Bromodeoxyuridine-labeled oligonucleotides as tools for oligonucleotide uptake studies

The mechanisms by which various oligonucleotides (ODNs) and their analogs enter cells are not fully understood. A common technique used in studies on cellular uptake of ODNs is their conjugation with fluorochromes. However, fluorescently labeled ODNs may vary from the parent compounds in charge and hydrophilicity, and they may interact differently with some components of cellular membranes. In this report, we present an alternative method based on the immunofluorescent detection of ODNs with incorporated 5-bromo-2'-deoxyuridine (BrdUrd). Localization of BrdUrd-modified ODNs has been achieved using FITC-labeled anti-BrdUrd antibodies. This technique allowed determination of the differences in cellular uptake of phosphodiester (PO) and phosphorothioate (PS) ODNs and their derivatives conjugated with cholesterol and menthol. The immunocytochemical method also has shown that the cellular uptake of some ODNs may be influenced by specific sequences that are responsible for the formation of higher-order structures.     

Met signaling mutants as tools for developmental studies

The Met receptor is widely expressed in embryonic and adult epithelial tissues; its ligand (hepatocyte growth factor/scatter factor, HGF/SF) is expressed in the mesenchymal component of various organs. The generation of hgf and met null mice has revealed an essential role for this ligand-receptor pair in the development of the placenta, liver, and limb muscles. However the early lethality of the null mutants has precluded analysis of Met function in late development. To extend the possible observation period, we generated mutant metalleles of different severity. This was done by impairing the ability of the receptor to transduce the HGF/SF signal, via mutation of consensus sequences in the multifunctional docking site present in the C-terminal tail of the receptor. Mice expressing a Met mutant still active as a kinase, but unable to recruit its effectors, died in mid-gestation with the same phenotype as the metknockout, proving the importance of phosphotyrosine-SH2 interactions in vivo. Mice expressing a Met receptor with partial loss of signaling function survived until birth and revealed novel aspects of HGF/SF-Met function during muscle development.     

Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials

Laccase is a ligninolytic enzyme widely distributed in wood-rotting fungi and which is also found in a variety of molds and insects as well as some plants and bacteria. Its biological roles range from depolmerization of lignin, coal and humic acids via the oxidation of various mono- and diaromatic structures, to polymerization reactions and pigment formation in microbial cells or spores. Apart from its action in catabolic, depolymerizing and polymerizing processes, laccases have also been shown to be powerful enzymes for coupling two different molecules to create new low-molecular-weight products in high yield. In addition to their homomolecular coupling capabilities, laccases are also able to couple a hydroxylated aromatic substrate with a nonlaccase substrate of variable structure to create new heteromolecular hybrid molecules. Thus, laccases are increasingly finding applications in biotechnology in the fields of environment-friendly synthesis of fine chemicals and for the gentle derivatization of biologically active compounds e.g., antibiotics, amino acids, antioxidants, and cytostatics. Finally, oligomerization and polymerization reactions can lead to new homo- or heteropolymers and biomaterials. These may be useful in a wide range of applications including the production of polymers with antioxidative properties, the copolymerizing of lignin components with low-molecular mass compounds, the coating of cellulosic cotton fibers or wool, the coloring of hair and leathers, or the cross-linking and oligomerization of peptides.     

Fungal laccases: versatile tools for lignocellulose transformation

Conversion of lignocellulosic materials to useful, high value products normally requires a pre-treatment step to transform or deconstruct the recalcitrant and heterogeneous lignin fraction. The development of "green tools" for the transformation of lignocellulosic feedstocks is in high demand for a sustainable exploitation of such resources. This multi-faceted challenge is being addressed by an ever-increasing suite of ligninolytic enzymes isolated from various sources. Among these, fungal laccases are known to play an important role in lignin degradation/modification processes. The white-rot fungus Pleurotus ostreatus expresses multiple laccase genes encoding isoenzymes with different properties. The availability of established recombinant expression systems for P.?ostreatus laccase isoenzymes has allowed to further enrich the panel of P.?ostreatus laccases by the construction of mutated, "better performing" enzymes through molecular evolution techniques. New oxidative catalysts with improved activity and stability either at high temperature and at acidic and alkaline pH have been isolated and characterized.     

Plant protoplasts as genetic tool: selectable markers for developmental studies.

Protoplasts have usually been presented as a methodological tool. Even as such, they make possible an impressive array of applications in plant biology. Here we report on the use of protoplast-derived selectable markers in the study of several disturbed genetic systems with obvious effects on plant development: (1) auxotrophic mutants and the control of amino acid biosynthesis and transport in vegetative and reproductive tissues; (2) introgression of alien genetic information across phylogenetic boundaries by protoplast fusion, a consequence of controlled dedifferentiation-redifferentiation processes and attenuated incompatibility reactions in cultured cells; (3) expression (in)stability of foreign genes in transgenic plants during successive meiotic generations and in crosses between independent transformants.     

Viral vectors as tools for studies of central cardiovascular control

During the last few years physiological genomics has been the most rapidly developing area of physiology. Given the current ease of obtaining information about nucleotide sequences found in genomes and the vast amount of readily available clones, one of the most pertinent tasks is to find out about the roles of the individual genes and their families under normal and pathological conditions. Viral gene delivery into the brain is a powerful tool, which can be used to address a wide range of questions posed by physiological genomics including central nervous mechanisms regulating the cardio-vascular system. In this paper, we will give a short overview of current data obtained in this field using viral vectors and then look critically at the technology of viral gene transfer.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 microm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and "patchability" of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66+/-0.07 microF/cm(2)) and conductance (23-44 microS/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Yarrowia lipolytica: a model organism for protein secretion studies.

This paper reviews the advantages of the yeast Yarrowia lipolytica as a tool in the study of protein secretion. Work has been focused on the early steps leading the polypeptide, from the cytoplasmic ribosomes where it is synthesized, to the lumen of the endoplasmic reticulum. Using a thermosensitive allele of the 7SL RNA, the first in vivo evidence for a co-translational translocation was shown. Genetic screens allowed the identification of several new components of the translocation apparatus: Sls1p, an ER lumenal component involved in both translocation and lumenal transit; Tsr1p, involved in SRP-ribosome targeting; Tsr3p. Major translocation partners were also identified by reverse genetics (Sec61p, Sec62p, Kar2p, Srp54p, Sec65p).     

Multiple sequence alignments as tools for protein structure and function prediction

Multiple sequence alignments have much to offer to the understanding of protein structure, evolution and function. We are developing approaches to use this information in predicting protein-binding specificity, intra-protein and protein-protein interactions, and in reconstructing protein interaction networks.     

Fluorescent proteins as tools to aid protein production

Fluorescent proteins are genetically encoded, highly versatile reporters useful for monitoring various aspects of recombinant protein production. In addition to the widely popular green fluorescent protein (GFP) from Aequorea victoria, a variety of other fluorescent proteins have been discovered that display a wide range of spectral properties. Synthetic variants have also been developed to overcome limitations associated with their wild-type counterparts. Having a large repertoire of fluorescent proteins with diverse traits opens new opportunities for rapid monitoring and optimization of recombinant protein production.     

Cellulase secretion by immobilized protoplasts of Trichoderma reesei

Protoplasts from Trichoderma reesei were immobilized in alginate and induced to produce cellulase (endoglucanase and β-glucosidase) enzymes. The specific activities of the synthesized enzymes were higher in immobilized protoplasts than in both free and immobilized mycelia. Immobilized protoplasts show an enhanced rate of exocellular β-glucosidase production compared to intact mycelia due to the lack of cell wall. The ratio of the exocellular/intracellular β-glucosidase was 5.9 for immobilized protoplasts and 0.32 for free mycelia.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation

Here we report the development of novel antibodies which specifically react with phosphothreonine residues [anti-(P-Thr)antibodies]. The specificity of the antibodies was assessed in radioimmunoassays where we could demonstrate that half-maximal and maximal binding of the antibodies to plates coated with BSA - P-Thr occurred at serum dilutions of 1:4000 and 1:1000, respectively. P-Thr inhibited antibody binding with a half-maximal effect at 40 microM. P-Ser was 200-fold less potent while P-Tyr was essentially ineffective. Anti-(P-Thr) antibodies could specifically bind to phosphothreonine-containing proteins on Western blots. Using such a procedure we could demonstrate enhanced threonine phosphorylation of the EGF receptor upon treatment of intact unlabeled A431 cells with EGF. We could further demonstrate antibodies binding to proteins present in extracts of rat hepatoma cells (Fao). P-Thr at 10 microM completely inhibited antibody binding while P-Ser, P-Tyr, Thr or Ser, each present at tenfold higher concentrations, had no such inhibitory effect. Anti-(P-Thr) antibodies were also capable of specifically immunoprecipitating 32P-labeled phosphoproteins present in Triton extracts of Fao cells. Immunoprecipitation of proteins of 38 kDa, 55 kDa, 85 kDa, 100 kDa and 155 kDa was inhibited by 1 mM P-Thr but not by P-Tyr. These findings suggest that anti-(P-Thr) antibodies could be powerful tools in studies aimed at monitoring alterations in threonine phosphorylation of specific proteins as they occur under physiological conditions in response to various extracellular stimuli. Identification of such proteins can be conveniently monitored by immunoblotting.     

Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies

Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts of this monocotyledonous genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermococcus kodakarensis as a host for gene expression and protein secretion

Taking advantage of the gene manipulation system developed in Thermococcus kodakarensis, here, we developed a system for gene expression and efficient protein secretion using this hyperthermophilic archaeon as a host cell. DNA fragments encoding the C-terminal domain of chitinase (ChiAΔ4), which exhibits endochitinase activity, and the putative signal sequence of a subtilisin-like protease (TK1675) were fused and positioned under the control of the strong constitutive promoter of the cell surface glycoprotein gene. This gene cassette was introduced into T. kodakarensis, and secretion of the ChiAΔ4 protein was examined. ChiAΔ4 was found exclusively in the culture supernatant and was not detected in the soluble and membrane fractions of the cell extract. The signal peptide was specifically cleaved at the C-terminal peptide bond following the Ala-Ser-Ala sequence. Efficient secretion of the orotidine-5'-monophosphate decarboxylase protein was also achieved with the same strategy. We next individually overexpressed two genes (TK1675 and TK1689) encoding proteases with putative signal sequences. By comparing protein degradation activities in the host cells and transformants in both solid and liquid media, as well as measuring peptidase activity using synthetic peptide substrates, we observed dramatic increases in protein degradation activity in the two transformants. This study displays an initial demonstration of cell engineering in hyperthermophiles.