Kinetics of petite mutation and thermal death in Saccharomyces cerevisiae growing at superoptimal temperatures.

Mass formation of petite mutants took place in a strain of Saccharomyces cerevisiae when grown at superoptimal temperatures. After an initial period of exponential growth, a second period followed during which exponential death and net exponential petite mutation concurred with exponential growth. The specific rates of the three exponential processes were of the same order of magnitude and varied with the temperature. Net exponential petite mutation did not occur during the deathless first period of growth at superoptimal temperatures nor at any time during growth at suboptimal temperatures. Mitochondria are discussed as possible targets of thermal death in mesophilic yeasts.     

Influence of pH and temperature on the substrate yield coefficient of yeast growth in a chemostat

The influence of pH and temperature on the substrate yield coefficient for growth of Saccharomyces cerevisiae in a chemostat under limited organic substrate conditions was studied. Mathematical analysis of the substrate yield coefficient as a function of pH and temperature in the near-optimal area was made. It was shown that the location of pH and temperature optima were independent of each other. The maximum substrate yield coefficient had the following coordinates: pH = 4.1, temperature = 28.5°C.     

Ethanol production by yeast at supraoptimal temperatures

Summary Experiments were performed to investigate growth and alcohol production by yeast cells at high temperatures. Raising the temperature from 30.0°C to 39.0°C resulted in the desired effect: less growth and higher ethanol productivity.At temperatures above 39.6oC,however, cell death predominates.     

Osmoregulation of the salt-tolerant yeast Debaryomyces hansenii grown in a chemostat at different salinities.

The intracellular solute composition of the salt-tolerant yeast Debaryomyces hansenii was studied in glucose-limited chemostat cultures at different concentrations of NaCl (4 mM, 0.68 M, and 1.35 M). A strong positive correlation between the total intracellular polyol concentration (glycerol and arabinitol) and medium salinity was demonstrated. The intracellular polyol concentration was sufficient to balance about 75% of the osmotic pressure of the medium in cultures with 0.68 and 1.35 M NaCl. The intracellular concentration of K+ and Na+, which at low external salinity gave a considerable contribution to the intracellular water potential, was only slightly enhanced with raised medium salinity. However, the ratio of intracellular K+ to Na+ decreased; but this decrease was less drastic in the cells than in the surrounding medium, i.e., the cells were able to select for K+ in favor of Na+. The turgor pressure, which was estimated on the basis of intracellular solute concentrations, was 2,200 kPa in cultures with 4 mM NaCl and decreased when the external salinity was raised, resulting in a value of about 500 kPa in cultures with 1.35 M NaCl. The maintenance of a positive turgor pressure at high salinity was mainly due to an increased production and accumulation of glycerol.     

Active plant growth at freezing temperatures

As a foundation for speculations in exobiology, we are attempting to understand plant responses to extreme environments on Earth. We have emphasized active plant growth at low temperatures and in response to ultraviolet light. We have studied the winter environment in the mountains near Logan, Utah and have found several plants that grow under the snow. We have measured chlorophyll synthesis, carbohydrate levels, and ion balances in these plants and established field experiments with hardy and nonhardy varieties of wheat. In the laboratory we have studied characteristics of three enzymes in two wheat varieties, finding a number of interesting differences in response to ultraviolet and low-temperature treatments. We have also examined cell ultrastructures of three grass species subjected to a range of temperatures. Chloroplasts were most affected at low temperatures, but other organelles were also influenced. Studies of ion balances substantiate the suggestion from ultrastructure work that membranes may exhibit the primary responses to low temperatures. Cytokinins are also implicated in the cold response. We are presently emphasizing the investigation of membranes. Lunar Science Institute Contribution.     

Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures.

Major heat shock proteins, such as the Escherichia coli DnaK protein, not only are required for cell growth after heat shock but seem to possess important functions in cellular metabolism at normal growth temperatures as well. E. coli delta dnaK52 mutants have severe cellular defects at 30 degrees C, one of which is in cell division (B. Bukau and G. C. Walker, J. Bacteriol, 171:2337-2346, 1989). Here we show that at 30 degrees C, delta dnaK52 mutants have defects in chromosome segregation and in maintenance of low-copy-number plasmids. Fluorescence microscopic analysis revealed that chromosomes were frequently lacking at peripheries of cell filaments of delta dnaK52 mutants and clustered at other locations. In other parts of the cell filaments, chromosomes were apparently normally distributed and they were also present in most of the small cells found in populations of delta dnaK52 cells. These defects might be at the level of DNA replication, since delta dnaK52 mutants have a threshold lower rate of DNA synthesis than wild-type cells. Chromosome segregation defects of delta dnaK52 mutants were also observed in an rnh dnaA mutant background, in which initiation of DNA replication is DnaA-oriC independent. We also found that low-copy-number P1 miniplasmids could not be stably maintained in delta dnaK52 mutants at 30 degrees C. delta par P1 miniplasmids that carry the P1-encoded rep functions required for their replication but lack the P1-encoded par functions required for faithful partitioning of the plasmids during cell division were also unstable in delta dnaK52 mutants. Taken together, our results indicate important, although not absolutely essential, functions for DnaK at 30 degrees C in one or more processes necessary for correct replication and/or partitioning of chromosomes and P1 miniplasmids. Furthermore, we found that P1 miniplasmids were also highly unstable in dnaJ259 mutants, indicating a role for the DnaJ heat shock protein in maintenance of these plasmids.     

On-line estimation of biomass concentration during transient growth on yeast chemostat culture using light reflectance

The application of light reflectance for estimating biomass concentration was investigated on oxidative chemostat culture of Saccharomyces cerevisiae. A correlation between light reflectance and dry weight was established for biomass concentrations from 0.5 to 10 g l^–1. The light reflectance signal was stable during the course of chemostat culture and proved to be sensitive to slight but fast changes in biomass concentration following shift-up in dilution rate, acetate pulse or during an oscillation. On-line estimated biomass revealed a larger time window of the biological response during spontaneous oscillations and could be used to predict carbohydrate storage.     

Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast

Mating-type switching in the fission yeast Schizosaccharomyces pombe is initiated by a strand-specific imprint located at the mating-type (mat1) locus. We show that the imprint corresponds to a single-strand DNA break (SSB), which is site- but not sequence-specific. We identified three novel cis-acting elements, involved in the formation and stability of the SSB. One of these elements is essential for a replication fork pause next to mat1 and interacts in vivo with the Swi1 protein. Another element is essential for maintaining the SSB during cell cycle progression. These results suggest that the DNA break appears during the S-phase and is actively protected against repair. Consequently, during the following round of replication, a polar double-strand break is formed. We show that when the replication fork encounters the SSB, the leading-strand DNA polymerase is able to synthesize DNA to the edge of the SSB, creating a blunt-ended recombination intermediate.     

Light effects in yeast: inhibition by visible light of growth and transport in Saccharomyces cerevisiae grown at low temperatures.

Growth rate, sugar transport, and amino acid transport of yeast cells grown at 12 degrees C were inhibited by cool-white fluorescent light. At light intensities below 1,250 lx, growth and membrane transport were only slightly inhibited. Above 1,250 lx, there was increasing inhibition of both processes. Transport of histidine was completely inhibited after 3 to 5 days in cultures grown at 12 degrees C under 3,500-lx illumination. Cells grown at 20 degrees C were not inhibited by light intensities that caused complete loss of viability and membrane transport activity in cells grown at 12 degrees C.     

Sister-chromatid exchange formation at supra-optimal growth temperatures

The present research was mainly focused on characterizing the formation of sister-chromatid exchanges at both optimal and supra-optimal growth temperatures. Under these conditions (25, 30 and 35 degrees C) meristem cells of Allium cepa L. exhibited a roughly constant cell-cycle time, and modifications in other cell-cycle parameters were negligible. Second-division chromosomes of cells incubated at 30 and 35 degrees C showed increased SCE yields as compared with those detected in cells maintained at the optimal temperature (25 degrees C). When cells with unifilarly BrdUrd-substituted DNA was damaged by irradiation with visible light, we obtained almost the same SCE yields at the various temperatures tested. We suggest that this production of SCEs could be the result of a reduced number of lesions produced by light (perhaps as a consequence of reduced intracellular free oxygen at high temperature) and/or of an increased efficiency in the repair capacity of the cells at these temperatures. The analysis of SCE formation in undamaged cells incubated at different temperatures during BrdUrd treatment has shown that the optimal growth temperature appears to be the experimental condition in which the cells are able to exhibit the lowest frequency of SCE.     

Growth kinetics of glucose-limited petunia hybrida cells in chemostat cultures: Determination of experimental values for growth and maintenance parameters

With glucose-limited continuous cultures of Petunia hybrida six steady states were obtained at specific growth rates varying from 0.0035 to 0.012 h(-1) (corresponding with culture residence times varying from 285 to 85 h). The macromolecular and the elemental biomass composition which were determined in four steady states showed no major differences over the range of growth rates examined. During all six steady states specific subtrate and oxygen consumption as well as biomass and extracellular product formation rates were monitored. Moreover the specific activities of the mitochondrial cytochrome and alternative pathway were determined and used to estimate specific adenosine triphosphate (ATP) production rates. Data thus obtained were used in the determination of maintenance and true growth yield parameters. For the maintenance on glucose and ATP values of 0.0070 C-mol/C-mol/h and 0.034 mol/C-mol/h were obtained, respectively. True yields of biomass on glucose and ATP were 0.50 C-mol/C-mol and 0.28 C-mol/mol, respectively. (c) 1995 John Wiley & Sons, Inc.