Mátyás Sipiczki

Latent homology and convergent regulatory evolution underlies the repeated emergence of yeasts

Convergent evolution is common throughout the tree of life, but the molecular mechanisms causing similar phenotypes to appear repeatedly are obscure. Yeasts have arisen in multiple fungal clades, but the genetic causes and consequences of their evolutionary origins are unknown. Here we show that the potential to develop yeast forms arose early in fungal evolution and became dominant independently in multiple clades, most likely via parallel diversification of Zn-cluster transcription factors, a fungal-specific family involved in regulating yeast-filamentous switches. Our results imply that convergent evolution can happen by the repeated deployment of a conserved genetic toolkit for the same function in distinct clades via regulatory evolution. We suggest that this mechanism might be a common source of evolutionary convergence even at large time scales.

Eleven novel sep genes of Schizosaccharomyces pombe required for efficient cell separation and sexual differentiation

Genetic analysis of 20 sterile mutants prone to form hyphae revealed 11 novel ste genes (sep6 to sep16) of Schizosaccharomyces pombe. None of the mutants was completely mycelial. Most mutants formed branching hyphae and showed normal septation. Aberrant septal structures and actin distribution were seen only at 36°C. sep9‐307, sep14‐576 and sep15‐598 showed genetic interactions with sep1‐1, a mutation in a forkhead transcription factor homologue. Additional genetic interactions were detected between sep6‐194, sep15‐598 and cdc16‐116, a mutant allele of an anaphase modulator of p34cdc2. sep9‐307 and sep15‐598 caused dikaryosis in wee1− background. In mating and sporulation tests, sep6−, sep7−, sep9−, sep10−, sep11− and sep15− proved to be defective in conjugation only, whereas sep8−, sep13− and sep16− were also defective in meiosis‐sporulation. sep12− and sep14− were only partially sterile. All mutants could produce M‐factor but sep8−, sep11−, sep15− and sep16− were defective in P‐factor production. The mutations in sep8, sep11 and sep16 suppressed the pat1‐114‐driven meiosis. All mutants were sensitive to the presence of higher concentrations of chloride in the medium and to short heat shocks. The diversity of the mutant phenotypes and the pleiotropic effects of the mutations suggest that these sep genes might act in, or interact with, a multiple overlapping network of regulatory modules. Copyright

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation of Schizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and β-mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of β-mercaptoethanol did not enhance transformation frequency.

Hybridization Studies by Crossing and Protoplast Fusion within the Genus Schizosaccharomyces Lindner

Summary: Hybridization studies based on the use of crossing and the protoplast fusion technique revealed the relationship between the various taxa of the yeast genus Schizosaccharomyces Lindner. From a series of intra- and interspecific crosses as well as protoplast fusions, we concluded that a revision of the genus is required. Being interfertile, S. pombe and S. malidevorans have to be included in one species S. pombe. From the protoplast fusion results, this new taxon seems to be closely related to S. octosporus. The results obtained are discussed in connection with the characteristics of the low temperature spectra of the cytochromes.

Role of Cell Shape in Determination of the Division Plane in Schizosaccharomyces pombe: Random Orientation of Septa in Spherical Cells

The establishment of growth polarity in Schizosaccharomyces pombe cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle.

Marker construction and cloning of a cut1-like sequence with ARS activity in the fission yeast Schizosaccharomyces japonicus

The dimorphic fission yeast Schizosaccharomyces japonicus has proved to be an excellent experimental model for the investigation of the eukaryotic cell. Here we show that it has a haplontic life cycle, in which the diploid phase is confined to the zygote. To make it amenable to genetic and molecular analysis, we generated genetic markers and cloned a genomic sequence which acts as ars when integrated into a plasmid. Diploids suitable for testing complementation and recombination between markers can be formed by protoplast fusion. The complementation tests and the recombination frequencies determined in octads of spores identified 28 non‐allelic groups (genes) of mutations of the auxotrophic and mycelium‐negative mutants. Two groups of linked markers were also identified. The cloned fragment, which expresses ars activity, encodes a putative amino acid sequence highly similar to a conserved domain of proteins Cut1 (Schizosaccharomyces pombe), BimB (Aspergillus nidulans) and Esp1 (Saccharomyces cerevisiae). Copyright

Non-introgressive genome chimerisation by malsegregation in autodiploidised allotetraploids during meiosis of Saccharomyces kudriavzevii x Saccharomyces uvarum hybrids

Saccharomyces strains with chimerical genomes consisting of mosaics of the genomes of different species (“natural hybrids”) occur quite frequently among industrial and wine strains. The most widely endorsed hypothesis is that the mosaics are introgressions acquired via hybridisation and repeated backcrosses of the hybrids with one of the parental species. However, the interspecies hybrids are sterile, unable to mate with their parents. Here, we show by analysing synthetic Saccharomyces kudriavzevii x Saccharomyces uvarum hybrids that mosaic (chimeric) genomes can arise without introgressive backcrosses. These species are biologically separated by a double sterility barrier (sterility of allodiploids and F1 sterility of allotetraploids). F1 sterility is due to the diploidisation of the tetraploid meiosis resulting in MATa/MATα heterozygosity which suppresses mating in the spores. This barrier can occasionally be broken down by malsegregation of autosyndetically paired chromosomes carrying the MAT loci (loss of MAT heterozygosity). Subsequent malsegregation of additional autosyndetically paired chromosomes and occasional allosyndetic interactions chimerise the hybrid genome. Chromosomes are preferentially lost from the S. kudriavzevii subgenome. The uniparental transmission of the mitochondrial DNA to the hybrids indicates that nucleo-mitochondrial interactions might affect the direction of the genomic changes. We propose the name GARMe (Genome AutoReduction in Meiosis) for this process of genome reduction and chimerisation which involves no introgressive backcrossings. It opens a way to transfer genetic information between species and thus to get one step ahead after hybridisation in the production of yeast strains with beneficial combinations of properties of different species.

Optimal conditions for mycelial growth of Schizosaccharomyces japonicus cells in liquid medium: it enables the molecular investigation of dimorphism

The non‐pathogenic dimorphic fission yeast, Schizosaccharomyces japonicus, could be a suitable model organism for investigation of the genetic background of mycelial growth, as it has a haploid chromosome set and its genome is sequenced. Since earlier results have suggested that its morphological transition required solid substrates, but molecular biological experiments would require hyphae production in a liquid medium, we wanted to find circumstances which would enable hyphae production in liquid media. Several external conditions were investigated, but the strongest inducer was fetal bovine serum (FBS). Its positive effect could be hampered by heat and was dependent on pH, temperature and concentration of the serum. Other protein‐containing compounds, such as peptone and bovine serum albumin or amino acids, proved to be ineffective or weak. Generally, the uninduced and induced mycelial growth of Sz. japonicus could be improved by lower external pH and higher temperature. Copyright