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.

Abstract: The present invention provides novel expression systems for producing desired polypeptides in certain strains of yeast. The present invention further provides methods for producing polypeptide products using such expression systems. The present invention also provides compositions relating to the same.

Abstract: Methods and materials are provided for stably introducing any gene into a specific locus in the genome of a microorganism such as yeast without the addition of any drug resistance genes. Specifically provided herein are new genetically engineered inositol-overproducing Saccharomyces cerevisiae strains obtained by using a novel set of yeast integration plasmids that allow the safe, stable, and controlled introduction of homologous as well as heterologous genes into the host genome. In particular, specific loci of the S. cerevisiae yeast genome can be targeted with single or multiple copies of a specific gene that is desired to be expressed or a given set of specific genes that the host can use without the addition of any drug resistance genes. The principles of this new methodology can also be used for the construction of other recombinant yeast and bacterial strains as well as higher eukaryotic cells.

Abstract: The invention provides shuttle vectors, and methods of using shuttle vectors, capable of expression in, at least, a mammalian cell. Furthermore, the shuttle vectors are capable of replication in at least yeast, and optionally, bacterial cells. Also provided is a method wherein yeast are transformed with a shuttle vector as provided herein. Heterologous nucleic acids flanked by 5′ and 3′ ends identical to a homologous recombination site within the shuttle vector are introduced to the transformed yeast and allowed to homologously recombine with the shuttle vector such that they are inserted into the vector by the yeast organism. The shuttle vector is then recovered and transferred to a mammalian cell for expression.

Abstract: Improved methods, compositions, and kits for oligonucleotide-mediated nucleic acid sequence alteration are presented.

Abstract: The present invention provides novel targets in eukaryotic cells for antibiotic agents and methods for identification of antibiotic agents affecting such targets and the use of the identified antibiotic agents in treatment of opportunistic infections in eukaryotic hosts. The invention is based upon the identification of species specific 5′ hinge and 3′ hinge regions of U3 small nucleolar ribonucleic acid (snoRNA). The present invention discloses that the external transcribed spacer (ETS) of the ribosomal RNA precursor (pre-rRNA) comprises sequences that are complementary to the 5′ and 3′ hinge regions of U3 snoRNA. The invention further discloses that sequence substitutions of the 5′ hinge or 3′ hinge region of U3 snoRNA severely compromise or fully inhibit the cleavage events at sites 1 and 2 in rRNA processing necessary to form mature 18S rRNA which is a vital component of the small subunit of ribosomes of all eukaryotes.

Abstract: The invention relates to processes for identifying inhibitors and activators of eukaryotic potassium channels, in which a mutated S. cerevisiae cell is used whose endogenous potassium channels TRK1, TRK2 and TOK1 are not expressed functionally, but which expresses heterologously a eukaryotic potassium channel to be studied. Other subject matters of the invention are mutated S. cerevisiae cells which do not express TRK1, TRK2 and TOK1, and the preparation and use of these mutated S. cerevisiae cells.

Abstract: A fungal microorganism can be engineered by means of genetic engineering to utilise L-arabinose. The genes of the L-arabinose pathway, which were unknown, i.e. L-arabinitol 4-dehydrogenase and L-xylulose reductase, were identified. These genes, together with the known genes of the L-arabinose pathway, form a functional pathway. This pathway can be introduced to a fungus, which is completely or partially lacking this pathway.

Abstract: The present invention relates to a method for producing a modified Saccaromyces cerevisiea having improved phytase activity, such a Saccaromyces cerevisiae, use of such a modified strain, as well as phytase production, and inositol isomers derived from use of such a modified strain.

Abstract: A fibrinolytically active metalloproteinase polypeptide (called “novel acting thrombolytic”) which is useful for blood clot lysis in vivo and methods and materials for its production by recombinant expression are described.

Abstract: Compositions and methods are disclosed for the production of polypeptides sensitive to proteolysis due to their content of arginine and lysine residues. The methods of the invention utilize yeast cells with reduced expression of either or both of the proteases encoded by YAP3 and MKC7. The methods of the invention also utilize yeast cells with reduced activity for either or both of the Yap3 and Mkc7 proteases.

Abstract: The present invention provides methods and compositions that enable the experimental determination as to whether any gene in the genome of a diploid pathogenic organism is essential, and whether it is required for virulence or pathogenicity. The methods involve the construction of genetic mutants in which one allele of a specific gene is inactivated while the other allele of the gene is placed under conditional expression. The identification of essential genes and those genes critical to the development of virulent infections, provides a basis for the development of screens for new drugs against such pathogenic organisms.

Abstract: The present invention describes a novel recombinant NADH recycling system that is used as a process for producing reduced compounds. In a specific embodiment, the reduced compounds include ethanol, succinate, lactate, a vitamin, a pharmaceutical and a biodegraded organic molecule. The NADH recycling system effects metabolic flux of reductive pathways in aerobic and anaerobic environments.

Abstract: The present invention makes available a rapid, reproducible, robust assay system for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular protein, e.g., a receptor or ion channel. The subject assay enables rapid screening of large numbers of compounds to identify those which act as an agonist or antagonist to the bioactivity of the cellular protein. In particular, the assay of the invention makes use of a cell that harbors a protein that is responsive to a cellular signal transduction pathway. The protein is operatively linked to a polypeptide which causes a detectable signal to be generated upon stimulation of the pathway, e.g., when a compound interacts with and modulates the activity of a cellular receptor or ion channel of the cell.

Abstract: Methods are provided for generating highly diverse libraries of expression vectors encoding fusion proteins such as single-chain antibodies via homologous recombination in yeast. The method comprises: transforming into yeast cells a linearized yeast expression vector having a 5′- and 3′-terminus sequence at the site of linearization and a library of insert nucleotide sequences that are linear and double-stranded; and having homologous recombination occur between the vector and the insert sequence such that the insert sequence is included in the vector in the transformed yeast cells.

Abstract: The invention involves a method of identifying nucleic acid sequences encoding signal peptide-containing proteins. The method features chimeric constructs containing a KRE9 gene that lacks a signal sequence. Yeast containing chimeric KRE9 plasmid constructs that encode signal sequences are selected based on their ability to grow on media in which sucrose is the sole carbon source.

Abstract: The invention relates to a strain of the yeast Saccharomyces cerevisiae which, owing to deletion of the genomic sequences, no longer synthesizes hexose transporters and, as a consequence, can no longer grow on substrates with hexoses as the only carbon source, and whose ability of growing on a substrate with a hexose as the only carbon source is restored when it expresses a GLUT4 gene.

Abstract: Disclosed in the present invention is a Zymomonas integrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into the Zymomonas in a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. The Zymomonas integrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media.

Abstract: The invention provides a method for the expression of exogenous DNA libraries in filamentous fungi. The fungi are capable of processing intron-containing eukaryotic genes, and also can carry out post-translational processing steps such as glyclosylation and protein folding. The invention provides for the use of fungi with altered morphology, which permits high-throughput screening and directed molecular evolution of expressed proteins. The same transformed fungi may be used to produce larger quantities of protein for isolation, characterization, and application testing, and may be suitable for commercial production of the protein as well.

Abstract: An object of the present invention is to provide a vector which can be integrated into a yeast chromosome in a high number of copies. Another object of the present invention is to provide a modified vector which can be integrated into the yeast chromosome in a high number of copies and of which expression units stably maintain on the chromosome. The vector according to the present invention comprises a marker gene for selecting transformants, a shortened promoter sequence which is operably linked to the marker gene and a sequence homologous to the chromosomal DNA of Candida utilis, and optionally a heterologous gene or a gene derived from C. utilis, wherein the vector is linearized by cleaving within said homologous DNA sequence or at both ends of the homologous DNA sequence with restriction enzymes, and wherein the heterologous gene or the gene derived from C. utilis can be integrated into the chromosomal DNA of C. utilis by homologous recombination.

Abstract: The present invention relates to a method for obtaining a recombinant yeast of Saccharomyces cerevisiae, which ferments lignocellulose raw materials to ethanol, including introducing DNA into a yeast so as to cause the yeast to have introduced genes encoding xylose reductase, xylitol dehydrogenase and xylulokinase.

Abstract: Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

Abstract: Methods and compositions for the expression of nucleotide sequences are provided. Compositions comprise the nucleic acid sequences of the transcriptional regulatory region of the PpSEC10 gene of Pichia pastoris. Methods are provided to chemically regulate the PpSEC10 transcriptional control region by modulating the iron concentration in the culturing medium. The methods find use in regulating expression of nucleotide sequences. Furthermore, the methods of the invention can be used to regulate polypeptide expression, more particularly in regulating heterologous polypeptide expression, particularly using a yeast host cell as the expression system.

Abstract: Novel genes have been isolated which encode cytochrome P450 and NADPH reductase enzymes of the &ohgr;-hydroxylase complex of C. tropicalis 20336. Vectors including these genes, transfected host cells and transformed host cells are provided. Methods of producing of cytochrome P450 and NADPH reductase enzymes are also provided which involve transforming a host cell with a gene encoding these enzymes and culturing the cells. Methods of increasing the production of a dicarboxylic acid and methods of increasing production of the aforementioned enzymes are also provided which involve increasing in the host cell the number of genes encoding these enzymes. A method for discriminating members of a gene family by quantifying the expression of genes is also provided.

Abstract: The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a promoter foreign to the nucleic acid sequence, wherein the promoter comprises a sequence selected from the group consisting of nucleotides 1 to 3949 of SEQ ID NO. 1, nucleotides 1 to 938 of SEQ ID NO. 2, and nucleotides 1 to 3060 of SEQ ID NO. 3, and a subsequence thereof; and mutant, hybrid, and tandem promoters thereof; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to the isolated promoter sequences and to constructs, vectors, and fungal host cells comprising the promoter sequences operably linked to nucleic acid sequences encoding polypeptides.

Abstract: The present invention relates to T-DNA vectors and methods for obtaining transgenic eukaryotes using said vectors. The transgenic eukaryotes are characterized in that they contain the T-DNA but not the illegitimately transferred vector backbone sequence. This is achieved by modifying the T-DNA borders such that they are more efficiently nicked or such that they allow elimination of illegitimately transferred vector backbone sequences by means of recombination.

Abstract: An invention is described which provides a diagnostic approach for diseases, such as HNPCC, that are associated with defects in MMR and provides a method for determining whether any specific genetic sequence of a gene associated with MMR that differs from a consensus sequence is a mutation (i.e., encodes a non-functional protein), a silent polymorphism (i.e., encodes a protein with normal protein function) or an efficiency polymorphism (i.e., encodes a protein with reduced efficiency in MMR). The invention allows the generation of databases of the functional significance of specific amino acid replacements on MMR protein function in vivo, which in turn will allow accurate and unambiguous interpretation of genetic tests of MMR.

Abstract: The present invention is directed to a method for selecting a prey polypeptide that is able to interact with a bait polypeptide of interest, to a prey polynucleotide encoding the prey polypeptide as well as to the prey polypeptide itself. The invention also concerns plasmids used for performing the method of the invention as well as prokaryotic or eukaryotic recombinant host organisms containing such plasmids and also a collection of said recombinant host organisms consisting in a DNA library, such as a collection of recombinant haploid Saccharomyces cerevisiae. Finally, the invention is also directed to a technical medium containing the whole information concerning the interactions between metabolically related bait and prey polypeptides and/or polynucleotides coding for bait and prey polypeptides.

Abstract: The invention relates to the modulation of fungal signaling pathways. More particularly, the invention relates to compounds that modulate such pathways and to methods for identifying such compounds. The invention provides novel modulators of fungal gene expression that can be used to regulate or modulate activities of specific signaling pathways and to identify and validate potential targets in drug discovery efforts.

Abstract: This invention relates to an isolated nucleic acid fragment encoding scorpion toxins that are K-channel modifiers. The invention also relates to the construction of a chimeric gene encoding all or a substantial portion of the K-channel modifier, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the K-channel modifier in a transformed host cell.

Abstract: The present invention relates to the use of a class of genes called oil body protein genes that have unique features. The discovery of these features allowed the invention of methods for the production of recombinant proteins wherein a protein of interest can be easily separated from other host cell components. The invention is further exemplified by methods for exploitation of the unique characteristics of the oil body proteins and oil body genes for expression of polypeptides of interest in many organisms, particularly plant seeds. Said polypeptides may include but are not limited to: seed storage proteins, enzymes, bioactive peptides, antibodies and the like. The invention can also be modified to recover recombinant polypeptides fused to oil body proteins from non-plant host cells. Additionally the invention provides a method of using recombinant proteins associated with seed oil bodies released during seed germination for expression of polypeptides that afford protection to seedlings from pathogens.

Abstract: The invention relates to nucleic acids which encode fungal polypeptides with the biological activity of acetoacetyl-CoA thiolase, to the polypeptides encoded by them and to their use as targets for fungicides and to their use for identifying new, fungicidally active compounds, and to methods of finding modulators of these polypeptides and, finally, to transgenic organisms containing sequences encoding fungal polypeptides with the function of an acetoacetyl-CoA thiolase.

Abstract: The invention concerns the integration of a gene of interest into the genome of a Yarrowia strain devoid of zeta sequences, by transforming said strain using a vector bearing zeta sequences.

Abstract: The present invention provides a universally usable knockout cassette that contains a selectable marker gene and is characterized by the specific design of the restriction cleavage sites. A method for preparing the knockout cassette is also provided as are methods of using the knockout cassette.

Abstract: Methods for the rapid repression of gene function in eucaryotic cells are disclosed including inducible means for both shutting down a targeted gene's transcription and rapidly removing a targeted gene's polypeptide product.

Abstract: The invention discloses a yeast with high biotin-productivity and the preparation method thereof. The yeast is transformed by an integrated plasmid, which includes a biotin synthase gene, an assistant DNA sequence for the integration of the plasmid into a host genome, a promoter sequence, and a selection marker.

Abstract: The present invention is directed to methods for producing gene targeting constructs by homologous recombination using mouse genomic libraries arrayed in shuttle vectors. The invention is also directed to methods of using targeting constructs made by the methods to generate transgenic animals.

Abstract: Cot-based cloning and sequencing (CBCS) is a method that permits the cloning and sequencing of an organism's sequence complexity at unprecedented efficiency. DNA renaturation kinetics (i.e., Cot) methods are used to fractionate genomic DNA into single-copy and repeat sequence components, each isolated kinetic component is used to construct a corresponding DNA library, and clones from each library are sequenced in numbers proportional to the complexity of the component from which they were derived. For some species, the number of clones that need to be sequenced in order to attain a specific level of coverage via CBCS is less than one-tenth the number required to achieve the same level of coverage using shotgun sequencing (the current means by which genomes are sequenced).

Abstract: This invention is related to yeast which is transformed to cDNA of deer velvet antler for production of efficacy substance.

Abstract: The invention concerns a process for the recombinant production and expression of eukaryotic alkaline phosphatase in yeast cells in which in particular a DNA coding for a eukaryotic highly active alkaline phosphatase having a specific activity above 3000 U/mg is used. The invention additionally concerns a process for inserting corresponding nucleic acid sequences into a vector for expression in methylotrophic yeast strains and it concerns appropriate vectors and host strains.

Abstract: The present invention provides novel expression systems for producing desired polypeptides in certain strains of yeast. The present invention further provides methods for producing polypeptide products using such expression systems. The present invention also provides compositions relating to the same.

Abstract: Surprisingly, the present inventors have discovered that Tn7, a prokaryotic transposon, carries mRNA 3′ end formation site information unique to eukaryotic genes. In vivo gene disruption by Sif, a Tn7-based transposon cassette, in eukaryotic cells can result in pre-mature termination of transcription, yet the resulting mRNA does not appear to rapidly decay as might be expected. These truncated messages are chimeric and polyadenylated. Sif transposons, therefore, can be used for in vitro transposition of selected genes and the resulting construct for in vivo gene replacement in fungi and other eukaryotes. Thus, the present invention provides a method for altering the expression of genes of interest in filamentous fungi and eukaryotes. The resulting mutant genes can be isolated and are useful for identification of functional domains in genes/proteins by methods including, but not limited to, yeast complementation assays or in vitro assays.

Abstract: The present invention provides methods of preparing gene targeted mammalian cells having a targeted gene mutation methods of making gene targeted mice, and gene targeting vectors that are useful in these methods.

Abstract: The present invention provides a novel dominant selectable marker system in yeast that is based on an aminoglycoside, nourseothricin (NST). This compound possesses a powerful antifungal activity against Candida albicans and S. cerevisiae. The invention provides a cognate drug resistance marker for use in gene transformation and disruption experimentation in Candida albicans and Saccharomyces cerevisiae. In particular, the invention presents: 1) direct utility for gene manipulations in both clinically and experimentally relevant strains regardless of genotype and without affecting growth rate, or hyphal formation; and 2) applicability to antifungal drug discovery, including target validation and various forms of drug screening assays.

Abstract: A method for detecting protein-protein interactions is provided, in which two fusion proteins are prepared and allowed to interact with each other in yeast cells. The interaction between the two fusion proteins leads to protein trans-splicing, generating an active and detectable reporter.

Abstract: The present invention relates, in general, to a novel genus of bacteria known as Ketogulonigenium. The present invention further relates to transformed Ketogulonigenium, and methods of transforming Ketogulonigenium. The present invention also relates to nucleic acid molecules, and vectors.

Abstract: A Pichia microorganism is transformed with DNA for the expression of a pertactin antigen whose amino acid sequence is at least 95% homologous with that set forth in FIGS. 1A, 1B, or 1C, or antigenic fragment thereof.

Abstract: An isolated DNA having the promoter sequence of the hex gene of P. chrysogenum or a DNA fragment that is hybridizable to the complement of the promoter sequence under stringent conditions and is capable of directing expression of DNA downstream of the fragment in P. chrysogenum. Also a process for promoting expression of a coding sequence of interest in a microorganism using the isolated DNA and a process to block expression of a gene of interest in a microorganism using the isolated DNA are disclosed.

Abstract: The present invention relates to an improved method of recombination for site-specific recombinase-mediated recombination using mutated recognition sequences. For this purpose a non-identical pair of recognition sequence mutants is used. Each of the recognition sequence mutants consists of two recognition sequences separated by a spacer. A mutation is introduced into one of the recognition sequences to create, after recombination by a sequence-specific recombinase, a recognition sequence mutant which is no longer recognized by the recombinase.

Abstract: This invention is directed to the transformation of the flavinogenic yeasts, Pichia guilliermondii and Candida famata, and mutants thereof, by electroporation (electrotransformation) and by spheroplast transformation. The invention is also directed to nucleic acid constructs such as vectors, plasmids, and ARS sequences which transform flavinogenic yeasts, and mutants thereof, at a high level and in a stable manner so as to result in stably transformed yeast host cells which express/produce recombinant products. This invention also is directed to flavinogenic yeasts, Pichia guilliermondii and Candida famata, and mutants and temperature sensitive mutants thereof, which produce or overproduce riboflavin.