Abstract: The present application generally discloses delivery of an agent which can be therapeutic or prophylactic and, more particularly, the preparation and use of attenuated bacteria, such as Salmonella, containing a bacteriophage in which the genome of the bacteriophage has been modified to encode for a gene product of interest, eg., an antigen or an anti-tumor protein. The bacteria functions as a vector for delivering the bacteriophage encoded gene product of interest to an appropriate site of action, e.g., the site of a solid tumor.

Abstract: A new fibrinogen binding protein or polypeptide originating from coagulase negative staphylococci, biotechnological methods for producing the protein or polypeptide having fibrinogen binding activity and a recombinant DNA molecule coding for the protein (or fragments thereof), and micro-organisms (including viruses) containing this recombinant DNA molecule. The present invention further comprises the therapeutic and diagnostic use of the protein and/or DNA, e.g., a diagnostic kit for determining the presence and/or type of coagulase negative staphylococci and a vaccine composition, comprising the protein or DNA.

Abstract: The invention relates to a recombinant NS gene of an influenza A virus comprising a functional RNA binding domain and a gene sequence modification after nucleotide position 400 of the NS1 gene segment, counted on the basis of influenza A/PR/8/34 Virus, wherein the modification bars transcription of the remaining portion of the NS1 gene segment. It further relates to embodiments, wherein the modification comprises deletions, insertions, or a shift of the open reading frame, and particularly to constructs comprising an insertion of an autocleavage site 2A, the nef gene from HIV-1 or the sequence encoding the ELDKWA-epitope of gp4l of HIV-1. The invention also relates to influenza virus transfectants that contain the modified NS gene and have an IFN inducing phenotype but which may or may not be sensitive towards IFN. The invention also relates to vaccines comprising such a chimeric virus.

Abstract: Phagemid vectors containing a sequence of features between a Col E1 origin and an f1 origin are useful for display of polypeptides or proteins, including antibody libraries.

Abstract: The present invention provides a isolated bacteriophage useful as a tool for studying biological, biochemical, physiological and genetic properties of actinomycetes and other organisms which comprises a novel strain of Saccharomonospora having certain specified characteristics. The invention also relates to a process for the isolation of the said bacteriophage and/or DNA phage and to a novel universal growth medium which is particularly useful in the said process. Another embodiment of the process relates to a clonong vector which comprises a plasmid or bacteriophage comprising the phage DNA of the invention.

Abstract: The present invention relates to (a) methods for improving a genetic stability of an insert nucleotide sequence in a recombinant single-stranded RNA virus vector, which comprises performing a mutagenesis of the foreign insert nucleotide sequence to provide even distribution of G/C content throughout the overall foreign insert nucleotide sequence and/or to increase G/C content of the foreign insert without substantially causing amino acids substitutions (b) a recombinant single-stranded RNA virus comprising an insert nucleotide sequence with improved genetic stability and (c) a recombinant poliovirus comprising an insert nucleotide sequence with improved genetic stability.

Abstract: A method for preparing water-stable semiconductor nanocrystal complexes that can be stably coupled to tertiary molecules using a self-assembled coating of diblock polymers. The diblock polymers have hydrophilic ends containing hydrophilic functional groups and hydrophobic ends containing hydrophobic functional groups. The diblock polymers are assembled around a semiconductor nanocrystal having a lyophilic surface outer layer. The diblock polymers are further crosslinked via bridging molecules that link adjacent diblock polymers through the hydrophilic functional groups of the hydrophilic ends of the diblock polymers to form a semiconductor nanocrystal complex. The functional groups present on the outer surface of the amphiphilic diblock polymer may serve as attachment sites for coupling tertiary molecules to the semiconductor nanocrystal complex.

Abstract: The present invention relates to the treatment of coronary heart disease by revascularization therapy, and more particularly to the intramyocardial injection of a pharmaceutical composition comprising a recombinant fibroblast growth factor-1 protein or a fragment of a recombinant fibroblast growth factor-1 protein, optionally, with a physiologic glue for inducing local neoangiogenesis in ischemic myocardium. Methods of producing the recombinant fibroblast growth factor 1 protein and fragments are also disclosed.

Abstract: We describe a process for generating multilayer particles comprising condensing a polymer with an oppositely charged polymer to form a particle and sequentially adding oppositely charged polymers to the particle forming at least three layers of polymers. The process is used to form a composition for delivering a biologically active compound to a cell.

Abstract: The invention described herein relates to compositions and methods for stimulating immune responses in vivo against a tolerogen. Novel biotechnological tools, pharmaceuticals, therapeutics and prophylactics, which concern chimeric or conjugated virus-like particles, and methods of use of the foregoing are provided for the study of B cell tolerance and the treatment or prevention of human diseases, which involve the onset of B cell tolerance, such as chronic viral infection, chronic inflammatory disease, and neoplasia.

Abstract: The present invention relates to chimeric biological vectors for gene trnasduction in eukaryotic cells. The invention further relates to a method for producing said chimerized vectors and a mehod for transduction of eukaryotic cells, in particular cells expressing integrin receptors.

Abstract: The present invention relates to methods for the identification of nucleic acid sequences encoding members of a multimeric (poly)peptide complex by screening for polyphage particles. Furthermore, the invention relates to products and uses thereof for the identification of nucleic acid sequences in accordance with the present invention.

Abstract: The invention provides a two component system for in vitro cloning of a heterologous polynucleotide into adenoviral DNA. The first component is an insert donor comprising a heterologous polynucleotide encoding a heterologous polypeptide. The second component is a vector donor comprising an adenovirus genome and an expression cassette. The insert donor and vector donor are adapted for site specific recombination using recombination sites from phage lambda for insertion of the heterologous polynucleotide into the expression cassette capable of forming an adenoviral expression clone in vitro in the presence of a suitable recombination mediator protein or proteins. The invention also provides a method of making recombinant adenovirus and use of the first and second components in such a method.

Abstract: The invention relates to coryneform bacteria which, instead of the singular copy of an open reading frame (ORF), gene or allele naturally present at the particular desired site (locus), have at least two copies of the open reading frame (ORF), gene or allele in question, preferably in tandem arrangement, and optionally at least a third copy of the open reading frame (ORF), gene or allele in question at a further gene site, and processes for the preparation of chemical compounds by fermentation of these bacteria.

Abstract: The present invention provides a method of transfer of a gene of interest from a first vector to a product vector comprising contacting a first and second vector in vitro with a site-specific recombinase so as to generate a co-integrate vector comprising the components of the first and second vector, and introducing the co-integrate vector to a prokaryotic host cell so as to generate a product vector by rolling circle replication, comprising the gene of interest.

Abstract: The present invention is directed to isolated transducing phages, methods of isolating transducing phages, and methods of using transducing phages including, for instance, transferring at least one nucleic acid fragment from a donor microbe to a recipient microbe, and producing a secondary metabolite from a microbe. The transducing phages typically have a broad host range, and transduce microbes in the Order Actinomycetales, in particular in the Family Streptomycetaceae, including Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae, Streptomyces avermitilis, and Saccharopolyspora erythraea. The transducing phages can be specialized transducing phages or generalized transducing phages.

Abstract: A method for introducing and expressing genes in animal cells is disclosed comprising infecting the animal cells with live invasive bacteria, wherein the bacteria contain a eukaryotic expression cassette encoding the gene. The gene may encode, e.g., a vaccine antigen, a therapeutic agent, an immunoregulatory agent or an anti-sense RNA or a catalytic RNA.

Abstract: The subject invention provides for a method of selectively cloning homoduplex nucleic acid molecules, in particular, by using a strain of host cells that contains a conditionally expressed and/or conditionally active mismatch-recognizing enzyme, e.g., a temperature sensitive variant of the gene encoding the endonuclease VII from phage T4. Using this host strain, the invention features a novel cloning method that selects for PCR products that are devoid of PCR-generated mutations.

Abstract: The subject invention provides for a strain of host cells that contains a temperature sensitive variant of the gene encoding the endonuclease VII from phage T4. Using this host strain, the invention features a novel cloning method that selects for PCR products that are devoid of PCR-generated mutations.

Abstract: The present invention relates to methods and compositions which enable the propagation of vectors containing cDNAs whose presence has hitherto been toxic to conventional bacterial strains. It is based, at least in part, on the discovery that a bacterial strain having an insertional mutation in the malT gene of Escherichia coli tolerated the propagation of a mec-4 cDNA-containing plasmid which was toxic to other bacterial strains. The methods and compositions of the invention may be particularly useful in the propagation of cDNAs encoding membrane proteins. The present invention also provides for ion channel assay systems comprising MEC-2, MEC-4, MEC-10 or variants thereof.

Abstract: The current invention provides method and vectors for selecting open reading frames. Open reading frames present in a fragment of DNA cloned into the vectors of the invention result in creation of a fusion protein between the amino acid sequence encoded by the fusion protein and a reporter protein. The vector further comprises recombination sites so that once a recombinant that comprises an open reading frame is identified, either the reporter sequence or the open reading frame can be removed from the vector.

Abstract: A method for modulating the activity of an AMP-forming enzyme (AFE) is disclosed. The method is based upon the novel observation that the activity of these enzymes is controlled by acetylation of the enzymes to inactivate them and de-acetylation of the enzymes to re-activate them. The acetylation of the enzyme occurs at a characteristic lysine residue. Various polypeptides, nucleic acids and other molecules that are related to modulating the AFE activity are also disclosed. Further disclosed are methods of modulating cellular acetyl-CoA or propionyl-CoA levels in bacterial hosts, methods of identifying agents that can modulate the activity of AFE acetylases, and methods of identifying AFE mutants that are insensitive to acetylation regulation.

Abstract: A method for selecting nucleic acid sequences encoding ligand and receptor molecules capable of specific binding to each other is disclosed in which nucleic acid encoding ligand or receptor molecules is expressed in a host microorganism in combination with a surface molecule, such as E. coli pili, so that the ligand or receptor are displayed on the surface of the host microorganism. A replicable genetic unit, such as a filamentous bacteriophage, is used to display candidate binding partners to the ligand or receptor, with the binding of the ligand or receptor to the candidate binding partner mediating the transfer of nucleic acid from the replicable genetic unit to the microorganism. The method can be highly selective as the host microorganism is modified so that it does not display the surface molecule other than as a fusion with the ligand receptor molecule. The method is rapid and simple and opens up new applications based on the detection of ligand and receptors where both are unknown.

Abstract: The present invention concerns a method for production of recombinant human proteins, in which Tetrahymena cells are transformed with recombinant DNA containing at least one functional gene that codes for a human protein, the recombinant Tetrahymena cells are cultured, in which the gene that codes for a human protein is expressed and the proteins are then isolated. The present invention also concerns a corresponding method, in which the gene that codes for a human protein contains a human leader sequence that leads to secretion of the expressed protein.

Abstract: The present invention concerns a method for production of genetically modified (recombinant) protists without using negative selection markers, in which an auxotrophic mutant of the protist is produced, this mutant is then transformed with recombinant DNA containing at least one gene for complementation of the corresponding auxotrophy, and the resulting recombinant protist is finally selected on a minimal medium that makes possible growth of only the correspondingly complemented protist. The present invention also concerns an efficient method for production of proteins by protists so modified, in which the gene for the protein being produced is coupled to the marker gene.

Abstract: Current technologies allow the generation of artificial DNA molecules and/or the ability to alter the DNA sequences of existing DNA molecules. With a careful coding scheme and arrangement, it is possible to encode important information as an artificial DNA strand and store it in a living host safely and permanently. This inventive technology can be used to identify origins and protect R&D investments. It can also be used in environmental research to track generations of organisms and observe the ecological impact of pollutants. Today, there are microorganisms that can survive under extreme conditions. As well, it is advantageous to consider multicellular organisms as hosts for stored information. These living organisms can provide as memory housing and protection for stored data or information. The present invention provides well for data storage in a living organism wherein at least one DNA sequence is encoded to represent data and incorporated into a living organism.

Abstract: The present invention provides a method for deriving an E. coli strain which produces L-theronine from E.coli strain VNIIgenetika 472T23 by transducing with bacteriophage P1 which bears a transposon.

Abstract: Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nuclei acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell sorter to identify said clones. More particularly, this is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; (ii) exposing said libraries to a particular substrate or substrates of interest; and (iii) screening said exposed libraries utilizing a fluorescence activated cell sorter to identify clones which react with the substrate or substrates.

Abstract: The present invention provides a safe immortalized bone marrow mesenchymal stem cell obtained by transferring a cell proliferation factor gene inserted between a pair of site-specific recombination sequences to a bone marrow mesenchymal stem cell.

Abstract: The present invention relates generally to the production of recombinant human uteroglobin (rhUG) for use as a therapeutic in the treatment of inflammation and fibrotic diseases. More particularly, the invention provides processes, including broadly the steps of bacterial expression and protein purification, for the scaled-up production of rhUG according to current Good Manufacturing Practices (cGMP). The invention further provides analytical assays for evaluating the relative strength of in vivo biological activity of rhUG produced via the scaled-up cGMP processes.

Abstract: To provide a promoter, a recombinant DNA, a gene expression vector, an expression vector, and a transformant, which are capable of expressing a gene without inducing gene expression with an inducer; and a method for producing a protein and a kit therefor, which can be operated easily and performed inexpensively by convenient and inexpensive steps.

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 invention relates to the identification of C1 bacteriophage genes that express protein involved in the lysis of bacterial cells during the phage life cycle, lysin and holin. The invention further relates to methods for lysing certain bacteria using lysin, which are useful for example in the treatment of an oral cavity bacterial infection.

Abstract: The invention features incapacitated whole cell bacterial immunogenic compositions produced by infecting a bacterium with Lys minus bacteriophage, which are deficient in the lysin protein. Lys minus bacteriophage retain activity in infection of its appropriate bacterial host, destruction of the bacterial genome, and replication, which are sufficient to inhibit bacterial growth and replication. The resulting, Lys minus-infected bacterium is provided in a state of bacteriostasis, and is not capable of replicating further (e.g., is “incapacitated”). The incapacitated bacterium can then be used as to elicit an immune response for prophylactic and/or therapeutic purposes. The invention thus also features incapacitated bacteria formulated appropriately for use in immunogenic compositions for eliciting an immune response, e.g., for production of antibodies in a non-human host or in a whole cell bacterial vaccine.

Abstract: The invention provides intracellular peptide toxins capable of killing bacterial and eukaryotic cells when present within the cell, while substantially lacking the ability to kill such cells when present externally. The invention also provides recombinant bacteriophage containing nucleic acid sequences encoding intracellular peptide toxins, and methods of using such bacteriophage to kill bacteria. Furthermore, the invention provides compositions, including pharmaceutical compositions, which can be used to kill bacteria or inhibit the growth of bacteria both in vitro and in vivo. Methods of treating a bacterial infection in a subject are also provided by the invention.

Abstract: The present invention is directed to isolated transducing phages, methods of isolating transducing phages, and methods of using transducing phages including, for instance, transferring at least one nucleic acid fragment from a donor microbe to a recipient microbe, and producing a secondary metabolite from a microbe. The transducing phages typically have a broad host range, and transduce microbes in the Order Actinomycetales, in particular in the Family Streptomycetaceae, including Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae, Streptomyces avermitilis, and Saccharopolyspora erythraea. The transducing phages can be specialized transducing phages or generalized transducing phages.

Abstract: Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.

Abstract: The rapamycin gene cluster is an example of a gene cluster which includes a gene (rapL) leading to the formation of a precursor compound (pipecolic acid, in this case) which is required for inclusion in the larger product (rapamycin) produced by the enzymes encoded by the cluster. We have produced a mutant strain containing a rapamycin gene cluster in which the rapL gene is disabled. The strain does not naturally produce rapamycin but does so if fed with pipecolic acid. By feeding with alternative carboxylic acids we have produced variants of rapamycins. Tests have shown biological activity.

Abstract: The invention relates to methods for targeting an exogenous polynucleotide or exogenous complementary polynucleotide pair to a predetermined endogenous DNA target sequence in a target cell by homologous pairing, particularly for altering an endogenous DNA sequence, such as a chromosomal DNA sequence, typically by targeted homologous recombination. In certain embodiments, the invention relates to methods for targeting an exogenous polynucleotide having a linked chemical substituent to a predetermined endogenous DNA sequence in a metabolically active target cell, generating a DNA sequence-specific targeting of one or more chemical substituents in an intact nucleus of a metabolically active target cell, generally for purposes of altering a predetermined endogenous DNA sequence in the cell.

Abstract: This invention describes the use of a method for the expression of proteins in bacteria, which is based on the inducer effect, for example, of a carbohydrate on the anti-terminating activity of a protein of the family of anti-terminators, B1gG/SacY. The procedure has been tested with the system derived from the operon lacTEGF, inducible by lactose in Lactobacillus casei, for the expression of proteins of different kinds. In the invention are included examples of expression in multicopy plasmids or of integration in the chromosomal operon lacTEGF, thus achieving the coordinated expression of the desired genes together with those that code the enzymes of the metabolism of the lactose.

Abstract: A histidine-tagged, deletion mutant of bacteriophage N4-coded, virion RNA polymerase (mini-vRNAP) which is active has been developed. The his-tagged mini-vRNAP has been cloned under the control of the pBAD promoter, is stable and is purified in a single step yielding large amounts (10 mg/liter of E. coli expressing cells). This RNA polymerase uses single-stranded DNA containing 17 bases (the promoter) upstream of the transcribed regions as a template. In the presence of E. coli SSB protein, it transcribes this template efficiently, providing a unique system to synthesize RNAs of the desired sequence using single-stranded DNA templates. The enzyme incorporates derivatized nucleoside triphosphates with high efficiency. A mutant of mini-vRNAP has been generated that incorporates deoxynucleoside triphosphates. In addition, the inventors have developed an in vivo system to express RNAs and proteins under mini vRNA polymerase promoter control.

Abstract: Variable domain murine T-cell receptor genes have been isolated and used to construct cloning and expression vectors. V&agr;, V&bgr;, and single chain V&agr;-V&bgr; fragments have been expressed as secreted domains in Escherichia coli using the vectors. The domains are secreted into the culture supernatant in milligram quantities. The single domains and the single chain T-cell receptors are folded into &bgr;-pleated sheet structures similar to those of immunoglobulin variable domains. The secreted fragments may be useful for immunization to generate anti-clonotypic antibodies, in vaccination or for high resolution structural studies. The genes encoding these domains may also serve as templates for in vitro mutagenesis and improvement of affinities of the TCR fragments for their interaction with cognate peptide-MHC complexes.

Abstract: The invention relates to novel human DNA sequences, targeting constructs, and methods for producing novel genes encoding thrombopoietin, DNase I, and &bgr;-interferon by homologous recombination. The targeting constructs comprise at least: a) a targeting sequence; b) a regulatory sequence; c) an exon; and d) a splice-donor site. The targeting constructs, which can undergo homologous recombination with endogenous cellular sequences to generate a novel gene, are introduced into cells to produce homologously recombinant cells. The homologously recombinant cells are then maintained under conditions which will permit transcription of the novel gene and translation of the mRNA produced, resulting in production of either thrombopoietin, DNase I, or &bgr;-interferon.

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: The invention relates to methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that encode proteins having photosynthetic carbon fixation activities, including Rubisco and Rubisco activase activities, which are useful for introduction into plant species, agronomically-important microorganisms, and other hosts, and related aspects.

Abstract: The present invention provides vectors that encode single-chain antigen-binding units in both prokaryotic and eukaryotic cells. The vectors are particularly useful for generating a genetically diverse repertoire of single-chain antigen-binding units to facilitate an in vivo screening of antigen-binding units that bind to a desired antigen inside a cell. The present invention also provides recombinant polynucleotides, host cells and kits comprising the vectors. Further provided by the invention are methods of using the subject vectors.

Abstract: The invention relates generally to methods and apparatuses that are adapted for the preparation of gene therapeutic compositions, as well as the compositions formed thereby. The invention also relates to methods adapted for making mixtures and condensate compositions. In the various embodiments, the present invention provides controlled and uniform mixing of gene therapy vectors and gene therapy vector vehicles for improved reproducibility, scaleability, stability, and pharmaceutical efficacy. Such compositions are suitable for use in mediation of disease.

Abstract: The present invention relates to the identification of novel metallo-proteases (MP) in Gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for Bacillus MP. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding MP. The present invention also provides host cells further comprising nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides cleaning compositions comprising an MP of the present invention.

Abstract: Among the inventions disclosed herein are: nucleic acid expression systems for bacteria having an intracytoplasmic membrane system, including, for example, methanotrophic bacteria; recombinant nucleic acid constructs comprising a pmo promoter operably linked to an expressible nucleic acid; cloning vectors suitable for making recombinant nucleic acid constructs and methods for the production of proteins such as membrane proteins.

Abstract: The present invention provides a isolated bacteriophage useful as a tool for studying biological, biochemical, physiological and genetic properties of actinomycetes and other organisms which comprises a novel strain of Saccharomonospora having certain specified characteristics. The invention also relates to a process for the isolation of the said bacteriophage and/or DNA phage and to a novel universal growth medium which is particularly useful in the said process. Another embodiment of the process relates to a clonong vector which comprises a plasmid or bacteriophage comprising the phage DNA of the invention.