeding from a buffer with magnesium ions to a buffer without magnesium ions although differences in chemical shifts and NOE intensities indicate changes in the conformation of the tRNA.

J Biol Chem, 1983 Jul 10, 258(13), 7954 - 9
Expression of the "split gene" cob in yeast mtDNA . Nuclear mutations specifically block the excision of different introns from its primary transcript; Pillar T et al.; Five nuclear mutants falling into five different complementation groups are shown to block the maturation of long form mitochondrial cob RNA at five different processing steps . At the same time they prevent complete processing of the oxi 3 RNA, thus exhibiting the same phenotype as mitochondrial box mutants (cyt b- and oxi 3-) . The different nuclear factors in question have varying ranges of specificity for the removal of introns from cob RNA, from only one to at the most three introns . Two mutated nuclear elements are shown to be specific for the processing of introns present only in the long form cob gene . One such mutation shows, as expected, no deleterious effect on the processing of the short form cob RNA exchanged into the mutant via cytoduction . The role of nuclear coded factors in the possible translation or activity of introncoded products ("maturases") is discussed for two mutants . Striking parallels are found between diverse polypeptide products, presumably translated from accumulated cob RNA intermediates, in pet- and mit- mutants blocked in the excision of the same intron.

J Biol Chem, 1983 Jul 10, 258(13), 8175 - 82
The histidine tRNA genes of yeast; del Rey F et al.; Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis . We have sequenced three of the histidine tRNA genes . The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis . None of the three yeast histidine tRNA genes has an intervening sequence . Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene . One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats . The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene . These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.

Biofizika, 1983 Jul-Aug, 28(4), 606 - 11
{The iron-sulfur center N-2 from NADH-dehydrogenase during direct and reverse electron transport in the mitochondria of rat liver and the yeast Endomyces magnusii}; Burbaev DSh et al.; A dramatic decrease of the rate of transport of reducing equivalents from NADH to coenzyme Q was observed in the 4th metabolic state (by Chance) . It was suggested that this decrease is due to the increase of total time of transition from the structural nonequilibrium state to the equilibrium one of the N-2 center . The structural nonequilibrium state of the center N-2 was observed only under the energy-dependent reverse electron transport, when the substrates for the reduction of coenzyme Q were used.

Z Naturforsch {C}, 1983 Jul-Aug, 38(7-8), 621 - 30
Hydrogen bond indices and tertiary structure of yeast tRNAPhe; de Giambiagi MS et al.; The rigidity and stability of the tertiary structure of yeast tRNAPhe is related to a bond index obtained in an IEHT (iterative extended Huckel theory) calculation . The index permits a quantitative estimate of the electron density along the hydrogen bond, having thus an appealing physical meaning . The results indicate that Hoogsteen-type bonds have, as expected, greater electronic population than Watson-Crick type ones . Other non-Watson-Crick pairs, the wobble pair and G15-C48, exhibit high values of the index for the NH...O bond . In the triples, the electron density of the hydrogen bridges does not weaken (compared with the one of the pairs involved) . Contour density maps are shown and dipolar moments of pairs and triples are qualitatively discussed.

Anal Biochem, 1983 Jul 1, 132(1), 225 - 8
Purification of three distinct enolase isoenzymes from yeast; Entian KD et al.; A method for the rapid isolation of yeast enolases, yielding three distinct isoenzymes, has been devised . In the first step anionic proteins were precipitated with polyethyleneimine, whereas hydrophobic enolase isoenzymes remained in the supernatant . Secondly, the supernatant was 45% saturated with ammonium sulfate and bound to phenyl-Sepharose CL-4B . Decreasing ammonium sulfate and simultaneously increasing ethylene glycol concentrations were used for elution . Finally, enolase isoenzymes were separated by chromatofocusing . The purified isoenzymes gave single bands after isoelectric focusing.

J Biochem (Tokyo), 1983 Jul, 94(1), 37 - 41
Physicochemical and immunochemical properties of a thermo-labile antigen (TLA a) from yeast cell surface; Tamai Y et al.; The physicochemical and immunochemical properties of a thermo-labile antigen (TLA a) which is located on the cell surface of Saccharomyces cerevisiae were studied . The sedimentation constant (S20,W) and molecular weight (sedimentation equilibrium method) were 6.26S and 68,800, respectively . The circular dichroic (CD) spectrum of TLA a had negative maxima at 210 and 221 nm, indicating the presence of alpha-structure of a polypeptide chain . The molar ratio of antibody to antigen which gave maximum precipitation was 2.7 . Approximately 50% of the antigenic activity of heat-denatured TLA a was recovered when denatured molecules were dissolved in 6 M guanidine hydrochloride followed by 25-fold dilution with H2O . The amount of TLA a existing on the yeast cell surface was estimated to be 37.5 micrograms per 10.5 mg of fresh cells, corresponding to 0.36% by weight of the fresh yeast.

J Biochem (Tokyo), 1983 Jul, 94(1), 283 - 90
Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria; Yoshida Y et al.; ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA . Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors . The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria . Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form . Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria . However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited . The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone . The inhibition by the uncoupler was not relieved by ATP added externally . It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4330 - 3
NMR study of slowly exchanging imino protons in yeast tRNAasp; Figueroa N et al.; We have monitored the exchange of imino and amino protons by NMR after quick transfer of yeast tRNAAsp in 2H2O solvent . When the concentration of exchange-catalyzing buffer is not too high, one imino proton exchanges considerably more slowly than any other (e.g., 100 hr versus 4 hr for the second-slowest imino proton at 18 degrees C in 15 mM Mg) . This provides excellent conditions for identification, by the nuclear Overhauser effect, of the slowest exchanging proton, which we show to be the imino proton of the U-8 . A-14 reverse Hoogsteen tertiary-structure base pair; other slowly exchanging protons are identified as imino protons from A . U-11 and G . psi-13 . In preliminary experiments, we find that the exchange of these protons is catalyzed by cacodylate or Tris buffer . The lifetimes of two other imino protons, ca . 10 min at 28 degrees C, are buffer independent . Slowly exchanging amino protons have also been observed . Correlation with the exchange of the uracil-8 imino proton suggests that they may be from adenine-14.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4417 - 21
Yeast recombination: the association between double-strand gap repair and crossing-over; Orr-Weaver TL et al.; In previous experiments, we have used yeast transformation to study the recombinogenic repair of double-strand breaks and gaps . A plasmid containing a double-strand gap within sequences homologous to the yeast genome integrates efficiently by crossing-over . During the process of integration, the double-strand gap is repaired, using chromosomal information as a template . This repair reaction results in the transfer of genetic information from one DNA duplex to another and is therefore a pathway for gene conversion . Because meiotic gene conversion is associated with a high frequency (up to 50%) of crossing-over, we wished to determine the degree of association of double-strand gap repair with crossing-over . Only the class of repair events resulting in crossovers (plasmid integration) were detected in our earlier experiments with nonreplicating plasmids . In this paper, we describe the outcome of double-strand gap repair in plasmids that are capable of autonomous replication and therefore allow recovery of both crossover and noncrossover products . After the correct repair of a double-strand gap, we recover approximately equal numbers of integrated and nonintegrated plasmids . Thus, gene conversion by double-strand gap repair can occur either with or without crossing-over, and it is similar in this respect to meiotic gene conversion . Circularization of linear plasmid DNA by ligation is also observed, suggesting that yeast has an additional repair pathway for double-strand breaks that is independent of recombination . Gap repair on replicating plasmids permits rapid cloning of chromosomal alleles.

Biochem Biophys Res Commun, 1983 Jun 29, 113(3), 751 - 6
Effect of tribenzylphosphate on the active phosphate transport and ATP synthesis in yeast mitochondria; Rigoulet M et al.; Tribenzylphosphate (TBP), a specific inhibitor of the high affinity system for Pi transport in yeast mitochondria, inhibits the active Pi transport measured by the energy-linked swelling . The dependence of the rate of oligomycin sensitive ATP synthesis as a function of the external Pi concentration shows two kinetic systems . The high affinity system, corresponds to the range of the external Pi concentration which stimulates the respiratory rate . TBP inhibits both this system and the state 4 leads to state 3 transition.

Biochemistry, 1983 Jun 21, 22(13), 3214 - 9
Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I; LaBonne SG et al.; We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms . We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold . It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase . This yeast protein, SSB-1, has a molecular weight of approximately 40 000 . At apparent saturation there is one protein molecule bound per 40 nucleotides . Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation . It binds to single-stranded RNA as strongly as to DNA . SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme . We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork . Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.

FEBS Lett, 1983 Jun 13, 156(2), 274 - 80
Simultaneous isolation of the yeast cytosol and well-preserved mitochondria with negligible contamination by vacuolar proteinases; Schwencke J et al.; Disruption of yeast spheroplasts by DEAE-dextran in isoosmotic conditions allows isolation of relatively undamaged subcellular fractions from yeast . The preservation of mitochondria and vacuoles permits the simultaneous isolation of the cytosol with negligible contamination by vacuolar proteinases and therefore, virtually eliminates proteolytic artefacts.

Nature, 1983 Jun 9-15, 303(5917), 543 - 6
Pb(II)-catalysed cleavage of the sugar-phosphate backbone of yeast tRNAPhe--implications for lead toxicity and self-splicing RNA; Brown RS et al.; Pb(II) is extremely efficient at depolymerizing RNA and studies on tRNAs have shown that site-specific cleavages in these molecules can be brought about by the action of Pb(II) . We have observed, by difference Fourier analysis, sugar-phosphate strand scission between residues 17 and 18 in crystals of yeast tRNAPhe soaked in dilute Pb(II) solution at pH 7.4 . We have also deduced the structure of the Pb(II)-tRNAPhe derivative at pH 5.0 where this cleavage reaction is considerably slower and report that, in this structure, the sugar-phosphate backbone remains intact . We have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction . From this crystallographic study, and associated biochemical work, we have formulated a possible mechanism for the cleavage reaction and also present here some general ideas on the action of metal ions on nucleic acids.

Biochemistry, 1983 Jun 7, 22(12), 2986 - 95
6-(p-toluidinyl)naphthalene-2-sulfonic acid as a fluorescent probe of yeast hexokinase: conformational states induced by sugar and nucleotide ligands; Ohning GV et al.; The fluorescent dye 6-(p-toluidinyl)naphthalene-2-sulfonic acid (2,6-TNS) has been shown to be a sensitive and nonperturbing probe of conformational states of yeast hexokinase . The binding of sugar ligands to hexokinase induced conformational states of the enzyme which could be distinguished by monitoring 2,6-TNS fluorescence and correlated well with their behavior during the catalytic reaction . The binding of five-carbon sugar inhibitors such as lyxose induced a conformational state of hexokinase that demonstrated a small quenching of 2,6-TNS fluorescence but an increased ability to bind metal-ligands when compared to free enzyme . The binding of good sugar substrates such as glucose produced a conformational state of hexokinase which demonstrated a large enhancement (37%) of bound 2,6-TNS fluorescence . This glucose-induced conformational state had an increased ability to bind metal-ATP ligands; however, the relative changes in the dissociation constants for the various metal-ATP ligands differ from those observed with hexokinase in the presence of lyxose . Hence, the lyxose-induced conformational state of hexokinase was concluded to be significantly different from the glucose-induced conformational state . The binding of poor sugar substrates such as 5-thioglucose induced a conformational state of hexokinase similar to the conformational state induced by glucose, but with a smaller enhancement of 2,6-TNS fluorescence (15%) and a lesser ability to increase the affinity for metal-ATP ligands . The six-carbon inhibitor with a bulky group on the 2-position, N-acetylglucosamine, gave minimal changes in 2,6-TNS fluorescence and effects on metal-nucleotide binding . These conformational states are interpreted in terms of the closure of the cleft between the two domains observed by X-ray crystallography . The binding of A1ATP to free hexokinase was not observed at concentrations up to 100 microM, which is consistent with the kinetic properties reported for this metal-ATP ligand . Although both CrATP and A1ATP have been reported to produce a slow burst-type transient in the progress curve of hexokinase, only CrATP demonstrated slow changes in 2,6-TNS fluorescence, indicating that the conformational state of hexokinase induced by A1ATP is different from the conformational state induced by CrATP.

Agents Actions, 1983 Jun, 13(4), 360 - 3
Non-acidic pyrazoles: inhibition of prostaglandin production, carrageenan oedema and yeast fever; Brune K et al.; Prostaglandin production from mouse peritoneal macrophages was elicited by the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) . The inhibitory potency (IC50) of metamizole and its major metabolites as well as other non-acidic pyrazoles was defined in this system . A reliable IC50-value could not be assigned to metamizole . Isopropylaminophenazone was as active as acetylsalicylic acid while aminophenazone and methylaminophenazone, the major metabolites of metamizole, were about 10 times and phenazone 100 times less potent than acetylsalicylic acid . The major excretion products of metamizole, 4-formyl- and 4-acetyl-aminophenazone were inactive . The IC50-values obtained agree with those necessary for manifestation of anti-inflammatory effects in rats but are up to 10 times higher than those measurable in human plasma after administration of analgesic-antipyretic doses.

Biochimie, 1983 Jun, 65(6), 355 - 60
Mitochondrial phenylalanyl t-RNA synthetase from yeast: formation of enzyme-substrate complexes shown by heat or SH reagent inactivation; Diatewa M et al.; The binding of substrates to purified mitochondrial phenylalanyl-tRNA synthetase from yeast was examined using the kinetics of heat or p-hydroxymercurybenzoate inactivation . Individually magnesium chloride and each of the substrates protect the enzyme against thermal denaturation and p-hydroxymercurybenzoate inhibition . No enzyme protection is observed with ATP alone against p-hydroxymercurybenzoate inhibition . The combinations of the various substrates induce a synergistic protection effect . Protection constants of 31 microM and 0.3 microM were found for L-Phe and mt tRNAPhe respectively, from heat inactivation studies . The inhibition of the enzyme activity by p-hydroxymercurybenzoate can be reverted by 2-mercaptoethanol or dithiothreitol.

J Inorg Biochem, 1983 Jun, 18(3), 195 - 211
Separation of biologically active chromium-containing complexes from yeast extracts and other sources of glucose tolerance factor (GTF) activity; Haylock SJ et al.; A procedure has been developed, based on ion-exchange chromatography, that readily allows the separation of eleven apparently homogeneous chromium-containing fractions from a brewer's yeast extract . Four of the fractions are amphoteric and show no glucose tolerance factor (GTF) activity, three are classified as negative (two of which are biologically inactive, while the third one shows a slight degree of GTF activity), whereas the four cationic chromium-containing fractions all show varying degrees of GTF activity . Application of the separation procedure to other biological sources of GTF activity resulted in a spectrum of cationic fractions, over the pH range 1.75 to 12, which suggests that GTF cannot be a single species . The cationic chromium-containing fraction from pork kidney powder and fraction P-3 from yeast appear to contain the most GTF-active material and P-3 shows saturation kinetics as expected for a biologically significant substance.

Arch Microbiol, 1983 Jun, 134(3), 193 - 203
Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes; Veenhuis M et al.; Inactivation of peroxisomal enzymes in the yeast Hansenula polymorpha was studied following transfer of cells into cultivation media in which their activity was no longer required for growth . After transfer of methanol-grown cells into media containing glucose - a substrate that fully represses alcohol oxidase synthesis - the rapid inactivation of alcohol oxidase and catalase was paralleled by a disappearance of alcohol oxidase and catalase protein . The rate and extent of this inactivation was dependent upon conditions of cultivation of cells prior to their transfer . This carbon catabolite inactivation of alcohol oxidase was paralleled by degradation of peroxisomes which occurred by means of an autophagic process that was initiated by the formation of a number of electron-dense membranes around the organelles to be degraded . Sequestration was confined to peroxisomes; other cell-components such as ribosomes were absent in the sequestered cell compartment . Also, cytochemically, hydrolytic enzymes could not be demonstrated in these autophagosomes . The vacuole played a major role in the subsequent peroxisomal breakdown since it provided the enzymes required for proteolysis . Two basically similar mechanisms were observed with respect to the administration of vacuolar enzymes into the sequestered cell compartment . The first mechanism involved incorporation of a small vacuolar vesicle into the sequestered cell compartment . The delimiting membrane of this vacuolar vesicle subsequently disrupted, thereby exposing the contents of the sequestered cell compartment to vacuolar hydrolases which then degraded the peroxisomal proteins . The second mechanism, observed in cells which already contained one or more autophagic vacuoles, included fusion of the delimiting membranes of an autophagosome with the membrane surrounding an autophagic vacuole which led to migration of the peroxisome inside the latter organelle . Peroxisomes of methanol-grown H . polymorpha were degraded individually . In one cell 2 or 3 peroxisomes might be subject to degradation at the same time, but they were never observed together in one autophagosome . However, fusions of autophagic vacuoles in one cell were frequently observed . After inhibition of the cell's energy-metabolism by cyanide ions or during anaerobic incubations the formation of autophagosomes was prevented and degradation was not observed.

Genetika, 1983 Jun, 19(6), 912 - 20
{Radiosensitivity of the nucleus and mitochondrion of the yeast cell: a study by cytoduction}; Stepanova VP et al.; We have crossed irradiated and unirradiated genetically marked haploid yeast cells and studied the influence of gamma-irradiation on the yield of nuclear-cytoplasmic haploid hybrids (cytoductants) . In the first cross, the cytoductants possessed the irradiated cytoplasm (mitochondrions) and unirradiated nuclei, in the second cross they possessed the irradiated nuclei and unirradiated mitochondria . After irradiation of nuclei, the yield of cytoductants dropped sharply in relation to doses of radiation . In contrast to this, no mitochondrion damage was observed after doses from 0 to 2000 Gy . A small number of cytoductants appeared after 8160 Gy applied to mitochondrion . We may suggest that the nuclear damage is the only cause of the cell inactivation after irradiation using doses up to 2000 Gy.

Arch Biochem Biophys, 1983 Jun, 223(2), 543 - 55
Factors affecting the oligomeric structure of yeast external invertase; Chu FK et al.; It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain . We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da . During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer . All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio . Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains . When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active . Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active . Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer . The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme . From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3292 - 6
Consistent association between sigma elements and tRNA genes in yeast; Brodeur GM et al.; Sigma is a recently described family of transposable elements in yeast (Saccharomyces cerevisiae) . The most striking feature of the seven sigma elements that have been previously identified is that all are located 16-18 base pairs upstream from tRNA-encoding regions . Because these cases were all encountered in the process of studying specific tRNA genes, the full extent of the association between sigma elements and tRNA genes could not be assessed . In this paper, we report a more global characterization of the sigma family in a typical laboratory yeast strain: of the 30 copies of sigma that we estimate to be present in the haploid genome, we have cloned and analyzed 25 loci . Although in two cases a pair of sigma elements were found within several kilobases of each other, the majority occur as individual elements at widely dispersed sites . Moreover, in all 25 cases analyzed, the sigma elements are closely associated with tRNA genes . Thus, the sigma transposable element has been shown to have an absolute association with another gene family.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3401 - 5
Deletions and single base pair changes in the yeast mating type locus that prevent homothallic mating type conversions; Weiffenbach B et al.; Several cis-acting mutations that prevent homothallic mating type conversions in Saccharomyces cerevisiae have been examined . Deletions within the mating type (MAT) locus were obtained by selecting for survivors among homothallic MAT alpha cells carrying the rad52 mutation . The survivors were unable to switch mating type, even in RAD+ derivatives . The deletions varied in size from fewer than 50 to more than 750 base pairs . All of the deletions removed a Hha I site at the border between the alpha-specific sequences (Y alpha) and the adjacent Z region . We also examined several spontaneous inc mutations that prevent MAT switching . Two of these mutations were cloned in recombinant DNA plasmids and their sequences were determined . The MAT alpha-inc 3-7 mutation proved to have an altered Hha I site at the Y alpha/Z border, by virtue of a single base pair substitution G . C leads to A . T in the second base pair of the Z region (Z2) . Restriction fragment analysis showed that two other independently isolated strains with MAT alpha-inc mutations had altered the same Hha I site . The MAT a-inc 4-28 mutation contains a single base pair substitution C . G leads to T . A at position Z6 . A base pair difference at position Z11 in two MATa strains does not affect MATa conversions . We conclude that the region near the Y/Z border is essential for the efficient switching of MAT alleles and constitutes an enzyme recognition site for a specific nucleolytic cleavage of MAT DNA.

Sabouraudia, 1983 Jun, 21(2), 167 - 70
Electron cytochemical demonstration of the capsule of yeast-like Sporothrix schenckii; Garrison RG et al.; Staining of yeast-like cells of Sporothrix schenckii by the cationic dye Alcian blue 8GX and lanthanum nitrate permitted the demonstration in thin sections of an electron opaque material localized at and along the outermost wall surface . This material was associated with the characteristic microfibrillar layer of the external cell wall . It was absent or largely removed when cells were grown in shaken liquid culture or after washing of cells from static cultures . It is believed that this loosely-bound material represents a capsular substance comprised, at least in part, of a mucopolysaccharide or a mucopolysaccharide-protein complex.

Antonie Van Leeuwenhoek, 1983 Jun, 49(2), 183 - 90
Extracellular glucose-producing exodextranase of the yeast Lipomyces lipofer; Ramos A et al.; A dextran-hydrolysing enzyme from Lipomyces lipofer IGC 4042 was purified from the supernatant of cultures grown on a mineral medium with dextran, by ultrafiltration and gel filtration on Bio Gel A-0.5 m . This preparation gave only one band by disc gel electrophoresis . Glucose was the only product of dextran hydrolysis . Optimum pH and temperature for the activity of the enzyme were pH 4.5-5.0 and 45 degrees C, respectively . The enzyme was most stable over a pH range of 4.5-6.0, and after 2 hours at 50 degrees C maintained over 60% of its original activity . The molecular weight was 29,000 daltons and the isoelectric point was at pH 7 . Km (45 degrees C, pH 5) for dextran T-40 was 1.2 X 10(-5) M . Glucose inhibited the enzyme competitively with a Ki (45 degrees C, pH 5) of 0.5 mM.

FEBS Lett, 1983 May 30, 156(1), 11 - 4
In vivo glucose activation of the yeast plasma membrane ATPase; Serrano R; The addition of glucose to yeast cells activates proton efflux mediated by the plasma membrane ATPase . Accordingly, the ATPase activity of purified plasma membranes is increased up to 10-fold . The activated ATPase has a more alkaline pH optimum, better affinity for ATP and greater sensitivity to vanadate than the non-activated enzyme . All these changes are reversed by washing the cells free of glucose . This suggests two states of the ATPase which are interconverted by a covalent modification . As glucose does not affect the phosphorylation of plasma membrane polypeptides, other type of covalent modification may be involved.

Nucleic Acids Res, 1983 May 25, 11(10), 3363 - 73
Melting of local ordered structures in yeast 5S ribosomal RNA in aqueous salts; Ohta S et al.; Equilibrium and kinetics of thermal melting of yeast 5S ribosomal RNA in aqueous NaCl with or without Mg2+ were investigated by differential thermal melting and temperature jump methods . Two peaks (1 and 2) and a shoulder were observed in each of melting curves at ionic strength I=0.002-0.5 and linearity between each of melting temperatures T1m and T2m and log I was found at I=0.01-0.5 in the Mg2+-free solution . The local structures were found to be stabilized considerably by Mg2+ . The temperature jump measurements gave the kinetic melting curve of the structure 1 at I=0.03 without Mg2+ or with 0.5 mM Mg2+ . The kinetic Tm coincided well with the corresponding static Tm . For the structure 1, various parameters were calculated from the kinetic data, which indicated a double helical character of the structure 1 . In terms of the values of Tm, G-C content, and enthalpy change of the transition of the structure 1 or 2, appropriateness of each of the secondary structure models of eukaryotic 5S RNA proposed previously was discussed.

Nucleic Acids Res, 1983 May 25, 11(10), 3269 - 82
Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast; Sellami M et al.; We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes . Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs . The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product) . The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors . As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs.

Nucleic Acids Res, 1983 May 25, 11(10), 3123 - 35
Cycloheximide resistance in yeast: the gene and its protein; Kaufer NF et al.; Mutations in the yeast gene CYH2 can lead to resistance to cycloheximide, an inhibitor of eukaryotic protein synthesis . The gene product of CYH2 is ribosomal protein L29, a component of the 60S ribosomal subunit . We have cloned the wild-type and resistance alleles of CYH2 and determined their nucleotide sequence . Transcription of CYH2 appears to initiate and terminate at multiple sites, as judged by S1 nuclease analysis . The gene is transcribed into an RNA molecule of about 1082 nucleotides, containing an intervening sequence of 510 nucleotides . The splice junction of the intron resides within a codon near the 5' end of the gene . In confirmation of peptide analysis by Stocklein et al . (1) we find that resistance to cycloheximide is due to a transversion mutation resulting in the replacement of a glutamine by glutamic acid in position 37 of L29.

J Biol Chem, 1983 May 25, 258(10), 5998 - 9
Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro; Gancedo JM et al.; Fructose-1,6-bisphosphatase purified from Saccharomyces cerevisiae is phosphorylated in vitro by a cAMP-dependent protein kinase . The phosphorylation reaction incorporates 1 mol of phosphate/mol of enzyme and is greatly stimulated by fructose 2,6-bisphosphate . Fructose 2,6-bisphosphate acts upon fructose-1,6-bisphosphatase, not on the protein kinase . The phosphorylation of fructose 1,6-bisphosphatase lowers its activity by about 50% . The characteristics of the phosphorylation reaction in vitro show that this modification is responsible for the inactivation of fructose-1,6-bisphosphatase observed in vivo.

Mycopathologia, 1983 May 22, 82(2), 83 - 8
Effects of pH, temperature, aeration and carbon source on the development of the mycelial or yeast forms of Sporothrix schenckii from conidia; Rodriguez-Del Valle N et al.; Culture conditions for the exclusive development of the mycelial and the yeast forms of Sporothrix schenckii from conidia in a rich, defined medium were established . Only the mycelial morphology developed when the pH of the medium was adjusted between 4.0 and 5.0 and the conidia were incubated at 25 degrees C, regardless of aeration . When the pH of the medium was adjusted between 6.5 and 8.0 and the conidia were incubated with aeration at 35 degrees C, only the yeast form was obtained . Using these culture conditions conidia were inoculated in a buffered-salts medium with vitamins with or without added carbohydrates as carbon sources . After incubation, the form obtained was recorded and the amount of growth determined on a dry weight basis . Development of the mycelial form was observed with all of the carbon sources tested (glucose, fructose, mannose, arabinose, maltose, sucrose and starch) while the yeast form developed only when glucose or another hexose was added to the medium . These observations indicated that in S . schenckii the development of conidia into a specific form is dependent on the interrelationship between the available nutrients and the culture conditions and that no specific parameter seemed to be exclusively determinant of the morphology obtained.

Eur J Biochem, 1983 May 16, 132(3), 629 - 37
Study of the interaction of yeast arginyl-tRNA synthetase with yeast tRNAArg2 and tRNAArg3 by partial digestions with cobra venom ribonuclease; Gangloff J et al.; Yeast tRNAArg2 and tRNAArg3 are two isoacceptors which show similar V and Km for yeast arginyl-tRNA synthetase despite important differences in their primary structures . Fragments resulting from the partial digestion of 3' or 5' end-labelled tRNAArg2 and tRNAArg3 in the presence or absence of arginyl-tRNA synthetase by cobra venom ribonuclease, an enzyme which cuts preferentially in double-stranded regions, were analysed by electrophoresis on polyacrylamide gels . In the absence of arginyl-tRNA synthetase, major cuts were observed in tRNAArg2 and tRNAArg3 at the end of the 3' part of the acceptor stem and in the 5' part of the anticodon stem, whereas the 5' part of the acceptor stem and the 3' part of the anticodon stem are only slightly cleaved . The D and the T stems are almost fully resistant to cobra venom ribonuclease attack confirming the strong tertiary structural organization of this region . In the presence of arginyl-tRNA synthetase the two or three last sites of the 3' halves of the acceptor stems and the sites in the 3' halves of the anticodon stems are almost completely protected against ribonuclease hydrolysis in both tRNAs; 31-69% protection of the sites located in the 5' halves of the anticodon stem is also observed . However, the cleavage levels are enhanced for the three head positions in the 3' halves of the acceptor stems and a new cut appears at the first position of this region in the case of tRNAArg3 . The similarity of the protection patterns of tRNAArg2 and tRNAArg3 suggests that both molecules interact in nearly the same manner with arginyl-tRNA synthetase, which in turn implies great similarities in their tertiary structure when involved in the complex . If this tertiary organization is like that described for tRNAPhe, all protected sites are located in the inside of its L-shaped model.

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