Klaus Wolf

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach.

Abstract In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

Biosorption of Metals

There are many interactions between living cells and metals. Essential metals must he taken up by the cells, and they must be stored at their destinations. When the metals are present in the medium in too low a concentration, they must be accumulated. If the ambient metal concentration is too high, even essential metals become toxic and mechanisms of detoxification must ensure survival of the cell. Non-essential metals may enter cells in several ways and cause damage to their metabolism. Therefore, these metals have to be detoxified. All living cells have developed mechanisms by which they take up, store, detoxify or dispose of metals. Surprisingly, some species have been found which are able to concentrate metals to an extent which far exceeds necessary concentrations (Gabriel et al. 1994, 1997; Muraledharand et al. 1995; Pillichshammer et al. 1995; Michelot et al. 1998). Even non-essential metals are concentrated by these organisms to a tremendously high extent. This property has attracted the attention of many researchers looking for cheap ways to concentrate metals from dilute solutions of various origins. This chapter deals with the use of fungal biomass for removal of metals from solution. We refer to the literature since 1990, as the older literature has been reviewed exhaustively by Volesky (1990). It is not the aim of this chapter to give a detailed description of the physicochemical process of sorption, or to discuss in detail the mechanisms by which intracellular metal concentrations are regulated. The physicochemistry of sorption has been discussed extensively by Volesky (1990, 1994), the industrial use of biosorption as well as different types of biosorbents by Volesky and Holan (1995), metal cation uptake by yeast by Blackwell et al. (1995), metal transport in Saccharomyces cerevisiae by Eide (1998), and metal dependent regulation of genes by Winge et al. (1998). More general reviews on the interaction of fungi with toxic metals have been given by Gadd (1993) and Gray (1998).

Determination of the xenoestrogens 4-nonylphenol and bisphenol A by high-performance liquid chromatography and fluorescence detection after derivatisation with dansyl chloride

An easily performable and highly selective method for the determination of the xenoestrogens bisphenol A (BPA) and technical 4-nonylphenol (mixture of isomers) from environmental samples was developed. The method consists of fluorigenic labelling of the substances by dansylation followed by HPLC separation of the derivatives. Specific wavelengths (lembda(ex)=354 nm, lambda(em)=545 nm) for detection of the dansylated phenols were determined in order to reduce the signals of interfering compounds. The applicablility of the method for environmental samples was demonstrated by using sewage sludge spiked with BPA and 4-n-nonylphenol (as internal standard).

A mitochondrial group-I intron in fission yeast encodes a maturase and is mobile in crosses.

The open reading frame in the first intron of the mitochondrial gene encoding subunit I of cytochrome c oxidase encodes a maturase and stimulates homologous recombination in Escherichia coli. In this paper, we demonstrate that this intron is mobile in crosses, indicating that it also encodes an endonuclease. This is the first report on an intron which possesses mobility and acts as a maturase.

Biosynthesis of phytochelatins in the fission yeast. Phytochelatin synthesis: a second role for the glutathione synthetase gene of Schizosaccharomyces pombe

By complementation screening of a cadmium‐sensitive Schizosaccharomyces pombe mutant deficient in phytochelatin synthesis, but with 44% of the wild‐type glutathione content, we cloned a DNA fragment involved in phytochelatin synthesis. Sequence analysis revealed that it encodes the second enzyme involved in glutathione (GSH) biosynthesis, glutathione synthetase (GSH2) (E.C.6.3.2.3, Wang and Oliver, 1997). The mutant allele shows a single base‐pair exchange at the 3′ end of the reading frame leading to a single amino acid change from glycine to aspartate. This mutation leads to a significant reduction of phytochelatin synthesis, whereas glutathione synthesis is impaired to a far lesser extent. Complementation with the Arabidopsis thaliana GSH2 cDNA led to a partial restoration of phytochelatin synthesis. These data strongly suggest that the GSH2 gene encodes a bifunctional enzyme that is able to catalyse both the synthesis of GSH by adding glycine to the dipeptide (γGlu‐Cys) and the synthesis of phytochelatins. The sequence has been submitted to EMBL, Accession No. Y08414. Copyright

Diversity and endemism of ciliates inhabiting Neotropical phytotelmata

While the diversity and distribution of macro-organisms living in phytotelmata (plant-container habitats) is well known, detailed taxonomic work on micro-organisms living in the same environments is limited. As a model clade of microbial eukaryotes, sampling of ciliates in Neotropical bromeliad tanks increased, and Neotropical phytotelmata such as bamboo stumps and tree holes were newly sampled. Thirty-three isolates from Brazil, Costa Rica, Dominican Republic, Jamaica and Mexico were sequenced for small subunit rDNA, and placed into a phylogenetic context using non-phytotelmata GenBank accessions. This and the morphological investigations discovered 45 undescribed, possibly endemic ciliate species. The potential endemics are from throughout most clades of the ciliate tree of life, and there is evidence of speciation within the Neotropical phytotelmata habitat. Our data show the number of potential Neotropical phytotelmata-endemic ciliate species increasing as more phytotelmata are sampled. While the new data show that the supposed endemics are mainly recruited from moss and ephemeral limnetic habitats, the bromeliad ciliate fauna is quite distinct from those of other limnetic habitats, lacking many typical and common freshwater genera, such as Coleps, Colpidium, Frontonia, Paramecium, Glaucoma, Nassula, Stylonychia and Trithigmostoma. There is no indication that specific ciliates are confined to specific bromeliads.

Characterization of the I-Spom I Endonuclease from Fission Yeast: Insights into the Evolution of a Group I Intron-Encoded Homing Endonuclease

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli . Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo , the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro . Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.

Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation.

Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.

SPINDLE MEMBRANES AND SPINDLE ARCHITECTURE IN INVERTEBRATES

Abstract Membranes are consistent components of spindles in plants and animals. Depending on the species, they either form layers at the periphery of the spindle, are scattered throughout the spindle area or can be observed in both locations. This review aims to provide a survey of the arrangements and functions of membranes associated with the spindle apparatus in diverse invertebrate groups. In order to make the reader familiar with the architecture of the spindle apparatus and the course of nuclear division, centrosomes, microtobules (MTs) and centromeres are briefly introduced, the issue of membrane structure is addressed in general terms and orthodox as well as unorthodox cases of genome separation are described. As regards function, spindle membranes are believed to play a role in the control of progression of nuclear division through the release and sequestration of Ca2+ ions. In fact, there is evidence that membrane-bound, intra-spindle compartments sequestrate Ca2+ ions and it is beyond doubt that spindle microtubules are sensitive to Ca2+ ions. It is tempting to correlate both features, but cogent evidence for this link has still to be produced. In invertebrates, there are findings that raise the possiblity of additional and alternative functions for spindle membranes: (i) the control of the interaction between MTs and chromosomes, (ii) the nucleation of MTs, (iii) a storage site for material used in later stages of development and (iv) a direct effect on the MT mass within the spindle through volume exclusion. Thereby, the concentration of tubulin monomer, the precursor of MTs, is raised and MT assembly is fostered. Finally, the possibility should not be overlooked that perispindle membranes prevent the entry of cytoplasmic elements into the spindle area and that the association with the spindle apparatus ensures the approximate bipartition of the cytoplasmic membrane inventory during cell division.

Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris

A novel surface-engineered strain of yeast Pichia pastoris was constructed that displays at its surface Kluyveromyces lactis Yellow Enzyme (KYE) fused to the C-terminal half of Saccharomyces cerevisiae α-agglutinin. The expression of the fusion protein was controlled by the AOX1-promoter. The new strain showed an increased sorption of the xenoestrogen Bisphenol A (BPA). It was shown that sorption of BPA depended on the presence of methanol in the growth medium and on the pH of the binding assays. The binding kinetics were typical for binding at a surface. The present results demonstrate that the α-agglutinin surface display system can be used in the yeast P. pastoris.