Biomedical subjects

M Sipiczki

Publications and source records attributed to M Sipiczki.

At least 37 records · Page 2Linked to original sources

PubMed2000

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.

Cell Division↗

PubMed1999

Hexavalent chromium uptake by sensitive and tolerant mutants of Schizosaccharomyces pombe.

Lysine and leucine auxotrophic, heterothallic (h+, h-) strains of Schizosaccharomyces pombe were used to obtain chromium (VI)-sensitive and -tolerant mutants by ultraviolet radiation-induced and nitrosoguanidine-induced mutagenesis. The minimal inhibitory concentrations of K2Cr2O7 on YEA media were 225 microM for the wild-type strain CW-6, 125 microM for the sensitive mutant CS-6.51 and 275 microM for the tolerant mutant CT-6.66. The mutants exhibited cross-sensitivity of various patterns to Cd2+, Cu2+, Ni2+, Zn2+ and VO3-(4). Cr(VI) was added to the actively growing cultures and the total chromium (TOCr) content of the cells was determined. The sensitive mutant exhibited a high bioaccumulation ability, with a dry biomass of 810 micrograms g-1 after 30 min, while the tolerant mutant had a significantly lower ability than the wild-type strain. In PIPES buffer, washed, lysine-starved biomasses were treated with 75 microM Cr(VI) and after 2 h, the TOCr and the organically bound chromium (OBCr) were determined. Under these conditions, the sensitive and tolerant mutants had the same TOCr content, 50% of which was OBCr. The wild-type strain exhibited a lower TOCr content than that of its mutants and only 35% of this was OBCr. The Cr(VI)-sensitivity was due to a significantly increased uptake of Cr(VI).

Chromium↗

PubMed1999

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 degrees 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.

Actins↗

PubMed1999

Scanning electron microscopy characterisation of colonies of Candida albicans morphological mutants.

The ultrastructures of colonies of two stable UV-induced morphological mutants and their parental strain of Candida albicans grown on glucose-containing solid medium were investigated by scanning electron microscopy. The structures and ultrastructures of these three types of colonies were determined not only in terms of the proportions of blastospores, hyphae and pseudohyphae, but also with regard to the mode of budding of blastospores and the positions of these particular cell types within the colonies. Hyphae with an atypical appearance and branching characters were observed both in regular-wrinkled and in irregular-wrinkled mutant colonies. Smooth colonies of the parental strain and the mutants exhibited the same hyphal network within the agar, suggesting that micro-environmental factors in the agar overcame the effects of these mutations.

Candida albicans↗

PubMed1999

Genetics, physiology and cytology of yeast-mycelial dimorphism in fission yeasts.

The order Schizosaccharomycetales contains a dimorphic and two yeast species. Sch. japonicus can form both yeast cells and mycelium, depending on the substrate and the culturing conditions. Sch. pombe is a strictly unicellular organism, but it can be forced to form mycelial cell chains by inactivating members of the sep gene family. The mutations in most of the sep genes confer pleitropic phenotypes indicating functional involvement in MAP-kinase-mediated signalling pathways. Two of them were found to encode transcription factor homologues of other eukaryotes.

Cell Polarity↗

PubMed1998

Cloning of caf1+, caf2+ and caf4+ from Schizosaccharomyces pombe: their involvement in multidrug resistance, UV and pH sensitivity.

We previously identified four nuclear genes (caf1+ to caf4+) in Schizosaccharomyces pombe, mutations in which can confer caffeine resistance. Here we report the cloning and sequencing of caf1+, caf2+ and caf4+. All three genes are allelic to genes (hba1+, crm1+ and trr1+, respectively) involved in multidrug resistance mechanisms or in stress response systems. In agreement with this the caffeine-resistant mutants caf1(hba1)-21, caf2(crm1)-3 and caf4(trr1)-83 are also resistant to brefeldin. Disruption of caf1(hba1)+ and caf4(trr1)+ makes cells sensitive to high pH. The overlapping ranges of pleiotropic effects and the genetic interaction detected between caf1(hba1)+ and caf2(crm1)+ suggest that the three genes function in interlinked systems.

Caffeine↗

PubMed1998

Growth polarity transitions in a dimorphic fission yeast.

Fission yeast cells grow by extension at the ends (poles) and divide by transverse fission. It has previously been reported that Schizosaccharomyces japonicus var. japonicus can switch to unipolar, filamentous growth. Here it is shown that the yeast-to-mycelium transition is a gradual process involving a changeover to unipolar growth associated with asymmetric divisions, the development of large polarly located vacuoles, the modifications of the actin and microtubular cytoskeleton and the repression of cell separation after division. High concentrations of glucose in the medium or supplementation of the medium with caffeine or cAMP support the bipolar yeast phase, inhibit the transition to the mycelial phase and induce the conversion of hyphae to yeasts. These effects suggest that cAMP may be involved in the regulation of dimorphism. Temperatures below 18 degrees C or over 35 degrees C are restrictive for the mycelial phase and provoke a return to yeast phase.

Caffeine↗

PubMed1998

Environmentally controlled dimorphic cycle in a fission yeast.

The fission yeast Schizosaccharomyces pombe shows bipolar growth and is a convenient model for studying cell polarity and polar growth. This paper shows that the related Schiz. japonicus var. japonicus can switch to unipolar growth and can exist in both yeast and mycelial phases. On solid media, the yeast phase is unstable and prone to switch to the mycelial form, which shows unipolar growth by tip elongation. The hyphae can colonize the body of the substrate (true mycelium) or just its surface (pseudo-mycelium). The yeast-to-mycelium transition and the growth of the mycelium are regulated by a nutritional gradient and are associated with extensive vacuolation. The mycelium can convert into arthroconidia or return to the yeast phase in response to environmental changes. These environmentally controlled morphological transitions make Schiz. japonicus var. japonicus an attractive model for the investigation of cell polarity and morphogenesis.

Cell Polarity↗

PubMed1998

Coordination of initiation of nuclear division and initiation of cell division in Schizosaccharomyces pombe: genetic interactions of mutations.

sep1+ encodes a Schizosaccharomyces pombe homolog of the HNF-3/forkhead family of the tissue-specific and developmental gene regulators identified in higher eukaryotes. Its mutant allele sep1-1 causes a defect in cytokinesis and confers a mycelial morphology. Here we report on genetic interactions of sep1-1 with the M-phase initiation mutations wee1-, cdc2-1w, and cdc25-22. The double mutants sep1-1 wee1- and sep1-1 cdc2-1w form dikaryon cells at high frequency, which is due to nuclear division in the absence of cell division. The dikaryosis is reversible and suppressible by cdc25-22. We propose that the genes wee1+, cdc2+, cdc25+, and sep1+ form a regulatory link between the initiation of mitosis and the initiation of cell division.

Actins↗

PubMed1997

sep1+ encodes a transcription-factor homologue of the HNF-3/forkhead DNA-binding-domain family in Schizosaccharomyces pombe.

We report on the cloning of sep1+, a gene whose mutation causes filamentous growth in Schizosaccharomyces pombe. Since cell growth and propagation are not affected by the mutation, it could not be cloned using selective conditions for the identification of the positive transformants. Instead, we cloned it from a cosmid of a contig (Hoheisel et al., Cell 73, 109-1120, 1993) supposed to cover the chromosomal region where the sep1-1 mutation mapped. The 1761 bp long ORF codes for a protein containing a sequence similar to the DNA-binding domains of the HNF-3/forkhead family of transcription factors.

Amino Acid Sequence↗

PubMed1997

Genome instability and chromosomal rearrangements in a heterothallic wine yeast.

Wine strains of Saccharomyces cerevisiae are usually homothallic diploids and show chromosome length polymorphism. We describe the analysis of a heterothallic strain heterozygous for the mating types a and alpha. Surveying cultures of the strain, we found a remarkable degree of heterogeneity in ploidy and in electrophoretic karyotype. The CHEF analysis of tetrads and dyads revealed an enormous variability of band patterns hampering the analysis of chromosome segregation. We propose that the instability of ploidy and chromosome polymorphism might be due to heterothallism that precludes the process "genome renewal" (MORTIMER et al. 1994) by selfdiploidization of spore clones.

Chromosomes, Fungal↗

PubMed1997

Caffeine-resistance in S. pombe: mutations in three novel caf genes increase caffeine tolerance and affect radiation sensitivity, fertility, and cell cycle.

Caffeine is a well known base analogue and is cytotoxic to both animal and yeast cells. There are two possible mechanisms by which yeast cells tolerate caffeine concentrations higher than normal, by mutation or by physiological adaptation. We have isolated novel caffeine-resistant mutants of S. pombe which define three distinct genes caf2, caf3 and caf4. These mutants achieved a level of caffeine resistance which is presumed to represent the upper limit attainable by mutation. The caf2-caf4 mutations, as well as the previously identified caf1 mutation, confer UV-sensitivity, caffeine-resistant UV repair, impaired fertility and sporulation, as well as a lengthened cell cycle. They are partially dominant for caffeine resistance and recessive for UV sensitivity. Some auxotrophic caf3-89 double mutants show drastically decreased caffeine resistance. The caf4 mutant is more resistant to gamma-radiation than wild-type cells and shows pH-sensitive growth. As each caf mutation can, individually, confer maximum caffeine resistance to the cells, all four genes are expected to operate in the same pathway. This pathway might also be responsible for the physiological adaptation since adaptation is lost in caf1-caf4 mutants.

Caffeine↗

PubMed1997

Polarity, spatial organisation of cytoskeleton, and nuclear division in morphologically altered cells of Schizosaccharomyces pombe.

To gain more information about the determination of cell polarity and its relationship to the organisation of cytoskeleton, we have examined the mycelial mutant sep1-1 and the multinucleate multipolar syncytia of the triple mutant sep1-1 spl1-1 cdc4-8 by indirect immunofluorescence techniques. We have found that polarity is predetermined by the shape of the cell. During transition from mitosis to interphase the microtubules of the arising cytoplasmic cytoskeleton gradually form a basket-like pattern that reflects the curvatures of the cell envelope. The presumable growing poles, where actin accumulates, usually correlates with sites where the cell tapers and the microtubules converge. However, no growth can be launched at these sites if the cell surface has not been properly processed. Mitosis and meiosis are not affected significantly by changes in cell morphology and polarity, but larger cells are less effective during sporulation. The azygotic asci produced by multinucleate syncytia frequently contain over 20 ascospores.

Cell Division↗

PubMed1995

Two novel genes involved in the sexual development of Schizosaccharomyces pombe.

We isolated two sterile mutants of Schizosaccharomyces pombe. One of them was mapped close to ste13 (8 cM). Since it turned out to be allelic with the hitherto unmapped ral2, its linkage with ste13 localizes ral2 on the right arm of chromosome II. The other mutant defines a novel class-I ste gene, ste15, closely linked to ste7 (4 cM) on chromosome I. ste15 is conjugation-specific and acts upstream of pat1 and ras1. During its genetic analysis, a phenotypic suppression of ste12-N9 was observed which was caused by mutations in the unlinked gene ssw1.

Epistasis, Genetic↗

PubMed1995

Phylogenesis of fission yeasts. Contradictions surrounding the origin of a century old genus.

The phylogenesis of fungi is controversial due to their simple morphology and poor fossilization. Traditional classification supported by morphological studies and physiological traits placed the fission yeasts in one group with ascomycetous yeasts. The rRNA sequence comparisons, however, revealed an enormous evolutionary gap between Saccharomyces and Schizosaccharomyces. As shown in this review, the protein sequences also show a large gap which is almost as large as that separating Schizosaccharomyces from higher animals. Since the two yeasts share features (both cytological and molecular) in common which are also characteristic of ascomycetous fungi, their separation must have taken place later than the sequence differences may suggest. Possible reasons for the paradox are discussed. The sequence data also suggest a slower evolutionary rate in the Schizosaccharomyces lineage than in the Saccharomyces branch. In the fission yeast lineage two ramifications can be supposed. First S. japonicus (Hasegawaea japonica) branched off, then S. octosporus (Octosporomyces octosporus) separated from S. pombe.

Base Sequence↗

PubMed1995

Quantitation of protein phosphatase 1 and 2A in extracts of the budding yeast and fission yeast.

Serine/threonine protein phosphatases are also involved in the control of cell division. The aim of the present study was to compare the activity of protein phosphatase 1 (PP1) and 2A (PP2A) in cell extracts of the budding and fission yeast, made at different phases of growth. The activities of PP1 and PP2A toward phosphorylase were similar in extracts of S. cerevisiae. In S. pombe extracts, PP1 was responsible for more than 80% of the phosphorylase phosphatase activity. Ammonium sulfate-ethanol treatment increased the specific activity of the phosphatases and the percentage of PP2A in S. cerevisiae extracts. No increase in the proportion of PP2A was observed upon the same treatment of S. pombe extracts. The above results were confirmed by fractionation of PP1 and PP2A activities on a heparin-Sepharose column. The proportion of PP1 and PP2A activities did not change significantly during exponential cell growth but cells from stationary phase exhibited lower phosphatase activities. These results may indicate a lower level of expression of the PP2A genes in S. pombe and/or differences in the structure of the holoenzymes or their regulators in the two genera.

Cell Division↗

PubMed1995

The rec8 gene of Schizosaccharomyces pombe is involved in linear element formation, chromosome pairing and sister-chromatid cohesion during meiosis.

The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.

Chromatids↗

PubMed1994

Suppression of yeast RNA polymerase III mutations by the URP2 gene encoding a protein homologous to the mammalian ribosomal protein S20.

URP2 was cloned as a multicopy suppressor of several temperature-sensitive mutations defective in RNA polymerase III-dependent transcription, but without effect on mutations affecting RNA polymerase I or II. This single-copy gene encodes a hydrophilic polypeptide of 121 amino acid residues with a predicted molecular mass of 13.9 kDa and a basic isoelectric point of 9.7. URP2 is a highly expressed gene, judging from its abundant messenger RNA and strong codon bias. The Urp2p protein is essential for cell growth, as shown by the lethal phenotype of the urp2::HIS3 null allele. Given its striking similarity to the S20 ribosomal polypeptide of rat (55% identical residues), Urp2p is in all likelihood the yeast form of this polypeptide. Both proteins are significantly related to S10, a component of the small ribosomal subunit of Escherichia coli that is known to operate as a transcriptional elongation factor. The latter observation suggests that the suppressor effect of URP2 may be due to a direct involvement of Urp2p in RNA polymerase III-dependent transcription. Alternatively, the overexpression of Urp2p could bypass a partial preribosomal RNA processing defect associated with RNA polymerase III mutants. URP2 was assigned to the left arm of chromosome VIII, and maps between DUR3 and YLF1. The latter gene product has homology to the E. coli gtp1 gene product, and may define a new family of putative GTP-binding proteins.

Amino Acid Sequence↗