Abstract: The present invention relates to the use of a novel enzyme and its encoding gene for transformation. More specifically, the invention relates to the use of a gene encoding an enzyme with acyl-CoA:diacylglycerol acyltransferase activity. This gene expressed alone in transgenic organisms will increase the total amount of oil (i.e. triacylglycerols) that is produced.

Abstract: The invention relates to new eukaryotic strains, preferably yeast strains, having the new fil phenotype, i.e. having the unexpected property of conserving good stress resistance in fermentation and/or growth phase, while conserving normal respiratory and fermentation metabolism on fermentable sugars such as glucose.

Abstract: The present invention intends to provide an IL-6R•IL-6 fusion protein and the like in which IL-6R and IL-6 are directly linked without a linker.

Abstract: The present invention generally relates to methods of modifying the glycosylation structures of recombinant proteins expressed in fungi or other lower eukaryotes, to more closely resemble the glycosylation of proteins from higher mammals, in particular humans. The present invention also relates to novel enzymes and, nucleic acids encoding them and, hosts engineered to express the enzymes, methods for producing modified glycoproteins in hosts and modified glycoproteins so produced.

Abstract: The invention concerns a method for the expression of a gene coding for a soluble proteinase K in yeast e.g. in Pichia pastoris with subsequent secretion into the culture medium. In addition a method for purifying the heterologously expressed and secreted proteinase K is described.

Abstract: The present invention provides a method for the propagation of lytic organisms which comprises the infection of the cells of a stable cell line within a hollow fibre bioreactor with a lytic organism, wherein after said infection, said organism multiplies within the cells and can be harvested, characteriscd in that the cell line can survive for at least ten days after said infection. The invention further provides a method as herein described wherein after harvest, the cell line is allowed to re-populate the bioreactor, and at least one subsequent harvest may be taken, with the cell line being able to repopulate the bioreactor after each harvest.

Abstract: The invention is directed to 5S rDNA vectors that can be used to transform yeast strains such as laboratory strains, industrial phototrophic strains, and wild-type strains. 5S rDNA vectors are formed from a 2.1 kb EcoRI-EcoRI S. cerevisiae rDNA fragment that includes the 5S gene and the NTS1 and NTS2 spacers. The p1-9g18 vector has the glycoamylase gene expression cassette of Aspergillus awamory inserted in the HpaI site of the NTS1 spacer. The pA-4 has the geneticin (G418) resistance gene inserted in the HpaI site of the NTS1 spacer, and the pGG7 vector has the geneticin (G418) resistance gene inserted in the HpaI site of the NTS1 spacer, and the glycoamylase gene expression cassette of Aspergillus awmory cloned in the HindIII site of the NTS1 spacer.

Abstract: The present invention relates to the use of proteins facilitating water diffusion or water transport through the cell membrane, preferably aquaporin or aquaporin related proteins to obtain freeze-tolerant eukaryotic cells, preferably yeast cells or plant cells. It relates further to a method for obtaining such cells, and to freeze-tolerant cells, characterized by an enhanced expression level of proteins facilitating water diffusion or water transport through the cell membrane.

Abstract: The present invention relates to a gene coding for a copolyester-synthesizing enzyme, a microorganism which utilizes said gene for the fermentative synthesis of a polyester, and a method of producing a polyester with the aid of said microorganism. More particularly, the present invention relates to a gene which functions in a host organism and is related to synthesize, by an enzyme, a plastic-like polymer degradable under the action of microorganisms in the natural environment (the soil, river or sea), a transformant derived from said host organism by transformation with said gene and having an improved ability to fermentatively synthesize a plastic-like polymer, and a method of producing a copolyester with the aid of said transformant.

Abstract: The invention refers to the production of recombinant gene products from cultures of the yeast Zygosaccharomyces bailii strains transformed with expression vectors bearing the gene coding for said proteins.

Abstract: A liposome for fusing with a yeast cell, the liposome characterised in that about 40-50 molar % of the liposome lipid bilayer is phosphatidyl choline (PC), about 10-20 molar % of the liposome lipid bilayer is a cationic amphiphile, about 10 molar % of the liposome lipid bilayer is a sterol and about 30 molar % of the liposome lipid bilayer is phosphatidyl ethanolamine (PE) and/or dioleoylphosphatidylethanolamine (DOPE).

Abstract: The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components.

Abstract: The present invention relates to methods for producing a biological substance, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the biological substance, wherein the fungal host cell comprises a first nucleic acid sequence encoding the biological substance operably linked to a second nucleic acid sequence comprising a promoter variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; and a subsequence thereof; and hybrid and tandem promoters thereof; and (b) isolating the biological substance from the cultivation medium. The present invention also relates to the isolated promoter variants and to constructs, vectors, and fungal host cells comprising the promoter variants operably linked to nucleic acid sequences encoding biological substances.

Abstract: A method of constructing and screening a library of polynucleotide sequences of interest in filamentous fungal cells by use of an episomal replicating AMA1-based plasmid vector, thus achieving a high frequency of transformation and a stable and standard uniformly high level of gene expression.

Abstract: The invention provides novel yeast promoters useful for controlling the expression of homologous and heterologous nucleic acid molecules in yeast cells. The yeast promoters are induced by a fermentable carbon source, such as glucose, or a non-fermentable carbon source, such as ethanol, or both. Therefore, expression of nucleic acid molecules encoding a polypeptide under the control of the novel yeast promoters may be regulated by varying the level of a fermentable carbon source, or a non-fermentable carbon source, or both.

Abstract: Polynucleotides that comprise regulatory regions of the yeast alcohol oxidase 1 promoter are provided. Isolated polynucleotides, recombinant polynucleotides, vectors, expression cassettes and transformed cells containing the regulatory regions are disclosed. Proteins produced by the transformed cells are also provided.

Abstract: The present invention provides a transformed cell in which a polynucleotide having a nucleotide sequence encoding an amino acid sequence of an osmosensing histidine kinase having no transmembrane region is introduced in a functional form into a cell deficient in at least one hybrid-sensor kinase, a method of assaying the antifungal activity of a test substance using the transformed cell, and a method of searching an antifungal compound using the method, and the like.

Abstract: The present invention relates to genetically modified yeast strains autonomously producing, from a simple carbon source, steroids. The invention also relates to a method for producing steroids from such yeast strains.

Abstract: In a method of constructing a eukaryotic host microorganism for production of a heterologous protein encoded by a trangenically introduced gene, production efficiency of the heterologous protein by the transformant obtained by introducing the gene encoding the heterologous protein into the host is improved.

Abstract: The invention concerns yeasts whereof the cold stress tolerance is enhanced by transformation with at least one gene coding for a protein selected among a group 2 LEA protein, a WALI protein, or a LTP.

Abstract: The present invention relates to methods of using synthetic lethal screening techniques to identify drug targets. The methods of the present invention use “barcoded” libraries of cells, where the library consists of a collection of different mutant clones, each mutant clone bearing a knock-out mutation of a different gene. Each mutant clone has a unique DNA identifier tag, or “barcode,” to allow for quick and convenient identification of the clone and its mutation. The use of such a library allows for rapid, quantitative, sensitive and simple identification of genes which interact with a mutated target gene. So identified genes are promising targets for drug screening.

Abstract: The infection of a mammalian host by a microorganism can be prevented or treated through the alteration of the C. albicans homologue of the high affinity phosphodiesterase, PDE2, gene and/or the adenylate cyclase-associated protein gene. These methods may be used in the identification, prevention or treatment of microbial infection of mammalian hosts such as immunocompromised or immunosuppressed humans, for example, those having AIDS or undergoing transplantation or anti-cancer therapy.

Abstract: The infection of a mammalian host by a microorganism can be prevented or treated through the disruption of the C. albicans homologue of adenylate cyclase-associated protein gene. These methods may be used in the identification, prevention or treatment of microbial infection of mammalian hosts such as immunocompromised or immunosuppressed humans, for example, those having AIDS or undergoing transplantation or anti-cancer therapy.

Abstract: The present invention provides for methods of screening agents for cancer therapeutic and prophylactic activity. In particular embodiments, cells of the cellular slime mold Dictyostelium discoideum are contacted with candidate agents and the expression of genes in the Nucleotide Excision Repair (NER) and Base Excision Repair (BER) pathways are examined. Such genes include the helicases repB and repD, and the apurinic-apyrmidinic endonuclease APE.

Abstract: Trichoderma spp. strains with high capacity for fungus biological control in wide ranges of temperature and pH are described, such strains being compatible with each other. Likewise, a process of selection of such strains through molecular markers is described. The described process reduce the number of necessary experiments to determine if a Trichoderma strain, not previously described, can display a biological activity more acceptable than those well known.

Abstract: The present invention provides recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed, in operable linkage therewith, wherein the transcription promoter comprises a region found upstream of the open reading frame of a highly expressed Phaffia gene, preferably a glycolytic pathway gene, more preferably the gene coding for Glyceraldehyde-3-Phosphate Dehydrogenase. Further preferred recombinant DNAs according to the invention contain promoters of ribosomal protein encoding genes, more preferably wherein the transcription promoter comprises a region found upstream of the open reading frame encoding a protein as represented by one of the amino acid sequences depicted in any one of SEQIDNOs: 24 to 50.

Abstract: Yeast cells are mutagenized to obtain desirable mutants. Mutagenesis is mediated by a defective mismatch repair system which can be enhanced using conventional exogenously applied mutagens. Yeast cells with the defective mismatch repair system are hypermutable, but after selection of desired mutant yeast strains, they can be be rendered genetically stable by restoring the mismatch repair system to proper functionality.

Abstract: An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having a flavanone-7-O-glucoside-2″-O-rhamnosyl-transferase catalytic activity and its uses.

Abstract: A method of cloning mammalian genes encoding proteins which can function in microorganisms, particularly yeast, and can modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the microorganism. It further relates to mammalian genes cloned by the present method, as well as to products encoded by such genes and antibodies which can bind the encoded proteins. More specifically, the present invention relates to a method of cloning mammalian genes which encode products which modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates or in a biochemical pathway which is controlled, directly or indirectly, by a RAS protein, to products (RNA, proteins) enocded by the mammalian genes cloned in this manner and to antibodies which can bind the encoded proteins.

Abstract: The present invention provides formaldehyde dehydrogenase genes (FLD) from methylotrophic yeasts. The FLD structural genes confer resistance to formaldehyde and are therefore useful as a selectable marker in methylotrophic yeasts. The FLD promoter sequences are strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Induction under either methanol, methylamine or both provides levels of heterologous gene expression comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1). The FLD promoter of Pichia pastoris (PFLD1)is an attractive alternative to PAOX1 for expression of foreign genes in P. pastoris, allowing regulation by carbon (methanol) or nitrogen (methylamine) source within the same expression strain.

Abstract: A DNA construct comprising: (1) a selective marker gene, (2) a galactose-inducible growth inhibition sequence, (3) a pair of FRT sequences in the same orientation flanking (1) and (2), and (4) a DNA fragment capable of recombining with a yeast chromosomal DNA located at each end of (3), wherein said FRT sequences contain the following sequence: 5′-GAAGTTCCTATAC TTTCTAGA GAATAGGAACTTC-3′ (SEQ ID NO: 1) inverted spacer inverted repeat (1) sequence repeat (2) or a sequence substantially identical to said sequence.

Abstract: Vectors for the expression in yeast of mammalian plasminogen derivatives such as microplasminogen and miniplasminogen are presented. Methods for expression of these proteins in a methylotrophic yeast expression system are disclosed as well as the activation and stabilisation of the recombinant proteins. The proteins of this invention are used In the treatment of focal cerebral ischemic infarction and other thrombotic diseases.

Abstract: Compositions, methods, and kits are provided for efficiently generating and screening humanized antibody with high affinity against a specific antigen. The library of humanized antibody is generated by mutagenizing a chimeric antibody template that combines human antibody framework and antigen binding sites of a non-human antibody. Alternatively, the library of humanized antibody is generated by grafting essential antigen-recognition segment(s) such as CDRs of the non-human antibody into the corresponding position(s) of each member of a human antibody library. This library of humanized antibody is then screened for high affinity binding toward a specific antigen in vivo in organism such as yeast or in vitro using techniques such as ribosome display or mRNA display. The overall process can be efficiently performed in a high throughput and automated manner, thus mimicking the natural process of antibody affinity maturation.

Abstract: Methods for producing modified polypeptides containing amino acid analogues are disclosed.

Abstract: The present invention provides genetically engineered strains of Pichia capable of producing proteins with reduced glycosylation. In particular, the genetically engineered strains of the present invention are capable of expressing either or both of an &agr;-1,2-mannosidase and glucosidase II. The genetically engineered strains of the present invention can be further modified such that the OCH1 gene is disrupted. Methods of producing glycoproteins with reduced glycosylation using such genetically engineered stains of Pichia are also provided.

Abstract: The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components.

Abstract: The invention pertains to methods, kits, molecules and cells to increase the rate or recombination and/or target recombination in dividing cells. In a particular aspect, the invention concerns methods and kits to induce targeted meiotic recombination.

Abstract: The present invention provides genetically engineered strains of methylotrophic yeast including Pichia and especially Pichia pastoris capable of producing proteins with reduced or modified glycosylation. Methods of producing glycoproteins with reduced and/or modified glycosylation using such genetically engineered strains of Pichia are also provided. Vectors, which comprise coding sequences for &agr;-1,2-mannosidase I, glucosidase II, GlcNAc-tranferase I and mannosidase II or comprising OCH1 disrupting sequence, for transforming methylotrophic yeasts are contemplated by the present invention. Kit for providing the comtemplated vectors are also included in this invention.

Abstract: The present invention describes novel transcriptional activators and activation systems. The activators of the present invention comprise a DNA binding moiety linked to a short peptide of novel sequence. Preferably, the peptide is substantially hydrophobic. Preferred peptides include at least one aromatic amino acid. The present invention also provides improved transcriptional activation systems, useful for the identification and characterization of protein-protein interactions. The invention also describes the production and use of certain TBP mutants that enhance transcriptional activation by some activators.

Abstract: Recombinant host cells that comprise recombinant DNA expression vectors that drive expression of a product and a precursor for biosynthesis of that product can be used to produce useful products such as polyketides in host cells that do not naturally produce the product or produce the product or precursor at low levels due to the absence of the precursor or the presence of the precursor in rate limiting amounts.

Abstract: A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

Abstract: The present invention relates to a method of identifying protein Constitutively Active Mutants (CAMs) and the use thereof.

Abstract: A method is described for the in vivo identification of epitopes of an intracellular antigen comprising the steps of :a) co-transforming of cells by a first vector including the nucleotide sequence encoding for the region of an antibody able to recognise and bind the intracellular antigen and by a second vector comprising the nucleotide sequence encoding for a peptide; b) growing co-transformed cells in such an environment that only cells wherein the antibody region and peptide recognise and interact each other are able to replicate and/or be recognised because: the antibody region able to recognise and bind the intracellular antigen is associated with a first molecule; the peptide is associated with a second molecule; the interaction of the first with the second molecule generates a selectable phenotype and/or recognisable signal; and the interaction of the first with the second molecule occurs only when the antibody region and peptide recognise and interact each-other; c) selecting the b) cells and identify

Abstract: Compositions and methods for the inducible expression of genes in eukaryotic cells. Expression of a nucleotide sequence of interest is controlled by a regulatory fusion protein that consists of a transcription blocking domain and a ligand-binding domain. When the cognate ligand for the ligand-binding domain is present, transcription of the nucleotide sequence of interest is blocked. Upon removal of the cognate ligand, the nucleotide sequence of interest is transcribed. The method is useful for large scale production of a desired product in eukaryotic cells.

Abstract: The invention provides, inter alia, a method of preparing a nucleic acid sequence that encodes a polypeptide that is displayed on a heterologous cell surface. The generally includes recombining a donor nucleic acid and an acceptor nucleic acid to form a recombined nucleic acid that encodes a polypeptide that is displayed. The recombination reaction is typically an in vivo reaction, in that at least the resolution of recombination intermediates occurs within a cell. Both site-specific recombination and homologous recombination can be used.

Abstract: The present invention relates to the use of Pseudozyma spp. for the production of recombinant polypeptides, proteins or peptides, by genetically transforming fungi with a plasmids or appropriate DNA expression vector.

Abstract: Disclosed herein are methods for generating recombinant DNA molecules in cells using homologous recombination mediated by recombinases and similar proteins. The methods promote high efficiency homologous recombination in bacterial cells, and in eukaryotic cells such as mammalian cells. The methods are useful for cloning, the generation of transgenic and knockout animals, and gene replacement. The methods are also useful for subcloning large DNA fragments without the need for restriction enzymes. The methods are also useful for repairing single or multiple base mutations to wild type or creating specific mutations in the genome. Also disclosed are bacterial strains and vectors which are useful for high-efficiency homologous recombination.

Abstract: Yeast cells are mutagenized to obtain desirable mutants. Mutagenesis is mediated by a defective mismatch repair system which can be enhanced using conventional exogenously applied mutagens. Yeast cells with the defective mismatch repair system are hypermutable, but after selection of desired mutant yeast strains, they can be be rendered genetically stable by restoring the mismatch repair system to proper functionality.

Abstract: Compositions, methods, and kits are provided for efficiently generating and screening a library of highly diverse protein complexes for their ability to bind to other proteins or oligonucleotide sequences. In one aspect of the invention, a library of expression vectors is provided for expressing the library of protein complexes. The library comprises a first nucleotide sequence encoding a first polypeptide subunit; and a second nucleotide sequence encoding a second polypeptide subunit. The first and second nucleotide sequences each independently vary within the library of expression vectors. In addition, the first and second polypeptide subunit are expressed as separate proteins which self-assemble to form a protein complex, such as a double-chain antibody fragment (dcFv or Fab) and a fully assembled antibody, in cells into which the library of expression vectors are introduced.

Abstract: Methods are presented for enhancing the efficiency of oligonucleotide-medidated repair or alteration of genetic information. The methods comprise using cells or cell-free extracts having altered levels or activity of at least one protein from the RAD52 epistasis group, the mismatch repair group or the nucleotide excision repair group. Kits and compositions are also presented.