Cycloheximide-resistant Mutants Research Articles

Powder Formulation of Antagonists Yeasts, Cryptococcus albidus and Cryptococcus terreus as Biofungicides

The research has following objectives: 1) to investigate compatibility of yeasts antagonist C. albidus and C. terreus, 2) to obtain suitable carrier  in powder formulation for those two yeasts, 3) to get appropriate formulation additives for those yeasts,  and 4) to obtain optimum powder formulation consisting yeasts, carrier materials and additives. Compatibility of C. albidus and  C. terreus  was tested through bio-assay against Lasiodiplodia theobromae on detached banana fruit and Alternaria solani on detached tomato leaves. Compatibility was indicated by no reduction of antagonistic activity.  Benomyl-resistant mutant of C. albidus  and cycloheximide-resistant mutant of  C. terreus were mixed with sterilized tapioca, talc and kaolin  to get initial yeasts density of  8.5 log cfu/g and water content of 15%,  then  packed by plastic bag and stored under room temperature. Survival of formulated yeasts was assessed monthly by plating on PDA medium containing 150 ppm cycloheximide for C. terreus, and 150 ppm benomyl for C. albidus. Yeasts population was expressed in log cfu/g materials.  To determine effect of carrier materials on antagonistic activity, bio-assay of formulated yeasts against pathogens was conducted after 3 months storage. Tested additives i.e. CaCl2, pure chitin and crab shell powder were added into suspension of C. albidus and C. terreus to get concentration of 1.25%, 0.5 % and 0. 1% (w/v). Sterilized aquadest and yeasts without additives was used as control. Then  the treatments was examined the antagonistic activity through  bio-asaay  on detached banana fruits and tomato leaves. Appropriate additive(s) was determined by its ability to increase antagonistic activity of yeasts. Storability and antagonistic activity of  C. albidus dan C. terreus in the mixture of best carrier and additive were examined. C. terreus was compatible to C. albidus. The best carrier materials supporting highest survival of C. terreus was tapioca and talc, those supported survival for four months storage. Talc was best carrier material for C. albidus formulation by maintaining its survival for five months storage. Additives which able to increase antagonistic activity of C. terreus were CaCl2 0.1 % , pure chitin  0.1 %  and crab shell powder  0.5%.  Additives providing best increase of antagonistic activity of C. albidus were CaCl2 0.5%, pure chitin  1.25 %  and crab shell 1.25%. Supplementation of chitin and crab shell, both at the rate of 1.25% into talc-base powder formulation increased survival of both yeasts at one months storage, however no significant effect subsequently.Key words: formulation, yeasts antagonists, Cryptococcus terreus, Cryptococcus albidus, carrier agent, talc, tapioca, kaolin, CaCl2, chitin, crab shell powder

酸味に特徴を有する酵母の選抜と試験醸造

Three different types of yeasts that can produce much malic acid were isolated in order to brew sake with a good sour taste. (1)The yeasts were strain A isolated from cycloheximide-resistant mutants of Kyokai yeast(K1401), strain B from 85kinds of sake mash in Ishikawa Prefecture, and strain C from cell fusants between K-901 and wine yeast(OC-2). (2)In a sake brewing test with a total of 500g of rice, three kinds of sake brewed with three selected yeasts respectively contained 1.5-2times more malic acid compared with one brewed with a control yeast, K-9. (3)In a sake brewing test scaled up using 3kg of rice, the high malic acid productivity of these three yeasts exhibited similar results to those described above without any decrease in ethanol productivity. Strains B and C gave sake a good sour, deep taste and a better evaluation by means of changing the water ratio of 130% to 120% in the mash. (4)Strain A was able to brew sake having a plus value in the sake meter, while strains B and C were able to brew sake having a minus value in this.

Kinetics of improved productivity of beta-galactosidase by a cycloheximide-resistant mutant of Kluyveromyces marxianus.

The maximum volumetric productivity of beta-galactosidase by a Kluyveromyces marxianus mutant, grown on lactose/corn steep liquor medium for 3 d, was 150 IU l(-1) h(-1) which is twice that of the parent organism. During product formation, mutated cells provided more resistance against thermal inactivation.

シクロヘキシミド耐性清酒酵母のリンゴ酸生成に関する遺伝子の発現解析及び実地醸造試験

シクロヘキシミド耐性清酒酵母のリンゴ酸生成に関する遺伝子の発現解析及び実地醸造試験

シクロヘキシミド耐性清酒酵母のリンゴ酸生成機構

A mutant that is a high malic acid producer (M7-14) was isolated from among cycloheximideresistant mutants of Kyokai 7 yeast (K-7).In a small scale sake brewing test using this strain, the sake produced had a sweet taste with a low alcohol concentration;sake meter -36 and 12% of ethanol.The production of malic acid by M7-14 was 2.8 times that of K-7.When sodium azide, which inhibits the tricarboxylic acid (TCA) metabolic cycle, was added to the M7-14 culture, malic acid production fell to half that of the same culture without sodium azide.From this result, it was proposed that a pathway other than the TCA cycle might be involved in malic acid production in M7-14.The mechanism of malic acid production by M7-14 was investigated by measurement of the enzyme activities in the TCA cycle and glyoxylic acid cycle.It was found that the activities of malate dehydrogenase and isocitrate lyase in M7-14 were higher than those of K-7.Thus, it is clear that the glyoxylic acid cycle is responsible for the high production of malic acid and succinic acid by M7-14.

Requirement for cytoplasmic protein synthesis during circadian peaks of transcription of chloroplast-encoded genes in Chlamydomonas.

Cycloheximide, an inhibitor of cytoplasmic translation, induced a rapid reduction of 70-80% in levels of mRNA for the chloroplast elongation factor Tu (tufA) in asynchronously growing Chlamydomonas. This effect was shown to be mainly transcriptional, and not restricted to tufA, as transcription of other chloroplast-encoded genes were cycloheximide-sensitive, although not all equally (psbA showed no more than 40% inhibition). Confirmatory evidence that the inhibition of chloroplast transcription was mainly due to blocking cytoplasmic translation was obtained with the cycloheximide-resistant mutant act1, and by using another translation inhibitor, anisomycin. In synchronously growing Chlamydomonas, chloroplast transcription is regulated by the circadian clock, with the daily peak occurring during the early light period. When cycloheximide was added during this period, transcription was inhibited, but not when it was added during the trough period (late light to early dark). Moreover, in synchronized cells switched to continuous light, the drug blocked the scheduled increase in tufA mRNA, but did not remove the pre-existing mRNA. These experiments define two functionally different types of chloroplast transcription in Chlamydomonas, basal (cycloheximide-insensitive) and clock-induced (cycloheximide-sensitive), and indicate that the relative contribution of each type to the overall transcription of a given gene are not identical for all genes. The results also provide evidence for nuclear regulation of chloroplast transcription, thereby obviating the need for an organellar clock, at least for these rhythms.

Cycloheximide耐性株から得られたリンゴ酸高生産性酵母

Several sake yeast strains with high malic acidproductivity were isolated from cycloheximideresistant mutants obtained by mutation of a haploid of sake yeast K-1001 with ethyl methanesulfonate. These strains had malic acid-producing ability six to nine times as high as the parent strain. In the sake brewed with yeast strains, the ratio of malic acid to total organic acid was 60 to 70%.

Endocytosis in Dictyostelium discoideum amoebae: Inhibition by cyclohexlmide and other inhibitors of protein synthesis

ABSTRACT Cycloheximide, an inhibitor of protein synthesis, inhibited endocytosis pathways in the vegetative amoebae of the cellular slime mold Dictyostelium discoideum. Half-maximal inhibition of fluid-phase pinocytosis in nutrient medium or in buffer occurred at 0.08 mM or 0.35 mM cycloheximide, respectively. Other inhibitors of eucaryotic protein synthesis inhibited fluid-phase pinocytosis, with the following IC50 in nutrient medium: 0.15 mM anisomycin, ImM emetine, 0.25 mM puromycin. Kinetic studies showed that the inhibitory effects of cycloheximide occurred rapidly, with a half-time of less than 10 min, and were fully reversible upon removal of the drug. Both fluid-phase pinocytic influx and efflux were inhibited by cycloheximide, which furthermore prevented the vanadate-induced efflux of fluorescein-labeled dextran, the fluid-phase marker used in these experiments. The massive secretion of lysosomal enzymes: acid phosphatase, N-acetylhexosaminidase or α-mannosidase, which follows overloading of the endosomal compartment by sucrose, was also inhibited by cycloheximide. All these phenomena were unimpaired by cycloheximide in the cycloheximide-resistant (cycAl) mutant strain X6.

The cytoplasmic ribosomes of Chlamydomonas reinhardtii: characterization of antibiotic sensitivity and cycloheximide-resistant mutants.

In vitro protein synthesis was used to characterize the antibiotic sensitivity of cytoplasmic ribosomes from wild-type and antibiotic-resistant strains of Chlamydomonas reinhardtii. Cytoplasmic ribosomes from two cycloheximide-resistant mutants, act-1 and act-2, were resistant to the antibiotic in vitro. The alteration effected by the act-1 mutation, which was dominant in diploids, was localized to the large subunit of the cytoplasmic ribosomes, but no ribosomal protein alterations were detected using two-dimensional gel electrophoresis. The act-2 mutation, which was semidominant in diploids, was frequently associated with a charge alteration in the large subunit ribosomal protein (r-protein) cyL38 that segregated independently from the antibiotic-resistant phenotype in crosses.

Pleiotropic changes in cycloheximide-resistant insect cell clones.

Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. From Aedes albopictus (mosquito) cells, cycloheximide-resistant mutants (Cx-705 and Cx-738) that were about 30-fold more resistant to cycloheximide than the parental cells have been obtained. The observation that protein synthesis in cell-free lysates from Cx-705 and Cx-738 cells was resistant to cycloheximide led us to suspect that the alteration in these mutants might affect the ribosome. The present studies show that the cycloheximide-resistant cells grow poorly and eventually die at 34.5 degrees C, a temperature at which wild-type cells grow normally. Relative to control cells, the cycloheximide-resistant cells show increased sensitivity to G-418, another antibiotic that interacts with the eukaryotic ribosome. However, there were no differences between cycloheximide-resistant cells and wild-type cells in sensitivity to puromycin, emetine, or cryptopleurine. Cx-705 cells were predominantly diploid; in contrast, the frequency of tetraploid nuclei in Cx-738 cells was about 40%.

Isolation and characterization of a mutant having an altered ribosomal protein in the yeast, Saccharomyces cerevisiae.

A cycloheximide-resistant mutant which also displays coreversion of leu2-1 UAA and met8-1 UAG mutations was isolated and characterized in the yeast, S. cerevisiae. One of the ribosomal protein (L23) differed electrophoretically in the mutant strain from that of the parental one. Diploid cells heterozygous for the mutation contained both altered and normal proteins and gave rise to tetrads of 2:2 segregation for the protein alteration, indicating that a single structural gene coding for L23 protein was mutated. In the meiotic progenies, however, alteration of the L23 protein did not cosegregate with any phenotypes of cycloheximide-resistance or reversion of the nonsense mutations. Trisomic analysis was performed to determine the genetic locus for the L23 protein, suggesting that the gene may not be on the chromosomes I through XVI examined.

THE EFFECTS OF INHIBITORS AFFECTING PROTEIN SYNTHESIS AND MEMBRANE ACTIVITY ON THECHLAMYDOMONAS REINHARDHIIPHOTOTACTIC RHYTHM

Cycloheximide and puromycin slowed the biological timing of the phototactic rhythm of Chlamydomonas reinhardii. However, actinomycin D, rifampicin, chloramphenicol, streptomycin, chloral hydrate, valinomycin, LiCl, ethanol, methanol, isopropanol, procaine, tetracaine, dibucaine, and sodium lauryl sulfate did not alter the period length.A cycloheximide resistant mutant (cyr-l) was isolated. The mutation is recessive since diploids produced from cyr-l and wild type are sensitive to cycloheximide. The phototactic rhythm of cyr-l, unlike that of the wild type, is not influenced by cycloheximide. This supports the view that the action of this drug in slowing the rhythm in the wild type is mediated by the inhibition of protein synthesis.

Genetic Evidence That Protein Synthesis Is Required for the Circadian Clock of Neurospora

Small doses of cycloheximide given at intervals (pulses) cause phase shifts of the circadian clock of Neurospora. The effects of this drug on the clock are mediated through its inhibition of protein synthesis, since two cycloheximide-resistant mutants whose 80S ribosomes are resistant to cycloheximide showed no phase shift after exposure to the durg.

Altered ribosomal protein L29 in a cycloheximide-resistant strain of Saccharomyces cerevisiae

A spontaneous high-level cycloheximide-resistant mutant of the yeast Saccharomyces cerevisiae (strain cy32) is found to have an altered protein of the large subunit (60S) of cytoplasmic ribosomes, namely protein L29. The resistance character segregates together with this biochemical defect and is semidominant in heterozygous diploids. Judged from in vitro susceptibility to inhibition by cycloheximide there are at least 50% resistant ribosomes present in such diploid strains. From these results it is concluded that cycloheximide resistance of mutant cy32 is caused by mutation of a single gene and that it is the structural gene for L29 which is affected.Preliminary genetic mapping data are also reported. They indicate a location of cyhx-32 marker on chromosome 7 near met13.

Isolation and characterization of a cycloheximide-resistant mutant of Acanthamoeba castellanii Neff

A cycloheximide-resistant mutant was isolated from the amoeba Acanthamoeba castellanii Neff. Drug resistance was found to be due to a ribosomal modification.

Cycloheximide resistance can be mediated through either ribosomal subunit.

Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug. The homozygous double mutant was more resistant than either single mutant. Cell-free extracts of wild type and of the three mutant strains, assayed for protein synthetic activity by both runoff of natural mRNA and poly(U)-dependent phenylalanine polymerization, demonstrated that in vitro the mutants were all more resistant than the wild type. Further fractionation of the cell-free systems into ribosomes and supernates localized cycloheximide resistance to the ribosome for both Chx-A and Chx-B homozygotes. Ribosome dissociation and pairwise subunit mixing in the in vitro system indicated that ribosome resistance was conferred by the 60S subunit from one strain whereas resistance in the other strain was mediated through the 40S subunit. This was further confirmed by reconstruction of all four cycloheximide-resistance "phenotypes" by mixing ribosomal subunits from appropriate strains. This finding suggests that the mechanisms by which these mutations confer resistance to cycloheximide are different.

Genetic and biochemical analysis of cycloheximide resistance in the fungus Podospora anserina.

Genetic analysis of cycloheximide-resistant mutants has shown that at least three genes control the resistance to cycloheximide in Podospora anserina and that the antibiotic resistance is recessive to sensitivity. In vitro and in vivo studies of protein synthesis indicated that for two mutants cycloheximide resistance is associated with the ribosomes. For one of these mutants, the elongation step in protein biosynthesis is insensitive to cycloheximide over a wide range of concentration. In this mutant the resistance to cycloheximide is a property of the 60S subunit.

Ribosomal proteins in the fungus Podospora anserina: evidence for an electrophoretically altered 60S protein in a cycloheximide resistant mutant.

Proteins of cytoplasmic ribosomes of the Podospora anserina were analyzed by two dimensional gel electrophoresis. The numbers of proteins were estimated to be 28 in the small subunit and 41 in the large subunit. The L21 protein of the large subunit was found to migrate differently in a cycloheximide resistant mutant.

Genetic studies on cycloheximide-resistant strains of Schizosaccharomyces pombe.

Cycloheximide-resistant mutants of Schizosaccharomyces pombe were isolated either as spontaneous mutants or after mutagenic treatment with nitrous acid, UV and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Twenty-three spontaneous mutants and 64 induced mutants were analysed genetically. Crosses revealed that at least four loci, designated cyh1, cyh2, cyh3 and cyh4 are responsible for resistance. Alleles of cyh1 show good growth on either high (100 mug/ml) or low (40 mug/ml) concentrations of cycloheximide whereas alleles at the cyh2, cyh3 and cyh4 loci gorw well on 40 mug/ml but poorly on 100 mug/ml. Some alleles at the cyh2 and cyh3 loci are also temperature sensitive (ts), the ts phenotype being conferred by the same gene as the resistance. In diploids, cyh1 and cyh4 are re-essive to wild type whereas cyh2 and cyh3 are semi-dominant. There was no intragenic complementation between three cyh1 alleles. Cross-resistance to trichodermin and anisomycin was shown by cyh2, cyh3 and cyh4 but not cyh1. Most cyh1 alleles, of spontaneous and UV origin only, were cold sensitive (cs) at 14 degrees and some of these were also cycloheximide dependent at the same temperature. It is suggested that the cyh1 and cyh4 genes are involved in ribosome formation or function and the other loci probably affect the uptake of cycloheximide by the cells.

Macronuclear genetics of Tetrahymena. II. Macronuclear location of somatic mutations to cycloheximide resistance.

Somatic cycloheximide-resistant mutants of syngen 1 of Tetrahymena pyriformis were isolated and genetically characterized. Two properties of the mutants were independently examined: (a) The transmission of the mutant phenotype during conjugation and (b) the kinetics of phenotypic assortment during vegetative propagation. The results of both studies strongly support the idea that these somatic mutations have a macronuclear location. The kinetics of assortment are consistent with the idea that the syngen 1 macronucleus contains about 45 assorting genetic units. The sib-selection method employed here, used in conjunction with the analysis of assortment kinetics and a previously described test for randomness of distribution, provides a probe of macronuclear genetics applicable to many ciliates, including those in which conjugation is not known to occur or is not under experimental control.