Georgios Lapathitis

Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization

The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.

Immunolocalization of Upstream Binding Factor and Pocket Protein p130 During Final Stages of Bovine Oocyte Growth

Abstract The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles (<1 mm). The entire nucleolus exhibited strong positivity for UBF (upstream binding factor, RNA polymerase I-specific transcription initiation factor), which displayed a dotted staining pattern. In contrast, protein p130 was diffusely distributed throughout the nucleus and excluded from nucleoli. In oocytes approaching the late period of growth (2–3-mm follicles), UBF localization shifted to the nucleolar periphery. Double staining of UBF-p130 revealed a gradual accumulation of p130 at the periphery shell around the nucleolus. In fully grown oocytes (>3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.

Accumulation of the Proteolytic Marker Peptide Ubiquitin in the Trophoblast of Mammalian Blastocysts

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrates lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Glucose- and K+-induced acidification in different yeast species

The process of acidification of the external medium after addition of glucose and subsequently of KCl to a suspension of yeast cells varies substantially from species to species. After glucose it is most pronounced inSaccharomyces cerevisiae andSchizosaccharomyces pombe but is very much lower inLodderomyces elongisporus, Dipodascus magnusii andRhodotorula gracilis. Both the buffering capacity and the varied effects of vanadate, suloctidil and erythrosin B indicate that the acidification is by about one-half due to the activity of plasma membrane H+-ATPase and by about one-half to the extrusion of acidic metabolites from cells. This is supported by the finding that a respiratory quotient greater than one (in various strains ofS. cerevisiae and inS. pombe) is indicative of a greater buffering capacity and overall acidification of the medium. Taking into account the virtually negligible buffering capacity of the medium in the pH range where the effect of K+ is observed, the effect of K+ is generally of a similar magnitude as that of adding glucose. It is clearly dependent on (anaerobic) production of metabolic energy, quite distinct from the dependence of the H+-ATPase-caused acidification.

Univalent cation fluxes in yeast.

Transport of H+, K+, Rb+ and Tl+ ions was studied in a wild‐type strain of Saccharomyces cerevisiae and in its mutants defective in the high‐affinity K+ transport system TRK1 and in the double mutant with an additional deletion in the TRK2 gene. In the absence of glucose K+, Rb+ and Tl+ elicited a more or less stoichiometric exchange outflow of H+, in the mutants K+ moved out of cells even in the presence of 10 mM KCl or KNO3. In the presence of glucose in the wild type, K+, Rb+ and Tl+ brought about a massive outflow of H+ while being transported inward against high concentration gradients. In the trklΔ mutant the exchange fluxes were reduced by 65‐85%, in the double mutant those of K+, Rb+ and Tl+ practically cease but outflow of H+ caused by Tl+ remained at the level of the trklΔ mutant. It appears that, in addition to the H+ export by the PMAl‐coded plasma membrane H+‐ATPase, at least three different univalent‐cation involving activities are present: the high‐affinity transport system for K+ (TRK1), another system (possibly TRK2) with different responses to K+ and Rb+, vs. Tl+, and an active system for K+ export. The first two are apparently active exchange systems for K+, Rb+, and Tl+ against H+. The source of energy for these highly active transports (acting against gradients of 1000:1 and 5000:1, respectively) is unclear.

Different sources of acidity in glucose‐elicited extracellular acidification in the yeast Saccharomyces cerevisiae

Three wild‐type strains of Saccharomyces cerevisiae, viz. K, Y55 and Σ1278b, two mutants lacking one or both of the putative K+ transporters, trklΔ and trklΔtrk2Δ, and a mutant in the plasma membrane H+‐ATPase, viz. pmal‐105, were compared in their extracellular acidification following addition of glucose and subsequent addition of KCl; in ATPase activity in purified plasma membranes; and in respiration on glucose. The glucose‐induced acidification was the greater the higher the respiratory quotient, i.e. the higher the anaerobic metabolism. A markedly lower acidification was found in the ATPase‐deficient pmal‐105 strain but also in the TRK‐deficient double mutant. The acidification pattern after addition of KCl corresponds to expectations in the TRK mutants; however, a similarly decreased acid production was found in the ATPase‐deficient mutant pmal‐105. The highest rate of ATP hydrolysis in vitro was found with the trklΔtrk2Δ mutant where glucose‐, as well as KCl‐induced acidification were lowest. Likewise, the pmal‐105 mutant with extremely low acidification showed only a minutely lower ATP hydrolysis than did its parent Y55 strain. Apparently, several different sources of acidity are involved in the glucose‐induced acidification (including extrusion of organic acids); in fact, contrary to the general belief, the H+‐ATPase may play a minor role in this process in some strains.

Structure of yeast plasma membrane H(+)-ATPase: comparison of activated and basal-level enzyme forms.

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolising cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Structure of the two enzyme forms and the effects of beta, gamma-imidoadenosine 5-triphosphate (AMP-PNP) and of diethylstilbestrol (DES) thereon were analysed by FT-IR spectroscopy. IR spectra revealed the presence of two populations of alpha-helices with different exposure to the solvent in both the A-ATPase and B-ATPase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two alpha-helix populations. Thermal denaturation experiments suggested a more stable structure in the B-form than in the A-form. AMP-PNP stabilised the A-ATPase structure while DES destabilised both enzyme forms. IR spectra showed that 60% of the amide hydrogens were exchanged for deuterium in both forms at 20 degrees C. The remaining 40% were exchanged at higher temperatures. The maximum amount of H/D exchange was observed at 50-55 degrees C for both enzyme forms, while in the presence of DES it was observed at lower temperatures. The data do not contradict the possibility that the activation of H(+)-ATPase is due to the C-terminus of the enzyme dissociating from the ATP-binding site which is covered by it in the less active form.

Critical findings on the activation cascade of yeast plasma membrane H+-ATPase

Strains of the yeast Saccharomyces cerevisiae, deficient in either of its two G-proteins, in the Snf3 and Rgt2 sensors, in the Gpr1 receptor and in various hexokinases were tested for their ability to start the activation cascade with a metabolizable monosaccharide that leads eventually to activation of plasma membrane H(+)-ATPase. The acidification rate after addition of glucose to glucose-grown cells and of galactose to galactose-grown ones, and the rate of ATP hydrolysis by purified plasma membranes in both types of cells were studied. It appears unequivocally that phosphorylation of the monosaccharide is essential for the activation; the role of the Gpa2 protein (possibly in combination with the Gpr1 receptor) is very probable while the two sensors appear to play somewhat ambiguous roles - in the absence of both the activation was actually higher than in the parent strain. The Gpa1 G-protein is not involved in acidification but may function in ATPase activity where, in addition to the phosphorylation step, other factors can play a role. There appear to be alternative pathways leading to the ultimate activation of the H(+)-ATPase, not necessarily involving G-proteins.

Extracellular acidification bySaccharomyces cerevisiae in normal and in heavy water

Titratable acidity of the extracellular medium was compared with that calculated from pH changes in a suspension ofSaccharomyces cerevisiae. After addition of cells to normal water the ratio of titratable acidity to the computed one was about 25, after addition of 50 mmol/Ld-glucose it was about 13, after subsequent addition of K+ ions it was only 2. In heavy water the respective values were 30, 9, and 1. Apparently, the principal buffer-generating processes have to do with glucose metabolism but little with the K+/H+ exchange observed after addition of K+. D2O appears to block processes producing the buffering capacity of the medium, among them possibly extrusion of organic acids.

Dicarbanonaborates in yeast respiration and membrane transport

Two derivatives of carborates, sodium 5,6‐dichloro‐7,8‐dicarbanonaborate (CB‐Cl) and sodium 5‐mercapto‐7,8‐dicarbanonaborate (CB‐SH) were found to inhibit endogenous as well as glucose‐induced respiration of the yeast Saccharomyces cerevisiae. Both substances slightly increased endogenous acid production, were neutral toward H+‐ATPase‐associated acidification but pronouncedly inhibited the K+‐stimulated acidification. The same effects were observed also with an ATPase‐deficient mutant of the yeast. The ATP‐hydrotyzing activity of yeast plasma membranes in vitro was severely reduced. The membrane potential was substantially increased toward more negative values. The H+‐symporting uptake of glutamic acid was considerably decreased, that of adenine was diminished much less. The effects of the dicarbanonaborates are obviously pleiotropic but their inhibition of ATP hydrolysis and of uptake of H+‐symported substances, on the one hand, and absolute lack of effect on ATPase‐catalyzed acidification, on the other, pose an unresolved problem.