FluMag-SELEX as an advantageous method for DNA aptamer selection.

Aptamers are ssDNA or RNA oligonucleotides with very high affinity for their target. They bind to the target with high selectivity and specificity because of their specific three-dimensional shape. They are developed by the so-called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. We have modified this method in two steps-use of fluorescent labels for DNA quantification and use of magnetic beads for target immobilization. Thus, radioactive labelling is avoided. Immobilization on magnetic beads enables easy handling, use of very small amounts of target for the aptamer selection, rapid and efficient separation of bound and unbound molecules, and stringent washing steps. We have called this modified SELEX technology FluMag-SELEX. With FluMag-SELEX we have provided a methodological background for our objective of being able to select DNA aptamers for targets with very different properties and size. These aptamers will be applied as new biosensor receptors. In this work selection of streptavidin-specific aptamers by FluMag-SELEX is described. The streptavidin-specific aptamers will be used to check the surface occupancy of streptavidin-coated magnetic beads with biotinylated molecules after immobilization procedures.

Halotolerance of the yeast Arxula adeninivorans LS3.

The non-pathogenic, dimorphic, ascomycetous yeast Arxula adeninivorans LS3 is halotolerant. It can grow in a minimal medium containing up to 20% NaCl. The growth parameters are only weakly influenced by 10% NaCl. However, NaCl in a concentration higher than 10% causes a decrease in the specific growth rate, a longer adaptation phase and a lower cell count in the stationary growth phase. Concentrations of glycerol and trehalose, which differed 100-fold in magnitude in a salt free medium, are also influenced differently by salt. NaCl induces accumulation of intracellular glycerol in exponentially growing cells but a reduced concentration of intracellular trehalose in stationary cells. Transcripts of the genes ARFC3, encoding a component of the replication factor C, and GAA, encoding a secretory glucoamylase, can be detected only in cells cultured in media with NaCl concentrations below 10%. Furthermore, NaCl in high concentration reduces the level of secreted proteins including glucoamylase end invertase.

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.

Molecular cloning and expression of the ARFC3 gene, a component of the replication factor C from the salt-tolerant, dimorphic yeast Arxula adeninivorans LS3.

The yeast replication factor C (RF-C) is a multi-polypeptide complex with five sub-units involved in chromosomal DNA replication. This factor, encoded by five genes, is well characterized for the yeast Saccharomyces cerevisiae. However, RF-C components from other yeast species have not been analysed yet. Here we report the cloning and characterization of ARFC3 from the dimorphic and osmo-tolerant yeast Arxula adeninivorans. This gene encodes one subunit of the RF-C complex. It is localized on chromosome 1 of the four Arxula chromosomes and comprises a coding region of 1014 bp, which corresponds to 338 amino acids. Two introns are contained within this gene. The ARFC3 transcript level is influenced by both salt and temperature. The latter also influences the morphological state (budding cells, mycelium). High salt concentration and high temperature result in a rapid decrease of the ARFC3 mRNA.

Long-term effects of restrictive culture conditions on Saccharomyces cerevisiae sec7 cells.

Long-term effects of restrictive conditions on the temperature-sensitive S. cerevisiae sec7 mutant were studied. By microscopic analysis no cell lysis could be detected of cells cultured for up to 19 days at 37 degrees C. The optical density as well as the cell number remained constant during the whole period under restrictive conditions. However, restrictive conditions decreased the incorporation of 35S-methionine into intracellular proteins in a reversible manner indicating that protein biosynthesis was inhibited whereas the cells remained alive. Northern blot experiments revealed that restrictive conditions did not markedly decrease the ratio of the mRNA levels to total RNA for the genes TEF1, TEF2, SUC2, and BGL2 up to 73 hours. However the content of total RNA decreased drastically with increasing incubation times at restrictive temperature. In spite of the reduced total RNA content, cells are capable of new synthesis of mRNA under restrictive conditions which was shown by incubation of the cells in the presence of actinomycin D--an inhibitor of the mRNA synthesis. Most of the cells which survived a long-term incubation at 37 degrees C are not able to divide and to form colonies immediately after their transfer to permissive conditions.

Genetic diversity of the yeast Candida utilis.

The electrophoretic karyotypes and some mtDNA restriction fragment patterns of 13 strains of Candida utilis and one strain of Hansenula jadinii were compared. PFGE separations revealed remarkable chromosome length polymorphisms between two groups of strains suggesting that perhaps they do not belong to the same species. However, all strains had the same or similar EcoRI, HindIII and BamHI mtDNA restriction patterns. The mtDNA genomes had an average size range of 55 kb. These results support the supposition that C. utilis is a yeast with a highly variable electrophoretic karyotype as already known for another imperfect yeast species, Candida albicans.