No description available.

Modelling studies were performed on a fermentation system using Saccharomyces cerevisiae NCYC 754. The production of fermentation product and cytochrome P-450 were studied under semi-anaerobic condition in batch cultures. The fermentation was carried out in a 5-litre fermenter and controlled at constant set-points which had been optimized by an earlier worker with respect to enzyme yield. An unstructured model was established to describe the biomass profile which comprised two growth phases; however the system did not demonstrate the classical diauxic growth as expected. Furthermore, against the general belief that glucose is the limiting substrate of the system; the maximum wet biomass seemed to depend on the concentration of peptone and yeast extract in the fermentation broth. Growth kinetics indicated that a second substrate was utilized before glucose metabolism began in spite of the presence of high levels of glucose. Luedeking and Piret type models, combined with ethanol inhibition, were derived to describe the profile of ethanol and cytochrome P-450 concentration. Later, it was demonstrated that a close correlation exists between initial glucose and cytochrome P-450 concentration. Viable count by agar plating techniques was used to test the proposed biomass model. The results were in line with the proposed model, even though the cell viability profile in the system was rather low. The Taguchi method was used to seek out the noise factor in the system, and optimize the operating conditions for a particular performance statistic. Contrary to earlier findings, the stirrer speed was found to have little effect on the yield of cytochrome P-450.

1. The inhibitory effect of glucosone on yeast fermentation was shown to the specific for the D-isomer, and exerted at some point before the breakdown of fructose-1:6-diphosphate. 2. The results of the fermentation experiments are analysed, kinetically, and glucosone shown to be a pseudo-irreversible inhibitor, of the type described by Ackermann and Potter (1949). It is shown that the effects produced by glucosone in animals can be correlated vd.th this finding. 3. The osone is shown to be phosphorylated by ATP in the presence of hexokinase. 4. It is suggested that the inhibitory effect produced by glucosone on yeast fermentation, is due to the slow dissociation of a glucosone phosphate from the hexokinase molecule. 5. The effect of hexokinase on a number of glucose analogues is reported, and indications are given of the high degree of specificity exhibited by the enzyme. 6. Methods are described for the preparation of an actively fermenting acetone-dried extract, and a maceration juice, from 'bakers' yeast. The preparation and some properties of cold-treated bakers' yeast are also described. 7. A method is described for the separation of the substrates .and products of the hexokinase reaction, on paper chromatograms.

Saccharomyces cerevisiae can assimilate ammonium by NADP-GDH or by GS-GOGAT. The aim of this project was to improve the efficiency of ammonium assimilation, and therefore substrate utilisation, of S. cerevisiae by elimination of the energy inefficient pathway (GS-GOGAT). GOGAT- mutants were isolated from a GDH- parent strain by their inability to use ammonium as sole nitrogen source. Two structural gene mutants were identified, one in each of the two structural genes encoding GOGAT. Constructs with different combinations of GDH- and GOGAT- mutations and corresponding wild type alleles were made, and their growth studied in medium supplemented with different levels of ammonium. The growth properties (as final culture density and growth rate) of GOGAT- and GOGAT+ strains transformed with the GDH1 gene, and grown with excess ammonium were very similar. It was concluded that, under the conditions used in this study, the loss of GOGAT does not improve the growth properties of the strain. Non-transformed constructs were grown with excess and limiting ammonium. Growth properties of the GDH- and GOGAT- strains suggest that GS-GOGAT functions in ammonium assimilation at very low ammonium levels. This conclusion needs further investigation because the GDH+ GOGAT- construct had lower NADP-GDH activity than the wild type. The physiology of ammonium assimilation by two industrial strains was compared to that of a laboratory wild type at different ammonium concentrations using shake-flask culture. All three strains possessed the three activities in MM+20mM NH4+, and the profiles of appearance/disappearance of activity were very similar. At lower ammonium concentrations, important differences between the strains became apparent. It is unclear if it is due to simple strain heterogeneity or represents significant differences between industrial and laboratory strains. On the basis of the enzyme data, GS-GOGAT appears to be important in ammonium assimilation by DCL1 at limiting concentrations.

In order to investigate the effect of the elimination of GOGAT activity in S. cerevisiae, the pool sizes of ammonia, glutamate and glutamine plus the specific activities of the enzymes involved in ammonia assimilation were determined for two genetically engineered strains (AR2 and AR5) and an haploid wild type ( Sigma 1278b). AR2 and AR5 strains carry the plasmid pCYG4 which directs about 5 fold more NADPH-GDH activity than wild type cells. AR5 strain is a double mutant, which lacks GOGAT activity. The studies were carried out using a microprocessor-controlled fermenter (PCS) which has the following features: 3 Main Boards (Central Processor Board, Memory Board and Analog/Digital - ON/OFF Switch Board). 4 Auxiliary Boards (pH, Oxygen, Temperature and Biomass Interface Boards). A connection block to link the PCS with the video terminal, with sensors from the fermenter, with a control box and with other microcomputer. AR2 and AR5 showed lower values of maximum specific growth rates than the wild type, determined either by batch mode or by washout kinetics. The reduction in the growth rate for AR2 and AR5 can be related to the added metabolic loads due to the plasmid encoded genes. Under carbon limitation there were no remarkable differences between the NADPH-GDH activities of AR2 (GOGAT+) and AR5 (GOGAT-). However, the concentrations of glutamate and glutamine for AR2 were higher (from 20 to 40 %) than those of AR5. The lack of the GOGAT activity also resulted in a decrease in the biomass concentration for AR5 compared to the GOGAT+ strains. Under nitrogen limitation NADPH-GDH activities were higher and intracellular ammonia concentrations lower than under carbon limited conditions. The intracellular concentrations of glutamate and glutamine were higher for the GOGAT+ strain than for the GOGAT- one. Although the biomass level was the same for the three strains, AR5 (GOGAT") cells changed from rounded to ellipsoidal form under nitrogen limited conditions. Oscillations were present in the NADPH-GDH activities of AR2 and AR5 strains growing under carbon and nitrogen limited media. They are probably due to segregational instability of the plasmid pCYG4 in these microorganisms.

No description available.

The aim of this study was to assess the feasibility of using the budding yeast Saccharomyces cerevisiae as a system in which to analyse plant promoters. The promoters chosen for study were the 19S and 35S promoters of cauliflower mosaic virus (CaMV) which, like cellular plant promoters, are transcribed in the plant nucleus by host cell RNA polymerase II. A complete CaMV genome was introduced into yeast on a 2 micron plasmid-based vector and using Northern blot analysis, several CaMV-hybridising transcripts were detected. More precise information on the activity of the promoters was obtained by constructing gene fusions in which the 19S and 35S promoters were linked to the bacterial lacZ gene. Biochemical assays for β-galactosidase showed that cells harbouring the 19S-lacZ gene expressed β-galactosidase but those harbouring the 35S-lacZ gene did not. The insertion of a yeast transcription termination signal upstream of the 19S promoter did not abolish or diminish expression of the 19S-lacZ gene. β-galactosidase was present at low levels in cells expressing 19S-lacZ, constituting less than 0.01% of total cell protein. The 5'ends of 19S-lacZ transcripts present in yeast were mapped by primer extension. The major RNA species initiated approximately 250bp upstream of the 19S-lacZ coding region, indicating the existence of a fortuitous promoter in this region of the CaMV DNA. Two less abundant RNA species initiated within the 19S-lacZ open reading frame at positions +9 and +25bp and may be produced from the genuine 19S promoter. There is evidence to suggest that one or both of these shorter transcripts is the functional mRNA for β-galactosidase. All three classes of RNA were polyadenylated. Coupling of the 19S-lacZ gene to a yeast enhancer (the GAL UAS) produced a 5-fold increase in β-galactosidase activity. At the transcriptional level, activation of the enhancer resulted in a massive increase in the level of the RNA initiating at -250bp but had a minor influence of the levels of the two RNA species initiating at +9 and +25. A series of deletion mutations within the 19S promoter was constructed using Ba131 nuclease. Analysis of these mutations in yeast revealed that sequences from -500 to -193bp and from -137 to -62bp were not required for 19S promoter function, but a deletion from -62 to -21bp (which removes the putative TATA box) severely reduced 19S-1acZ gene expression. Transgenic tobacco plants containing the 19S promoter deletions fused to a CAT gene were produced by A.tumefaciens-mediated gene transfer but the analysis of these plants was not completed.

Part I of this thesis describes the digestion of bakers' yeast s-RNA at 0° and at room temperature in the presence of various concentrations of magnesium ions and at several different s-RNA : enzyme ratios (w/w). Digestion of s-RNA with pancreatic ribonuclease (ratio, 2000:1, (w/w) ) in the presence of 0. 2 M magnesium and at 0°, followed by chromatographic analysis, indicated that about 75% of the ultraviolet absorbing material was of medium to high molecular weight. Analysis of the nucleotide composition of the fraction containing the intermediate size oligonucleotides showed that fraction to be enriched in the odd bases ψ, T, 1-methylG, N, N-dimethylG and several other unidentified nucleotides. This result was incompatible with the 'hairpin' models for s-RNA, proposed by McCully and Cantoni (1962). The presence of spermine in the digestion mixture clearly masked the catalytic action of pancreatic ribonuclease, albeit to a lesser extent than magnesium ions, contrary to a report by Thomas and Hubst (1963) in their study of the affect of spermine on the catalytic activity of E. coli ribonuclease toward E. coli ribosomal RNA. The studies described in Part II of this thesis are concerned with the fractionation of mixed s-RNA from yeast. Part II-A describes a new chromatographic material consisting of a bifunctional mercuri-dioxane derivative attached through a thiol group to the cellulose matrix. A previous report (Eldjarn and Jellum, 1963) had shown that HS-proteins could be fractionated on an organomercurial-cellulose (Material I) derived from aminoethyl-cellulose. The studies reported, here suggest that ¹⁴C-cysteinyl-s-RNA cannot be retained on Material I because the salt concentration required to overcome its ion-exchange properties preclude any interaction between the mercury of Material I and the sulfhydryl group of ¹⁴C-cysteinyl-s-RNA. The present study describes how this disadvantage was overcome by preparing an organomercurial-cellulose devoid of ion-exchange properties (Material II). Preliminary studies showed that Material II can retain the radioactivity associated with ¹⁴C-cysteinyl-s-RNA. These studies suggested that Material II might fractionate nucleic acids according to their base composition. Part II-B describes some of the attempts to fractionate yeast s-RNA by column partition chromatography. In general, resolution of acceptor activities of s-RNA, as well as the recovery of biological activity, was poor. Part II-C describes some of the author's contributions to the studies carried out in this laboratory on the fractionation of yeast s-RNA on another new chromatographic material, benzoylated-DEAE-cellulose (Material III). Certain variables (such as, magnesium ion concentration and pH), which can be manipulated during rechromatography of s-RNA on Material III, are discussed in light of the chromatographic behaviour of glycine and other acceptor RNA's. The forces responsible for this fractionation are also discussed. Part II-D describes a chemical procedure for the isolation of glycine s-RNA which, when combined with the chromatographic procedures described in Part II-C, afforded glycine-specific s-RNA, in high purity. Observations on the effect of magnesium ion during enzymatic synthesis of glycyl-s-RNA are discussed. The advantages and possible consequences of using purified aminoacyl-s-RNA synthetase enzymes for the preparation of the aminoacyl-s-RNA's are also discussed. The need to prepare large quantities of aminoacyl-s-RNA synthetases derives from their indispensable role in the development of chemical methods for purifying amino acid-specific s-RNA's (see Part II-D). Grinding of yeast cells with glass beads, which is the usual method employed for disrupting yeast cells, was found to be inadequate. Lysis of yeast cells with toluene at 37° was found to be a partial solution to this problem. Although previous studies showed that most of the synthetase enzymes were destroyed by this treatment (von Tigerstrom and Tener, 1967), investigations described in Part III of this thesis, show that most of the synthetase activities can be preserved by controlling the pH of the toluene-yeast mixture. However, there were still several synthetase activities missing in the cell-free extracts prepared by the warm toluene method. Preparation of large quantities of yeast cell-free extract containing all of the aminoacyl-s-RNA synthetases was finally achieved by treatment of the yeast cells with toluene containing excess dry ice followed by an incubation period at 3-5°. Partial purification of the aminoacyl-s-RNA synthetases was achieved by chromatographing the cell-free preparation on hydroxyl-apatite. The presence of 40% glycerol in the eluting buffers was found to be essential for the preservation of most of the synthetase activities. When the cell-free preparations were chromatographed on hydroxy-apatite, most of the protein, but only a few of the synthetases were eluted by a linear gradient of phosphate buffer to 0.20 M. Most of the synthetases were eluted only when the column was washed with ammonium sulfate. The significance of this observation is discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The transcription factors Msn2p and Msn4p are major components of the Saccharomyces cerevisiae environmental stress response. Their transient activation/deactivation is dependent on a regulatory network centering on nucleocytoplasmic shuttling but also includes a range of other mechanisms. The acetyltransferase complex, NuA4 has been implicated in repression of Msn2p yet the mechanism is largely unknown. Gds1p is an uncharacterized yeast protein identified in a recent study as a physical interactor and acetylation target of NuA4. Gds1 protein level is dependant both on NuA4 and environmental stress and our analysis shows it to be involved in the nuclear exclusion of Msn2p in the absence of stress. Unstressed cells lacking GDS1 exhibit increased nuclear accumulation Msn2p and an increase in transcription of the stress reporter gene, HSP12. My work supports a model in which Gds1 and NuA4 can work independently to inhibit the Msn2/4 dependant yeast stress response in the absence of stress.

A three part study was carried out to examine rheological, colloidal and floe microstructural aspects of industrial brewing yeast strains. Following a review of the literature, the rheological properties of four yeast strains (two flocculent ale and lager types and their non-flocculent variants) were examined. In related colloidal studies, orthokinetic flocculation rates of these strains as well as their cell surface charge were determined. Floc microstructure was characterized using both light and scanning electron microscopy. In a summary chapter, the cell floc model (a modification of Hunter's elastic floc model) was used to the explain the rheological and colloidal behaviour of brewing yeast suspensions. Flow behaviour studies of the commercial yeast suspensions suspended in a calcium-containing sodium acetate buffer revealed that yeast flocculent characteristics had an important influence on their suspension flow behaviour. As cell concentrations increased, suspension flow properties become increasingly non-Newtonian and could be described by the Casson model at low rates of shear and the Bingham model at shear rates above 100 s⁻¹. The cell floc model was proposed to explain the Bingham flow behaviour of these csuspensions. The Bingham yield stress in these suspensions was believed to be a function of the orthokinetic capture coefficient, cell volume and the energy to break up doublet cells. Increasing temperature tended to lower the Bingham yield stress in lager strains and increase the yield stress in ale strains. A semi-empirical explanation for the viscosity increase of deflocculated cell suspensions and the estimation of pseudo-capture coefficients was presented. Furthermore, studies of the flow behaviour of yeast strains suspended in decarbonated ale and lager beer revealed that: 1) suspensions of flocculent strains show higher yield stress values than their non-flocculent variants, 2) ale strain suspensions tended to have higher yield values than the lager strains and 3) yeast dispersed in beer had higher yield stress values than when suspended in buffered calcium suspensions. This last observation was believed to reflect the influence of ethanol on the cell binding process which has important implications for future measurements of yeast flocculation. Colloidal studies revealed for the first time, that the orthokinetic rate of flocculation of brewing yeast cells could be modelled by a first order equation, as predicted by fundamental colloid theory. While subject to considerable variation, measured rate constants led to the calculation of orthokinetic capture coefficients. Yeast cell zeta potential values generally agreed with literature data but could not be employed in the DLVO model of colloid flocculation to explain measured orthokinetic capture coefficient values. Examination of the cell zeta potential data indicated that the data had non-normal distributions. SEM examination of the four industrial yeast strains suggested that a number of distinct structures mediated cell-to-cell interaction and that intra-strain differences occurred. These findings, along with the observation of non-normal surface charge distributions, indicated that these industrially pure strains had undergone substantial variation. Treatment of the flocculent cells with pronase tended to reduce cell-to-cell contacts. In the summary chapter the cell floe model was employed to describe the rheological behaviour of the yeast suspensions. Estimation of the force needed to separate doublet yeast cells were made using critical shear rate data (i.e., the point at which Bingham flow begins). This estimate was similar to that reported for single antibody bonds and may be due to the presence of lectin-like structures on the yeast cell wall. / Land and Food Systems, Faculty of / Graduate