‘Red pigment’ from ADE‐2 mutants of <i>S. cerevisiae</i> prevents DNA cleavage by restriction endonucleases

Meškauskas, Arturas; Ksenzenko, V. N.; Shlyapnikov, M. G.; Kryukov, V. A.; Čitavičius, Donaldas

doi:10.1016/0014-5793(85)80344-6

Cited by 5 publications

(3 citation statements)

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“…Of the purines, adenine is particularly critical for cell viability, maintenance, and adaptation to environmental changes due to its direct structural role in the nucleotide moieties of DNA, RNA, and high‐energy intermediates and to its participation in stress‐induced regulatory pathways. Mutations in the purine biosynthetic pathway in yeast at the ADE1 and ADE2 loci have been studied (Crowley and Kaback, 1984; Meskauskas et al, 1985; Myasnikov et al, 1991). These mutations are used to examine purine metabolism and turnover in yeast and are general nutritional markers for recombinant strains.…”

Section: Introductionmentioning

confidence: 99%

Adenine Quantitation in Yeast Extracts and Fermentation Media and Its Relationship to Protein Expression and Cell Growth in Adenine Auxotrophs of Saccharomyces cerevisiae

et al. 1997

Biotechnol. Prog.

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We have described a method to reliably measure the free adenine content of yeast extract powders or the adenine concentrations found in chemically-defined and complex fermentation samples. This method relies on the selective precolumn derivatization of adenine with chloroacetaldehyde to form the fluorescent adenine adduct 1,N6-ethenoadenine. The derivatized adenine can then be resolved from other components found in samples with reverse phase HPLC and selectively monitored with fluorescence. This method was then used to study the adenine nutritional requirements of adenine auxotrophs of recombinant Saccharomyces cerevisiae. The adenine content of individual yeast extract powders was examined in relation to the cell mass (dry cell weight, DCW) achieved in culture media formulated with these powders. A general increase in DCW was observed with increasing adenine concentration in the yeast extract. Conversely, we observed that as adenine concentration increased in complex media the expression levels of a heterologous protein decreased. This method also allowed us to examine the adenine/DCW ratio in both steady-state continuous culture and batch culture. In both cases, the total in vivo adenine content as measured by the amount of adenine utilized from the culture media was estimated to be ca. 25-40 mg/g DCW. However, data suggest that this value is in excess of what is strictly required for cell growth and represents the quantity of adenine required to saturate intracellular pools of adenine or adenine metabolites. A minimum requirement for cell growth is at least as low as 12.5 mg of adenine/g of cells.

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Section: Introductionmentioning

confidence: 99%

Adenine Quantitation in Yeast Extracts and Fermentation Media and Its Relationship to Protein Expression and Cell Growth in Adenine Auxotrophs of Saccharomyces cerevisiae

et al. 1997

Biotechnol. Prog.

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“…These data indicated that the original mutants were respiratory-deficient. The white colony phenotype can be attributed to respiratory deficiency because the development of a pink color in the ade2 genetic background found in the eki1⌬ (KS101) and cki1⌬ eki1⌬ (KS106) mutants and the parent W303-1B strain would be dependent on oxidative phosphorylation (73). Another characteristic common to respiratory-deficient mutants is the inability to grow at elevated temperatures (74).…”

Section: Resultsmentioning

confidence: 99%

Respiratory Deficiency Mediates the Regulation of CHO1-encoded Phosphatidylserine Synthase by mRNA Stability in Saccharomyces cerevisiae

Choi

Carman

2007

Journal of Biological Chemistry

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The CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) is one of the most highly regulated phospholipid biosynthetic enzymes in the yeast Saccharomyces cerevisiae. CHO1 expression is regulated by nutrient availability through a regulatory circuit involving a UAS INO cis-acting element in the CHO1 promoter, the positive transcription factors Ino2p and Ino4p, and the transcriptional repressor Opi1p. In this work, we examined the post-transcriptional regulation of CHO1 by mRNA stability. CHO1 mRNA was stabilized in mutants defective in deadenylation (ccr4⌬), mRNA decapping (dcp1), and the 5-3-exonuclease (xrn1), indicating that the CHO1 transcript is primarily degraded through the general 5-3 mRNA decay pathway. In respiratorysufficient cells, the CHO1 transcript was moderately stable with a half-life of 12 min. However, the CHO1 transcript was stabilized to a half-life of >45 min in respiratory-deficient (rho ؊ and rho o ) cells, the cox4⌬ mutant defective in the cytochrome c oxidase, and wild type cells treated with KCN (a cytochrome c oxidase inhibitor). The increased CHO1 mRNA stability in response to respiratory deficiency caused increases in CHO1 mRNA abundance, phosphatidylserine synthase protein and activity, and the synthesis of phosphatidylserine in vivo. Respiratory deficiency also caused increases in the activities of CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, and the phospholipid methyltransferases. Phosphatidylinositol synthase and choline kinase activities were not affected by respiratory deficiency. This work advances our understanding of phosphatidylserine synthase regulation and underscores the importance of mitochondrial respiration to the regulation of phospholipid synthesis in S. cerevisiae.3 is one of the most highly regulated enzymes of phospholipid synthesis in the yeast Saccharomyces cerevisiae (4 -6). PS synthase is an integral membrane protein that is localized to the endoplasmic reticulum (7). It catalyzes the formation of PS by a Mn 2ϩ -dependent sequential reaction by displacing CMP from CDP-DAG with serine (8). The reaction product PS is a major structural component of yeast membranes accounting for 4 -18% of total phospholipids depending on growth conditions (9 -11). PS also serves as the precursor for the synthesis of the most abundant membrane phospholipids PE (20 -32%) and PC (35-55%) that are synthesized by the de novo CDP-DAG pathway ( Fig. 1) (5, 9). cho1 mutants defective in PS synthase activity can still synthesize PE and PC if they are supplemented with ethanolamine or choline; indeed, cho1 mutants are ethanolamine/choline auxotrophs (12, 13). Ethanolamine is used for PE synthesis via the CDP-ethanolamine branch of the Kennedy pathway (Fig. 1). The PE synthesized by the Kennedy pathway may be methylated to PC via the CDP-DAG pathway (Fig. 1). Choline is used for PC synthesis via the CDP-choline branch of the Kennedy pathway (Fig. 1). In wild type cells, both the CDP-DAG and Kennedy pathways contrib...

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“…This mutation leads to the accumulation of red pigment, the product of polymerization of aminoimidazole ribotide (AIR) (Smirnov et al , 1967). The red pigment can be involved in interesting physiological effects, such as modification of DNA cleavage (Meskauskas et al , 1985).…”

Section: Introductionmentioning

confidence: 99%

The effect of red pigment on the amyloidization of yeast proteins

Nevzglyadova

Kuznetsova

Михайлова

et al. 2011

Yeast

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The intensity of amyloid-bound thioflavine T fluorescence was studied in crude lysates of yeast strains carrying mutations in the ADE1 or ADE2 genes and accumulating the red pigment (a result of polymerization of aminoimidazoleribotide), and in white isogenic strains-either adenine prototrophs or carrying mutations at the first stages of purine biosynthesis. We found that the red pigment leads to a drop of amyloid content. This result, along with the data on separation of protein polymers of white and red strains in PAGE, suggests that the red pigment inhibits amyloid fibril formation. The differences in transmission of the thioflavine T fluorescence pattern by cytoduction and in blot-hybridization of pellet proteins of red and white [PSI + ] strains with Sup35p antibodies confirmed this conclusion. Purified red pigment treatment also led to a decrease of fluorescence intensity of thioflavine T bound to insulin fibrils and to yeast pellet protein aggregates from [PSI + ] strains. This suggests red pigment interaction with amyloid fibrils. Comparison of pellet proteins from red and white isogenic strains separated by 2D-electrophoresis followed by MALDI analysis has allowed us to identify 48 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, closely corresponding to prion-dependent proteins that we characterized previously. Also present were some proteins involved in stress response and proteolysis. We suppose that the red pigment acts by blocking certain sites on amyloid fibrils that, in some cases, can lead in vivo to interfere with their contacts with chaperones and the generation of prion seeds.

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‘Red pigment’ from ADE‐2 mutants of S. cerevisiae prevents DNA cleavage by restriction endonucleases

Cited by 5 publications

References 5 publications

Adenine Quantitation in Yeast Extracts and Fermentation Media and Its Relationship to Protein Expression and Cell Growth in Adenine Auxotrophs of Saccharomyces cerevisiae

Adenine Quantitation in Yeast Extracts and Fermentation Media and Its Relationship to Protein Expression and Cell Growth in Adenine Auxotrophs of Saccharomyces cerevisiae

Respiratory Deficiency Mediates the Regulation of CHO1-encoded Phosphatidylserine Synthase by mRNA Stability in Saccharomyces cerevisiae

The effect of red pigment on the amyloidization of yeast proteins

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