Abstract: Methods are provided for isolation of DNA sequences encoding proteins with properties of interest by means of expression cloning in filamentous fungal host cells. The isolated DNA sequences are useful in processes for producing the proteins of interest.

Abstract: The present invention includes compositions and methods for site-specific polynucleotide replacement in eukaryotic cells. These methods include single polynucleotide replacement as well as gene stacking methods. Preferred eukaryotic cells for use in the present invention are plant cells and mammalian cells.

Abstract: A Bifidobacterium comprising a genome that is customized so as to lack an operable functional gene is disclosed. A method of making such cells is also disclosed. The method is used to make Bifidobacterium cells that lack certain functional antibiotic resistance genes, such as tetW, and are sensitive to antibiotics such as tetracycline.

Abstract: The present invention concerns to recombinant influenza viruses and modified Vaccinia Ankara viruses (MVA), and to a process for construction of recombinant influenza viruses and modified vaccinia Ankara viruses (MVA) with genes that encode for the T. gondii parasite SAGI (MVA) and SAG2 (MVA and influenza) proteins, by means of a homologous recombination technique between two transfer vectors (for construction of MVA virus) and reverse genetics (for construction of influenza virus). Additionally, the present invention describes a vaccine composition using recombinant influenza viruses and modified vaccinia Ankara viruses (MVA), or recombinant adenoviruses and modified vaccinia Ankara viruses (MVA), for immunization against infections caused by the T. gondii parasite.

Abstract: The present invention relates to a process for allowing homologous recombination between non-identical DNA sequences of an organism and various applications thereof.

Abstract: The invention uses recombinant technology to create infectious molecular clones that capture the sequence diversity of viral genes found in natural populations of mixed genotype viruses, such as arises during HIV infections and many other viral diseases. The invention captures the sequence diversity of different genes in these “quasi-species” populations by recombining them into in a constant genetic “backbone” for each viral species by backcrossing PCR products derived from quasispecies gene variants into this backbone in an E. coli BAC plasmid.

Abstract: A method of double crossover homologous recombination in a host cell comprising: a first homologous recombination event between a donor DNA molecule comprising a first element of a selectable allele and an acceptor DNA molecule comprising a second element of the selectable allele in the host cell, thereby to form a product of the first homologous recombination event in the host cell; and a second homologous recombination event within the product of the first homologous recombination event, thereby to form a product of the second homologous recombination event in the host cell which confers a selectable phenotype on the host cell, wherein the selectable phenotype arises following and in dependency on the formation of a selectable allele from the first and second elements of the selectable allele.

Abstract: A circular DNA molecule, useful for gene therapy, comprising at least one nucleic acid sequence of interest, characterised in that the region allowing the replication thereof has an origin of replication with a functionality in a host cell that requires the presence of at least one specific protein foreign to said host cell. A method for preparing same, cells incorporating said DNA molecules and uses thereof in gene therapy are also described.

Abstract: The present invention is directed to methods for producing and selecting new mutant strains of B. fragilis that constitutively express a particular capsular polysaccharide or only selected capsular polysaccharides; compositions directed to the new mutant strains of B. fragilis that constitutively express a particular capsular polysaccharide or only selected capsular polysaccharides; improved methods for purification of individual capsular polysaccharides; and compositions directed to new res02 and inv19 genes and their gene products. Significantly, the present invention provides methods and compositions for overexpressing and purifying immunomodulatory capsular polysaccharide A (PSA) in high yield.

Abstract: A nucleic acid molecule encoding the gilvocarcin V gene cluster and subunits thereof. Recombinant vectors and host cells comprising a nucleic acid compound encoding the gilvocarcin V gene cluster or subunits thereof. Host cells comprising recombinant vectors encoding the gilvocarcin polyketide synthase and gilvocarcin post-PKS modifying enzymes from Streptomyces griseoflavus can be used to produce gilvocarcin and functional gilvocarcin mutants, analogs and derivatives thereof with application as antibiotics, anticancer agents, immunosuppressants, antivirals, and neuroprotective agents.

Abstract: An isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, with the L-aspartic acid at position 5 of the amino acid sequence replaced by another proteinogenic amino acid, and possesses citrate synthase activity. In addition, a vector comprises the polynucleotide and a bacterium comprises the vector. An isolated polynucleotide comprises a nucleotide sequence comprising, from position 1 to 39, the nucleotide sequence corresponding to position 1 to 39 of SEQ ID NO: 11, from position 40 to 105, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12, with each proteinogenic amino acid except L-aspartic acid being present at position 5. A method of producing an L-amino acids is also described.

Abstract: The invention relates to recombination systems and methods for eliminating nucleic acid sequences from the chromosomal DNA of eukaryotic organisms, and to transgenic organisms—preferably plants—which comprise these systems or were generated using these methods.

Abstract: An enteric bacterial strain was engineered to over-produce L-tyrosine using a one-step method. The pheA-tyrA chromosomal region of the bacterial genome was replaced with an engineered chromosomal segment, resulting in inactivation of the pheA coding region and strong expression of the tyrA coding region, resulting in high levels of L-tyrosine production.

Abstract: The present invention relates to methods and compositions for engineering Clostridia species. In particular, embodiments of the present invention relate to the expression of recombinant resolvase proteins in Clostridia species.

Abstract: The present invention is directed to methods for producing and selecting novel mutant strains of B. fragilis that constitutively express a particular capsular polysaccharide or only selected capsular polysaccharides; compositions directed to the novel mutant strains of B. fragilis that constitutively express a particular capsular polysaccharide or only selected capsular polysaccharides; improved methods for purification of individual capsular polysaccharides; and compositions directed to novel res02 and inv19 genes and their gene products. Significantly, the present invention provides methods and compositions for overexpressing and purifying immunomodulatory capsular polysaccharide A (PSA) in high yield.

Abstract: The present invention relates to genetic modification for industrial applications of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic. The present invention comprises a method for modifying moderately thermophilic Bacillus species that are facultative anaerobic and homolactic by genetic engineering comprising: introducing a DNA cloned in a thermosensitive plasmid system containing a pSH71 replicon or a homologue thereof into cells of a moderately thermophilic Bacillus species that is facultative anaerobic and homolactic; culturing the cells on a selective medium at a permissive temperature for plasmid replication to select transformed cells capable of growing on said selective medium at said permissive temperature; culturing said transformed cells on a selective medium at a non-permissive temperature for plasmid replication to select transformed cells capable of growing on said selective medium at said non-permissive temperature.

Abstract: The invention relates to nucleic acid modifications for a directed expression modulation by the targeted insertion or removal of CpG dinucleotides. The invention also relates to modified nucleic acids and expression vectors.

Abstract: The present invention relates generally to the fields of gene therapy, immunology, and vaccine technology. More specifically, the invention relates to a novel system that can rapidly generate high titers of adenovirus vectors that are free of replication-competent adenovirus (RCA). Also provided are methods of generating these RCA-free adenoviral vectors, immunogenic or vaccine compositions comprising these RCA-free adenovirus vectors, methods of expressing a heterologous nucleic acid of interest in these adenovirus vectors and methods of eliciting immunogenic responses using these adenovirus vectors.

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 rendered genetically stable by restoring the mismatch repair system to proper functionality.

Abstract: Novel genes encoding P. pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 are disclosed. A method for inactivating alternately at least two biosynthetic pathways in a methylotrophic yeast is provided. A method for producing and selecting yeast strains characterized as being capable of genetic integration of heterologous sequences into the host genome using the genes involved in the biosynthetic pathways is also disclosed.

Abstract: The construction of a mutant in the phoP gene by means of homologous recombination from a clinically isolated Mycobacterium tuberculosis reduces the virulence thereof in mouse bone marrow macrophage. Moreover, the phoP mutant reduces the virulence thereof in the experimental mouse model. Said phoP mutant can persist without being eliminated both in the macrophage and in the mouse. Mice inoculated with the phoP mutant are protected against M. tuberculosis infection. The use of mutants of mycobacteria in which the phoP gene or the genes regulated by phoP have been inactivated are candidates for vaccines against human and animal tuberculosis as well as possible recombinant vaccines against other pathogens.

Abstract: The present invention provides a process for producing yeast excellent in cell productivity and gene manipulation of which is easy, being added with nutritional requirement by disrupting only a specific gene, and a transformant thereof. Moreover, the present invention also provides a process for producing a gene expression product, particularly a polyhydroxyalkanoic acid. In the present invention, yeast in which a plurality of genes is disrupted is produced using the homologous recombination. Moreover, a transformant is obtained by introducing a plurality of enzyme genes involved with polyhydroxyalkanoic acid synthesis such as a polyhydroxyalkanoic acid synthase gene and an acetoacetyl CoA reductase gene into said gene-disrupted yeast. Furthermore, said transformant is cultured, copolyesters comprising a polyhydroxyalkanoic acid are efficiently accumulated within the cells, and a polymer is harvested from the cultured product.

Abstract: The application relates for method for cloning the gene comprising the steps of: 1. Providing a replication-deficient baculovirus vector, 2. Providing a rescue vector comprising (a) nucleic acid sequence which is capable of restoring replication in the replication-deficient baculovirus vector and (b) at least one gene to be cloned; 3. Causing the replication-deficient baculovirus vector and rescue vector to recombine to produce a replication-enabled baculovirus vector comprising the at least one gene to be cloned; and 4. Growing the replication-enabled baculovirus vector within a suitable invertebrate cell, such as an insect cell. Preferably the baculovirus vector is based upon AcMNPV. Also disclosed are replication-deficient baculovirus vectors, rescue vectors, cells containing such vectors and kits comprising such vectors.

Abstract: The present invention provides a novel method of significantly promoting the ratio of homologous recombination in desirable cells. In order to enhance the ratio of homologous recombination in desirable cells such as eukaryotic cells, mutation is introduced into genes encoding factors necessary for non-homologous recombination, such as KU70 or KU80, or the above genes are disrupted, so as to cause the loss of the functions thereof. At the time, foreign DNA is introduced into the cells via the electroshock method or the like, so as to carry out homologous recombination, thereby promoting the frequency of homologous recombination in the cells.

Abstract: A recombinant, attenuated strain of Brucella suis or Brucella melitensis with a deficiency in carboxyl-terminal protease activity or tail-specific protease activity can be used as a vaccine for the prevention or treatment of Brucellosis. Prior exposure to the Brucella species is identified by detecting a genetic sequence for carboxyl-terminal (i.e. tail-specific) protease activity in a biological sample.

Abstract: The present invention relates to mutants cells comprising a marker-free modification of a gene, and methods for obtaining and using such mutant cells.

Abstract: An electronic sensor is provided for detecting the presence of one or more targets of interest in a sample. The sensor preferably comprises a special type of field effect transistor in which conductance is enhanced by target binding to recognition elements in the active region. An array of sensors may be formed to analyze a sample for multiple targets. The sensor may be used, for example, to detect the presence of pathogens, polypeptides, nucleic acids, toxins and other biochemical and chemical agents. The sensor is useful in a wide variety of applications including medical diagnostics, agriculture, public health, environmental monitoring and biomedical research.

Abstract: An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: Antisense compounds, compositions and methods are provided for modulating the expression of kinesin-like 1. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding kinesin-like 1. Methods of using these compounds for modulation of kinesin-like 1 expression and for treatment of diseases associated with expression of kinesin-like 1 are provided.

Abstract: The present invention provides adapter-directed display systems for expressing exogenous polypeptide within a host cell and/or displaying the exogenous polypeptide on the outer surface of a genetic package. This subject systems are particularly useful for displaying a genetically diverse repertoire of monomeric and multimeric polypeptides. The invention also provides both expression and helper vectors and kits containing components of the subject display systems. Also provided are genetic packages displaying the exogenous polypeptides of particular interest. Further provided by the invention are methods of using the subject display systems.

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 present invention provides an improved method of making eukaryotic gene transfer vectors comprising homologous recombining lambdid vectors with a second DNA in a bacterium to generate novel recombinant eukaryotic viral gene transfer vectors as well as a novel lambdid vector used in the inventive method and an inventive system comprising the novel lambdid vector.

Abstract: The present invention relates to bacterial luciferase transposon cassettes suitable for conferring bioluminescence properties on a Gram-positive bacteria, Gram-negative bacteria, and other organisms of interest. The invention further includes cells transformed with vectors carrying the transposon cassettes, cells whose genomes have been modified by introduction of such cassettes, and methods of making and using such transposon cassettes, transposon cassette vectors, and cells containing the transposons.

Abstract: The present invention relates to mutant MVA vaccinia viruses, which are used for the generation of recombinant MVA viruses, as well as host cells, which have been infected with these mutant MVA viruses. The present invention further relates to DNA-vector constructs, and a method for the generation of recombinant MVA by using the mutant MVA viruses and the DNA-vector constructs. The mutant MVA vaccinia viruses of the present invention are characterized in that the MVA ORF 050L gene or a functional part thereof has been inactivated in the viral genome.

Abstract: Methods for preparing yeast with improved biotin productivity using integrating plasmids encoding biotin synthase. The yeast is transformed by an integrating plasmid, which includes a Candida utilis biotin synthase gene BIO2, an assistant DNA sequence to promote integration of the plasmid into the C. utilis genome, a promoter sequence, and a selection marker. Other embodiments include Saccharomyces cerevisiae integrating plasmids.

Abstract: The invention provides a novel Adenovirus backbone plasmid, which when co-transfected with a shuttle vector, allows for production of recombinant viruses quickly and easily. The present invention also provides host cells and a cloning system for generating recombinant adenoviruses.

Abstract: This invention relates to the screening of nucleic acids. More particularly, the present invention provides a dysfunctional viral genome capable of both expressing libraries of exogenous nucleic acids and selecting the sequences having a predefined characteristic or function within the cell, such as nucleic acids encoding signal peptides, secreted proteins, membrane bound proteins, proteases and drug-resistance proteins. The invention further provides a method and a kit for selecting nucleic acids having a desired feature, wherein production of a viral particle is dependent on insertion of an exogenous nucleic acid having the desired feature into a dysfunctional viral genome or into a viral genome exposed to a substance inhibiting viral packaging function(s).

Abstract: The invention encompasses Drosophila Recombination Associated Protein (DRAP) isolated D. melanogaster and a nucleic acid sequence encoding DRAP. The Drosophila Recombination Associated Protein, its homologues from other organisms or active peptides derived therefrom, as well as DNA encoding such protein are useful for homology-dependent pairing of three DNA strands. The combination of strand-transfer and topoisomerase activities associated with DRAP permits directed pairing and cleavage at defined site(s) within DNA. This in turn makes possible the isolation and/or removal of a defined segment of DNA. DRAP is also useful in cloning, genomic cloning and gene mapping, in promoting gene disruptions or “knockout” mutations, in carrying out targeted mutagenesis of specific genes and in generating transgenic animals.

Abstract: The present invention provides compositions, including vectors, and methods for the rapid subcloning of nucleic acid sequences in vivo and in vitro. In particular, the invention provides vectors used to contain a gene of interest that comprise a sequence-specific recombinase target site. These vectors are used to rapidly transfer the gene or genes of interest into any vector that contains a sequence-specific recombinase target site located downstream of a regulatory element so that the gene of interest may be regulated.

Abstract: The present invention solves the problem of integrating multiple copies of a gene of interest by homologous recombination into well defined positions adjacent to conditionally essential genes in a bacterial host strain chromosome, which already comprises at least one copy of the gene of interest in a different position.

Abstract: The invention relates to a method for increasing the copy number of a chromosomally integrated expression cassette in a microbial strain without leaving antibiotic resistance markers behind in the strain, the necessary genetic constructs, and the strains resulting from the method of the invention.

Abstract: The invention relates to a nucleic acid preparation with a content of below 1% protein, preferably below 0.1% protein, free of ethidium bromide, phenol, caesium chloride and detergents based on octyl phenol poly(ethylene glycol other)n and with a content of below 1 EU/mg DNA of endotoxins. Said preparation is suitable as a drug particularly in gene therapy.

Abstract: This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the &PHgr;C31 integrase, that can mediate recombination between the recombination sites, but not between hybrid recombination sites that are formed upon the recombination. Thus, the recombination is irreversible in the absence of additional factors. Eukaryotic cells that contain the recombinase polypeptides, or genes that encode the recombinases, are also provided.

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: 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: The invention concerns a new tool for efficient mutagenesis enabling the generation of a collection of mutants in fungi by random insertion of a characterized Fusarium oxysporum Impala transposon in the genome of said fungi. The invention also concerns the resulting mutants.

Abstract: An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: A enterotoxin-deficient mutant of a member strain of the Bacillus cereus group does not produce HBL enterotoxin, which has been regarded as a human pathogen found in member strains. An enterotoxin-deficient mutant is suitable for use as a biocontrol agent. Methods for making the mutant and for using the mutant are described.