Studies on Substances Which Increase RNA Content of Yeast Cells

Active substances which increased RNA content and RNA productivity in yeast culture without affecting the growth rate of yeast were investigated.

The remarkable effect of zinc ion on RNA accumulation was found in flask cultures of Candida utilis.

The active substance of culture of Saccharomyces cerevisiae was isolated from the culture filtrate of Streptomyces sp. S–22 and it was identified as anisomycin, an antiprotozoal and antifungal antibiotic. The effect of anisomycin on the enhancement of yeast RNA formation was shown only with the Saccharomyces genus, which was more sensitive to the antibiotic than other genus. This phenomenon was exhibited only in the case of anisomycin and cycloheximide, whose modes of action were similar among various antibiotics. The ratio of four nucleotides in RNA fraction was almost equal to that of ribosomal RNA.     

Effects of two TMV strains on the synthesis and stability of chloroplast ribosomal RNA in tobacco leaves

Summary Multiplication of TMV-strains vulgare (light-green/dark-green mosaic symptoms) and flavum (severe yellow/green mosaic) had different effects on the ribosomal RNA of tobacco leaf chloroplasts. Vulgare inhibited chloroplast ribosomal RNA synthesis while having no effect on cytoplasmic ribosomal RNA synthesis (Fig. 2). Flavum inhibited chloroplast ribosomal RNA synthesis more severely than vulgare, and caused an earlier degradation of chloroplast ribosomal RNA than in control or vulgare-infected leaves (Fig. 1). Flavum also inhibited cytoplasmic ribosomal RNA synthesis. A connection between these differing effects on chloroplast ribosomal RNA metabolism and severity of visible symptoms is suggested, and discussed in relation to a possible influence on symptoms of denatured virus coat protein.Abbreviations TMV Tobacco Mosaic Virus - RNA Ribonucleic acid - DNA Deoxyribonucleic acid - m millions (in molecular weight values)     

Synthesis of macromolecules in the yeastCandida utilis as influenced by a specific factor inducing cell division

From the yeastCandida utilis a compound was isolated which uncouples the process of cell growth and division by accelerating the cell division without influencing outgrowth of cell mass when applied on another population of identical yeasts. This compound accelerated the initiation of DNA synthesis and had no influence on the synthesis of RNA and protein. Moreover, in the presence of division inducing factor the yeasts started multiplication before the content per cell reached the control level. The stimulating effect of division inducing factor was not obvious when the proteosynthesis of yeast cells was inhibited. We concluded that the division inducing factor regulates the formation of a protein which is synthesized in a very small amount and has a role in the initiation of DNA replication.     

tmRNA of Streptomyces collinus and Streptomyces griseus during the growth and in the presence of antibiotics

Streptomycetes are soil microorganisms with the potential to produce a broad spectrum of secondary metabolities. The production of antibiotics is accompanied by a decrease in protein synthesis, which raises the question of how these bacteria survived the transition from the primary to the secondary metabolism. Translating ribosomes incapable to properly elongate or terminate polypeptide chain activate bacterial trans‐translation system. Abundance and stability of the tmRNA during growth of Streptomyces collinus and Streptomyces griseus producing kirromycin and streptomycin, respectively, was analysed. The level of tmRNA is mostly proportional to the activity of the translational system. We demonstrate that the addition of sub‐inhibitory concentrations of produced antibiotics to the cultures from the beginning of the exponential phase of growth leads to an increase in tmRNA levels and to an incorporation of amino acids into the tag‐peptides at trans‐translation of stalled ribosomes. These findings suggest that produced antibiotics induce tmRNA that facilitate reactivation of stalled complex of ribosomes and maintain viability. The effect of antibiotics that inhibit the cell‐wall turnover, DNA, RNA or protein synthesis on the level of tmRNA was examined. Antibiotics interfering with ribosomal target sites are more effective at stimulation of the tmRNA level in streptomycetes examined than those affecting the synthesis of DNA, RNA or the cell wall.     

Growth and physiology of a yeast cultivated in batch and continuous culture systems

Inoculum size has been found to affect significantly the maximum attainable specific growth rate during batch cultivation ofCandida utilis. Lower inoculum size resulted in an increased growth rate and relatively longer lag. The culture is found to be most active in the beginning of the exponential phase as regards its RNA synthesis rate. Batch data were used for predicting the conditions of the yeast population in single-stage continuous culture system. Predicted and the experimental values showed a reasonable agreement. In single-stage chemostat the physiology of the yeast was studied on the basis RNA, DNA and protein synthesis rates at various growth rates. The results indicate that the productivity of cells and the rate of synthesis of macromolecules is highest at the dilution rate values of 0.33 to 0.35 hr⁻¹. In order to attain so-called unrestricted conditions of growth a pluristage pluristream continuous system was employed. It is assumed that under such conditions the specific growth rate and the synthetic activity of yeasts may reach its maximum on a given medium. The results presented do not show such conditions of growth under the experimental conditions employed (D ₁=0.35 hr⁻¹ andD ₂=0.2 to 1.7 hr⁻¹) withCandida utilis cultivated on beet molasses medium. Second stage of a two-stage two-stream continuous system is constantly fed with the cells from the foregoing stage; this category of cells on entering the new conditions of the second stage is expected to show some adaptation period. Experiments are reported to this effect.     

Acid treatment for RNA removal from yeast

Summary A method for RNA reduction from Candida utilis NRRL Y-660 by means of HCl-treatment was developed, and performs well with regard to protein recovery and aminoacid pattern.     

A Comparison of the Inhibition of Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells by Nine Different Macrolide Antibiotics

Nine structurally similar macrolide antibiotics were tested at a concentration of 0.5 μg/ml for their relative inhibitory effects on ribosome functions in Staphylococcus aureus cells. Eight of the compounds examined inhibited protein synthesis at this concentration. Seven of the nine compounds were also effective in blocking formation of the 50S ribosomal subunit. Roxithromycin and 14-hydroxy clarithromycin inhibited protein synthesis to a greater extent than they affected 50S subunit formation. Conversely, the compound 11,12-carbonate-3 deoxy-clarithromycin affected 50S assembly more than translation. Only clarithromycin had any effect on 30S ribosomal subunit assembly. The decline in growth rate and cell number was proportional to the effect on ribosome formation or function by each compound. These inhibitory activities can be related to structural differences between these macrolide antibiotics. Received: 6 May 1998 / Accepted: 27 July 1998     

Continuous culture ofCandida utilis growing under cell-cycle-period changes of aeration

Periodic interruption of oxygen supply in a cell-cycle period decreases the RNA content in the biomass ofCandida utilis. The effect was observed in a mineral medium in slowly as well as in rapidly growing cultures.     

SENESCENCE: HORMONAL CONTROL OF RNA AND PROTEIN SYNTHESIS IN EXCISED BEAN POD TISSUE

The kinetics of degradation of RNA, total protein, ribosomal and soluble (R-S) protein and DNA were followed over 24 hours in excised segments of Kentucky Wonder pole beans (Phaseolus vulgaris). In absence of exogenous auxin, RNA degradation proceeded from about zero time. By 15 hours, after about 20% of the RNA had degraded, the degradation of DNA, total protein and R-S protein was initiated. Exogenous auxin (25 ppm α-napthaleneacetic acid) prevented these degradations. The addition of kinetin had little or no effect. Auxin enhanced incorporation of orotic acid into RNA two to several times more than it did amino acids into protein. Actinomycin D repressed RNA synthesis about 75% including a repression of auxin-induced synthesis. It had no effect on the basal level of protein synthesis but did repress auxin-induced synthesis. The results indicate that the primary action of auxin in preventing senescence of bean endocarp tissue is at the site of RNA synthesis, and the effect of auxin on DNA and protein is a consequence of the effect of auxin on RNA metabolism.     

Nucleic acid reduction from yeast activation of intracellular rnase

The study of a heat-shock process for RNA reduction was carried out for different yeast strains. Different results were obtained from each of them. Candida utilis NRRL Y-660 shows its best performance after a 8-s. heat-shock in the presence of 3% NaCl. For commercial baker's yeast Saccharomyces cerevisiae and Kluyveromyces fragilis L-1930, similar results were obtained with only 1% of NaCl. The latter needed longer heat-shock periods. e.g. 15s. to give such an RNA reduction. Biomass recovery ranged from 60 to 75%, being higher for C. utilis and K. fragilis while excessive losses were observed in S. cerevisiae cells. No significant protein deterioration was obtained in the best performance samples. The aminoacid profile appears to be improved in comparison to the starting material in these strains after RNA reduction.     

Russian Article pp. 255–266

The growth of the yeast Candida utilis VKM-Y-2332 was investigated during the cultivation on a mineral medium with addition of ethanol in the regime of the chemostat and the pH-auxostat. The composition of the macroelements of the biomass produced by Candida utilis VKM-Y-2332 was studied. Stoichiometric equations of the growth process of the examined yeast strain on ethanol were presented. The growth of Candida utilis on ethanol and glucose was also characterized by the way of comparision.     

Effects of ribonucleic acid removal methods on proteolytic activity and protein solubility in Candida utilis

Candida utilis was grown under controlled conditions and nucleic acids were removed from whole cells and homogenates by alkaline hydrolysis techniques, en-zymatic methods, and washing with buffer. Homogenization released hydrolytic enzymes and proteolytic activity increased with incubation at elevated temperatures under acidic conditions. Slight proteolysis occurred in all incubated samples and this may contribute to protein insolubilization. Very little protein was lost during incu-bation when compared to similar processes using bakers' yeast. This can be due to lower levels of protease activities in C. utilis. Alkaline hydrolysis methods resulted in hydrolysis of some proteins and irreversible insolubilization of the protein. These methods also destroyed any residual enzymatic activities. Heat denaturation studies suggest that protein insolubilization occurs at neutral pH when heat treatments equivalent to or greater than 85° C for 15 min are used. SDS-PAGE methods were used to characterize and monitor changes in protein. Eighteen proteins and/or sub-units were present at levels of 1% or greater. Results may help to explain changesin functional properties of sample preparations which accompany RNA removal.     

Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics

Of thirteen ribosome-directed antibiotics surveyed for their inhibitory effect on viral protein synthesis in Escherichia coli infected with coliphage MS2, three antibiotics, kasugamycin, tetracycline and chloramphenicol, were found to exert differential inhibition, with synthesis of maturation protein being more sensitive than coat protein synthesis. Differential effects of kasugamycin and tetracycline were also observed in vitro. Such differential inhibition might reflect the presence of cistron-speciflc ribosomes or the induction of functional ribosomal heterogeneity by these antibiotic ligands.     

Isolation of aCandida utilis variant by using a nitrogen-limited continuous culture

During continuous culture ofCandida utilis the appearance of a morphologic variant yeast was detected. The new microorganism developed systematically whenever it was changed from normal to stressed propagation conditions. A simple system was used for the isolation of the yeast variant, which was defective in cellular division and showed improved kinetic parameters and oxygen uptake rate. An asynchronic nitrogen-limited continuous culture ofCandida utilis allowed us to enrich the population in the chemostat with the modified yeast and isolate it in a defined medium. Assimilation and fermentation tests indicated it to be a variant ofCandida utilis that showed stable morphologic and physiologic differences with the parental yeast.Candida utilis growing in this nitrogen-limited continuous culture also showed a high mutation rate.     

Nalidixic acid causes a transient G1 arrest in the yeast Saccharomyces cerevisiae.

Summary The addition of nalidixic acid to growing cells of the yeast Saccharomyces cerevisiae resulted in a transient depression in the rate of ribosomal precursor RNA production and a transient arrest of cells in G1. Protein synthesis rates were less affected. Lower concentrations of nalidixic acid also affected RNA synthesis and progression through G1 but had no effect on protein synthesis rates. We suggest that nalidixic acid has a primary effect on RNA synthesis leading to a G1 arrest.     

Inhibition of Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells by 11 Different Ketolide Antibiotics

Eleven structurally similar ketolide antibiotics were tested at a concentration of 1 μg/ml for their relative inhibitory effects on growth and ribosome activities in Staphylococcus aureus cells. Ten of the compounds examined had an inhibitory effect on protein synthesis at this concentration and eight of the 11 compounds were also effective inhibitors of the formation of the 50S ribosomal subunit. All of the drugs tested inhibited protein synthesis to a greater extent than they affected 50S subunit formation. The decline in growth rate and cell number was proportional to the effect on ribosome formation and function. The growth of an ermC erythromycin-resistant strain of S. aureus was also significantly inhibited by nine ketolide compounds, suggesting that they were not inducers of methylase gene expression. These inhibitory activities can be related to structural differences between these ketolide antibiotics. Received: 6 May 1998 / Accepted: 27 July 1998     

产朊假丝酵母细胞壁33 ku蛋白的功能研究

通过胰蛋白酶和枯草杆菌蛋白酶对产朊假丝酵母Candida utilis细胞壁的酶解,发现一种分子质量为33 ku的酵母细胞壁主要结构蛋白. 研究显示,在细胞壁上这种蛋白质与细胞壁绝大多数蛋白质成分不同, 它不被胰蛋白酶水解,但对枯草杆菌蛋白酶的作用敏感.33 ku蛋白存在于酵母菌整个对数生长期的细胞壁中,特别是在对数早期细胞壁中,它是唯一的对胰蛋白酶作用不敏感的蛋白质成分.实验证明,该蛋白质对维系酵母细胞壁骨架成分葡聚糖的相互连接和细胞壁的完整结构,具有重要作用,是一种重要的酵母细胞壁嵌合蛋白.     

Preparation of some pyrazoline derivatives and evaluation of their antifungal activities

The synthesis of a new series of 1-[(benzazole-2-yl)thioacetyl]-3,5-diaryl-2-pyrazoline derivatives was obtained by reacting 1-(chloroacetyl)-3,5-diaryl-pyrazolines with 2-mercaptobenzimidazole/benzoxazole/benzothiazole. The chemical structures of the compounds were elucidated by ¹H-NMR, ¹³C-NMR, and FAB⁺-MS spectral data. Their antifungal activities against Candida albicans, Candida glabrata, Candida utilis, Candida tropicalis, Candida krusei, and Candida parapsilosis were investigated. A significant level of activity was observed.