Prothymosin α Is Not Found in Yeast

According to published accounts, prothymosin α exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A.FEBS Lett.257, 247–250, 1989). We report here our failure to find evidence for prothymosin α in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human prothymosin α coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human prothymosin α coding region sequences, and isolation of yeast proteins essentially as described in the publication above. A survey of theSaccharomyces cerevisiaecomplete genome database using the program BLASTp verified our findings: there is no prothymosin α-homologue in yeast. Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes. Therefore, the presence of a prothymosin α gene in animals other than mammals is highly unlikely.     

Simulation of the distribution of parental strains’ genomes in RC strains of mice

Recombinant Congenic strains (RC strains) were developed to facilitate mapping of genes influencing complex traits controlled by multiple genes. They were produced by inbreeding of the progeny derived from a second backcross from a common `donor' inbred strain to a common `background' inbred strain. Each RC strain contains a random subset of approximately 12.5% of genes from the donor strain and 87.5% of genes from the background strain. In this way the genetic control of a complex disease may be dissected into its individual components. We simulated the production of the RC strains to study to what extent they have to be characterized in order to obtain sufficient information about the distribution of the parental strains' genomes in these strains and to acquire insight into parameters influencing their effectiveness in mapping quantitative trait loci (QTLs). The donor strain genome in the RC strains is fragmented into many segments. Genetic characterization of these strains with one polymorphic marker per 3.3 centiMorgans (cM) is needed to detect 95% of the donor strain genome. The probability of a donor strain segment being located entirely in between two markers of background strain origin that are 3 cM apart (and hence escaping detection) is 0.003. Although the donor strain genome in the RC strains is split into many segments, the largest part still occurs in relatively long stretches that are mostly concentrated in fewer than 13 autosomes, the median being 9 autosomes. Thus, in mapping QTLs, the use of RC strains facilitates the detection of linkage. Received: 20 December 1996 / Accepted: 23 July 1997     

Characterization of Mamalian GS-α Proteins Expressed in Yeast

Abstract

The guanine nucleotide regulatory protein, G_s, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the Mp 42-45,000, short form (S), and M₁= 46-52,000, long form (L), of the a-subunits of rat G_s were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP 1 promoter and transformed into Saccharomvces cerevisiae. In the presence of 100 pM CuSOq, the transformed yeast expressed Gs-a mRNAs and proteins. In reconstitution experiments, rat Gs-a(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol, and guanine nudeotidedependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous G_s-a. G_s-a(S) demonstrated twice the activity of Gs-a(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of Gs-a(S) expressed in yeast with Gs purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as puriiied G,. Addition of bovine brain py subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for Gq-a(S and L) obtained from yeaa. In contrast, transducin py only enhanced agonist-stimulated adenylyl cyclase activity for Gs-a(S and L) following reconstitution. These results demonstrate that the expression of functional mammalian Gs-a subunits in yeast may be useful for their biochemical characterization.     

Involvement of the Yeast DNA Polymerase δ in DNA Repair in Vivo

The POL3 encoded catalytic subunit of DNA polymerase δ possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24°, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase δ. SDP5 is most probably the p55 subunit of Polδ of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair.     

Accumulation of Ade+ reversions in isoauxotrophic strains ofSaccharomyces cerevisiœ allelic inRAD6 during adenine starvation

A comparative method based on an analysis of accumulation of starvation-induced Ade⁺ reversions and cell death during adenine starvation was developed and exploited for estimating the role ofRAD6 in the starvation-induced reversions. It was shown that inactivation ofRAD6 function inSaccharomyces cerevisiœ markedly enhances the accumulation of Ade⁺ reversions, and therefore it is likely that this gene is taking part in maintaining the low level of starvation-induced mutations in yeast cells. This work was supported by a grant 204-1080-1993 from GACR to the last author andCharles University grant 274/1996 to the first author.     

Studies onBacillus thuringiensis H-14 strains isolated in Egypt—I. Screening for active strains

Screening for spore-forming bacteria to use against dipterous insects, namelyMusca domestica, Aedes aegypti andCulex pipiens from Egypt, led to two isolates, M1 and S128, being found which were highly toxic to bothAedes andCulex strains. They had calculated international Toxicity Units (ITU) valdes of 17,467 and 20,065 ITU/mg, respectively, as compared with 15,000 ITU/mg forBacillus thuringlensis H-14 IPS-82 Reference Standard. They were taxonomically classilled asBacllus thuringlensis H-14. Their sensitivity to a number ofBacillus phages is also described.

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Résumé Le criblage pour des bactéries sporulantes à utiliser contre des insecles diptères, notammentMusea domestica, Aedes aegypti, etCulex pipiens d'Egypte, a conduit à deux isolats, M1 et S128, dont on a montré la forte toxicité tant pour des souches deAedes que deCulex. Elles avaient des valeurs calculées d'Unités Internationales de Toxicité (UIT) respectivement de 17 467 et de 20 065 UIT par mg, ce qu'il faut comparer aux 15 000 UIT par mg chezBacillus thurigiensis H-14, standard de référence IPS-82. On les a classifiées sur le plan taxonomique commeBacillus thurigiensis H-14. On décrit également leur sensibillté à un certain nombre de phages deBacillus.

     

Yeast and cancer cells – common principles in lipid metabolism

One of the paradigms in cancer pathogenesis is the requirement of a cell to undergo transformation from respiration to aerobic glycolysis – the Warburg effect – to become malignant. The demands of a rapidly proliferating cell for carbon metabolites for the synthesis of biomass, energy and redox equivalents, are fundamentally different from the requirements of a differentiated, quiescent cell, but it remains open whether this metabolic switch is a cause or a consequence of malignant transformation. One of the major requirements is the synthesis of lipids for membrane formation to allow for cell proliferation, cell cycle progression and cytokinesis. Enzymes involved in lipid metabolism were indeed found to play a major role in cancer cell proliferation, and most of these enzymes are conserved in the yeast, Saccharomyces cerevisiae. Most notably, cancer cell physiology and metabolic fluxes are very similar to those in the fermenting and rapidly proliferating yeast. Both types of cells display highly active pathways for the synthesis of fatty acids and their incorporation into complex lipids, and imbalances in synthesis or turnover of lipids affect growth and viability of both yeast and cancer cells. Thus, understanding lipid metabolism in S. cerevisiae during cell cycle progression and cell proliferation may complement recent efforts to understand the importance and fundamental regulatory mechanisms of these pathways in cancer.     

Comparison of β-glucosidase activities in different Debaryomyces strains

Summary β-Glucosidase production by Debaryomyces vanrigii and Debaryomyces hansenii was studied using two media. Cellobiose was found to stimulate the biosynthesis of the enzyme, while NH₄NO₃ (1.0 g/l) and NH₄Cl (1.26 g/l) were the best nitrogen sources for D. hansenii and D. vanrigii respectively. Optimal conditions for enzyme activity were established in relation to pH, temperature and enzyme stability. Thermal and pH stability studies show that β-glucosidase from D. vanrigii was more stable at pH 4.5–5.0 at 50°C, while that enzyme from D. hansenii was stable at pH 6.5 at 35°C. This feature may be advantageous in the commercial application by hydrolysing cellobiose, the potent inhibitor of cellulose solubilizing enzymes.     

Symbiotic efficiency of Hup+ and Hup− strains ofRhizobium japonicum

Summary Relative efficiency of Hup⁺ and Hup^– R. japonicum strains with Pusa 16 cultivar of soybean was studied. Inoculation with the Hup⁺ strain (A 1014) reduced the protein content in grain as compared to uninoculated control.     

Spatially periodic discrete contact regions in polylysine-induced erythrocyte—Yeast adhesion

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 μm) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.     

Activation of Yeast Mitochondrial Translation: Who Is in Charge?

Mitochondrial genome has undergone significant reduction in a course of evolution; however, it still contains a set of protein-encoding genes and requires translational machinery for their expression. Mitochondrial translation is of the prokaryotic type with several remarkable differences. This review is dedicated to one of the most puzzling features of mitochondrial protein synthesis, namely, the system of translational activators, i.e., proteins that specifically regulate translation of individual mitochondrial mRNAs and couple protein biosynthesis with the assembly of mitochondrial respiratory chain complexes. The review does not claim to be a comprehensive analysis of all published data; it is rather focused on the idea of the “core component” of the translational activator system.     

PLK2 Modulates α-Synuclein Aggregation in Yeast and Mammalian Cells

Phosphorylation of α-synuclein (aSyn) on serine 129 is one of the major post-translation modifications found in Lewy bodies, the typical pathological hallmark of Parkinson’s disease. Here, we found that both PLK2 and PLK3 phosphorylate aSyn on serine 129 in yeast. However, only PLK2 increased aSyn cytotoxicity and the percentage of cells presenting cytoplasmic foci. Consistently, in mammalian cells, PLK2 induced aSyn phosphorylation on serine 129 and induced an increase in the size of the inclusions. Our study supports a role for PLK2 in the generation of aSyn inclusions by a mechanism that does not depend directly on serine 129 phosphorylation.     

Ochratoxin A in Brewer’s Yeast Used as Nutrient Supplement

Brewer’s yeast comprises different strains ofSaccharomyces cerevisiae used for beermaking. It is additionally used as a nutrient supplement to increase the intake of B vitamins and is recommended primarily for children in growth, women during pregnancy and lactation and persons during convalescence. A total of 51 samples of brewer’s yeast from the German market were analysed for the occurrence of ochratoxin A (OTA) by means of immunoaffinity clean up and HPLC with fluorescence detection. Thirty-two samples (63%) were found to be naturally contaminated with OTA in the range from the detection limit (0.03) to 1.53 ng/g. Mean values of the positive samples varied between 0.10 ng/g (powder) and 1.2 ng/g (dragees). In a worst case scenario, the consumption of brewer’s yeast could enhance the calculated daily intake for the German population by 10 to 14 ng OTA/day and person and increase the intake particularly for children from 1.3 up to about 1.9 ng/kg body weight.Thus, the results document that food supplements consisting of natural brewer’s yeast from the brewing process are a yet unknown source for the intake of ochratoxin A and a potential exposure risk. The screening of brewer’s yeast food supplements for OTA is therefore recommended in the context of food safety and quality control.