Cryopreservation of filamentous micromycetes and yeasts using perlite.

The viability, growth and morphology of 48 strains of Ascomycota (including 17 yeasts) and 20 strains of Zygomycota were determined after a 2-d and then after 1-year storage in liquid nitrogen using a new cryopreservation method with perlite as a particulate solid carrier. In case of Ascomycota, 45 strains (94 %) out of 48 survived both 2-d and 1-year storage in liquid nitrogen, respectively. In case of Zygomycota, all 20 strains survived both storage. In addition, 3 strains of Basidiomycota counted among yeasts were tested and all survived the 1 year storage. In all surviving cultures no negative effects of cryopreservation by this method have been observed after 1-year of storage in liquid nitrogen. The results indicate that the perlite protocol can be successfully used for cryopreservation of taxonomically different groups of fungi and also for fungi which failed to survive other routinely used preservation procedures.

Immunity to killer toxin K1 is connected with the Golgi-to-vacuole protein degradation pathway.

Killer strains of Saccharomyces cerevisiae producing killer toxin K1 kill sensitive cells but are resistant to their own toxin. It is assumed that in the producer, an effective interaction between the external toxin and its plasma membrane receptor or the final effector is not possible on the grounds of a conformation change of the receptor or its absence in a membrane. Therefore, it is possible that some mutants with defects in intracellular protein transport and degradation can show a suicidal phenotype during K1 toxin production. We have examined these mutants in a collection of S. cerevisiae strains with deletions in various genes transformed by the pYX213+M1 vector carrying cDNA coding for the K1 toxin under the control of the GAL1 promoter. Determination of the quantity of dead cells in colony population showed that (1) the toxin production from the vector did not support full immunity of producing cells, (2) the suicidal phenotype was not connected with a defect in endocytosis or autophagy, (3) deletants in genes VPS1, VPS23, VPS51 and VAC8 required for the protein degradation pathway between the Golgi body and the vacuole exhibited the highest mortality. These results suggest that interacting molecule(s) on the plasma membrane in the producer might be diverted from the secretion pathway to degradation in the vacuole.

Isolation and characterization of the dsRNA virus from the yeast Endomyces magnusii.

Virus-like particles (VLPs) have been isolated from the yeast Endomyces magnusii. The VLPs measure 43 nm in diameter and contain six species of dsRNA (0.78, 0.83, 1.77, 1.84, 2.64, 4.30 kb respectively). E. magnusii produces a 'toxic' protein, which reduces the growth, and changes the colony morphology, of sensitive strains of Hansenula sp. growing on solid media. All strains of E. magnusii tested produced the 'toxin' and contained the VLPs. Current procedures of curing failed to destroy the ability to produce the 'toxin'.

The use of a killer factor in the selection of hybrid yeast strains.

A new selection method of yeast cell hybrids, based on the application of yeast killer factors, has been developed in the model system of induced protoplast fusion of Saccharomyces cerevisiae superkiller strain T158C (alpha, his-) and different sensitive strains. The method is comparable in its efficiency to the currently used selection techniques based on the auxotrophy or respiration deficiency of the parental cells. Selection by means of a killer factor, when compared to other methods, has the following advantages: (1) Sensitivity to a killer factor is a current natural property of most of the yeast strains. (2) Killer strains of different species of yeast are available and the killer character can be easily transmitted from strain to strain. (3) Hybrid cells produce a killer factor (K+) and are resistant to it (R+). Both these properties are easily and independently testable. (4) Hybrid cells can be cured of the "killer character" by growth at elevated temperatures. Therefore the killer character can also serve as a temporary marker for the purposes of selection of hybrid cells and can be eliminated when no longer desired. On an example of interspecific fusion of strain T158C of S. cerevisiae and strain P9 of S. uvarum the procedure has been shown to be advantageous particularly for the construction of new industrial yeast strains because mutagenesis of the original industrial strain is not required.

Effect of glucose on the biochemical properties of the bacterial cytoplasmic membrane.

The cytoplasmic membrane isolated from cells of Citrobacter freundii growing in a cultivation medium with glucose was found to exhibit a decreased ability to oxidize formate and succinate and a decreased activity of formate, succinate and lactate dehydrogenase as compared with the cytoplasmic membrane of cells growing on galactose. The activation energy for dehydrogenation and oxidation of succinate simultaneously increases. The quantitative content of cytochromes and quinones in both types of membranes does not differ considerably. The two types of membranes also do not mutually differ in the qualitative and quantitative representation of fatty acids in lipids and in the sensitivity to the effect of low concentrations of detergents. The mass ratio of proteins and lipids is lower in the membranes of cells grown on glucose.

Direct selection shuttle plasmid vector, pPW264, used for cloning the alpha-amylase gene of Schwanniomyces occidentalis.

We constructed a novel cloning system with positive selection for inserted fragments. The gene for tetracycline resistance (tetR) originally used in plasmid pTR262 was replaced with the gene for chloramphenicol acetyltransferase (cat) and terminator sequences were introduced downstream of the cat gene. The terminator sequences stop transcription originating on strong PR promoter that would otherwise proceed through the region of replication origin and interfere with plasmid replication. Thus the copy number of recombinant plasmid molecules is stabilized. The cloning system has been constructed in a new YEp type shuttle vector, pPW264. The 8.1 kb-vector carries two unique cloning sites, BglII and HindIII. The maintenance of the vector and selection in yeast is ensured by URA3 Saccharomyces cerevisiae gene. The vector was employed in cloning of the gene for alpha-amylase from Schwanniomyces occidentalis.

Accumulation of Ade+ reversions in isoauxotrophic stains of Saccharomyces cerevisiae allelic in RAD6 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 of RAD6 in the starvation-induced reversions. It was shown that inactivation of RAD6 function in Saccharomyces cerevisiae 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.

Accumulation of Ade(+) reversions in isoauxotrophic strains ofSaccharomyces cerevisioe 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 cerevisioe 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.

Changes in plasma membrane fluidity lower the sensitivity of S. cerevisiae to killer toxin K1.

The possible correlation between plasma membrane fluidity changes induced by modified cultivation conditions and cell sensitivity to the killer toxin K1 of Saccharomyces cerevisiae were investigated. Cells grown under standard conditions exhibited high toxin sensitivity. Both a membrane fluidity drop and fluidity rise brought about markedly reduced sensitivity to the toxin. These results do not fit the hypothesis of physiological relevance of direct toxin-lipid interaction, suggesting that the essential event in killer toxin action is interaction with membrane protein(s) that can be negatively influenced by any changes of membrane fluidity.

Ammonia mediates communication between yeast colonies.

Under certain growth conditions unicellular organisms behave as highly organized multicellular structures. For example, the fruiting bodies of myxobacteria and of the slime mould Dictyostelium discoideum form structures composed of non-dividing motile cells. Although non-motile, yeasts can create organized structures, colonies in which cells communicate and act in a coordinated fashion. Colony morphologies are characteristic for different species and strains. Here we describe that, in addition to short-range intracolony cell-cell communication, yeasts exhibit long-distance signals between neighbouring colonies. The volatile alkaline compound ammonia, transmitted by yeast colonies in pulses, has been identified as a substance mediating the intercolony signal. The first alkaline pulse produced by neighbouring colonies is non-directed and is followed by acidification of the medium. The second pulse seems to be enhanced and is oriented towards the neighbour colony. Ammonia signalling results in growth inhibition of the facing parts of both colonies. This phenomenon is observed in different yeast genera. The presence of amino acids in the medium is required for ammonia production. Colonies derived from the yeast Saccharomyces cerevisiae shr3 mutant, defective in localization of amino-acid permeases, do not produce detectable amounts of ammonia and do not exhibit asymmetric growth inhibition.

The involvement of the RAD6 gene in starvation-induced reverse mutation in Saccharomyces cerevisiae.

The accumulation of Ade+ revertants during adenine starvation and Trp+ revertants during tryptophan starvation in haploid polyauxotrophic strains of Saccharomyces cerevisiae occurs in a time-dependent manner. Accumulation of revertants is enhanced in Rad6- strains, suggesting that starvation-induced reversion is influenced by some of the RAD6 gene functions. The higher frequency of adaptive reversions in Rad6- strains is somewhat influenced by, but does not totally depend on, the genetic background. Therefore, the RAD6 gene product is involved in maintaining a low level not only of spontaneous mutation but also of starvation-induced reversion. The starvation-induced Ade+ and Trp+ reversions both appear to be adaptive. The analysis of growth characteristics and the genotype of revertants shows a difference between early and late-appearing revertants. These results support the hypothesis that the adaptivity of starvation-induced reversion is based on the selective fixation of random mutations, and particularly on transcription-enhanced repair and/or mutagenesis processes.