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: The present invention relates to methods of identifying putative antibiotic resistance genes. According to one embodiment, this is carried out by first isolating a microbial DNA molecule or cDNA which encodes a putative antibiotic resistance protein or polypeptide and then determining whether the microbial DNA molecule or cDNA confers resistance against an antibiotic agent. According to another embodiment, this is carried out by first determining whether a microbial DNA molecule or cDNA confers resistance against an antibiotic agent when the microbial DNA molecule is expressed in its native cell following transformation of the cell and then isolating the microbial DNA molecule or cDNA which confers resistance against the antibiotic agent.

Abstract: The present invention relates generally to a Plasmid Maintenance System for the stabilization of expression plasmids encoding foreign antigens, and methods for making and using the Plasmid Maintenance System. The invention optimizes the maintenance of expression plasmids at two independent levels by: (1) removing sole dependence on balanced lethal maintenance functions; and (2) incorporating at least one plasmid partition function to prevent random segregation of expression plasmids, thereby enhancing their inheritance and stability. The Plasmid Maintenance System may be employed within a plasmid which has been recombinantly engineered to express a variety of expression products.

Abstract: The present invention provides an advance in phage display technology by permitting the uncoupling of the propagation of phages containing inserted sequences encoding heterologous polypeptides from the expression of said polypeptides. The invention provides phage constructs and methods for their use to permit phage coat protein expression, and thus phage propagation, in the absence of display of heterologous polypeptides, which may be expressed as a fusion with said coat protein in a regulated manner.

Abstract: It can be difficult to achieve efficient transformation of many strains of bacterial cells due in part to the presence of one or more restriction and modification (R-M) systems in the cells that restricts unmodified transforming DNA. Phage T7 OCR protein is a potent inhibitor of Type I R-M systems. Methods are disclosed for improving transformation efficiency of Eubacterial and Archaebacterial cells having an R-M system by introducing into the cells an inhibitor of the restriction activity. For example, addition of 1–5 micrograms of T7 OCR protein to 50 microliters of electrocompetent cells having a Type I R-M system prior to electroporation significantly increased transformation efficiency by unmodified plasmids, fosmid clones, and artificial transposons comprising synaptic complexes.

Abstract: Novel Shine-Dalgarno (ribosome binding site) sequences, vectors containing such sequences, and host cells transformed with these vectors are provided. Methods of use of such sequences, vectors, and host cells for the efficient production of proteins and fragments thereof in prokaryotic systems are also provided. In particular embodiments of the invention, compounds and methods for high efficiency production of soluble protein in prokaryotic systems are provided.

Abstract: The present invention provides methods of introducing a polynucleotide into a target cell, wherein the method employs a light generating protein coding sequence acting as a reporter. An important advantage of the methods described herein is that drug resistant target cells or target cells having no useful auxotrophic markers can be effectively transformed. The present invention also includes transformed cells produced by the methods described herein. Also described are light generating protein coding sequence modifications, a variety of vectors, and methods of using the transformed cells of the present invention.

Abstract: The present invention provides a recombinant plasmid vector comprising a kanamycin resistance gene, a promoter, an endoxylanase signal sequence, a nucleotide sequence coding for an oligopeptide consisting of 13 amino acids including 6 consecutive histidine residues, and a human granulocyte colony stimulating factor(hG-CSF) gene; an E. coli transformed with the said vector; and, a process for producing complete hG-CSF protein with high purity from the protein pool secreted by the said microorganism.

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: 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.

Abstract: Methods are provided for the rapid identification of essential or conditionally essential DNA segments in any species of haploid cell (one copy chromosome per cell) that is capable of being transformed by artificial means and is capable of undergoing DNA recombination. This system offers an enhanced means of identifying essential function genes in diploid pathogens, such as gram-negative and gram-positive bacteria.

Abstract: The present invention is directed to a recombinant vector for transforming yeast and a process for transforming yeast thereby, more particularly to a recombinant vector comprising a gene encoding a mutated L41 protein having cycloheximide-resistant activity and a ribosomal DNA. The recombinant vector and the process for transforming thereby of the present invention is applicable to the efficient and stable integration of desired foreign DNA into yeast genome, thus providing useful tools for the production of a natural pigment, astaxanthin.

Abstract: The present invention relates generally to a Plasmid Maintenance System for the stabilization of expression plasmids encoding foreign antigens, and methods for making and using the Plasmid Maintenance System. The invention optimizes the maintenance of expression plasmids at two independent levels by: (1) removing sole dependence on balanced lethal maintenance functions; and (2) incorporating at least one plasmid partition function to prevent random segregation of expression plasmids, thereby enhancing their inheritance and stability. The Plasmid Maintenance System may be employed within a plasmid which has been recombinantly engineered to express a variety of expression products.

Abstract: The invention provides origin of replication sequences and replication genes and proteins for a plasmid functional in Fusobacterium (e.g., F. nucleatum) and related species. Provided by the invention are also plasmids and vectors that can replicate in Fusobacterium. Further, the invention provides shuttle vectors that can replicate in Fusobacterium and in other microorganisms, such as E. coli. Still further, the present invention provides host cells comprising the plasmids and shuttle vectors, and methods for transformation of the host cells with the plasmid and shuttle vectors of the invention.

Abstract: High throughput DNA sequencing vectors for generating nested deletions using enzymatic techniques and/or transposition-based techniques are disclosed. Methods of constructing contigs of long DNA sequences and methods of generating nested deletions are also disclosed. A truncated lacZ derivative useful in measuring the copy number of the lacZ derivative in a host cell is also disclosed.

Abstract: Polypeptides, in particular the polypeptide of formula I: Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-Val-Ser-Thr-Lys- Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala- Met-Leu-Asn-Pro-Pro-Asn-Arg-Cys-Leu-Lys-Asp-Thr- Asp-Cys-Pro-Gly-Ile-Lys-Lys-Cys-Cys-Glu-Gly-Ser- Cys-Gly-Met-Ala-Cys-Phe-Val-Pro-Gln and analogues thereof which possess inhibitory activity against human leukocyte elastase. The polypeptides may be obtained by expression using plasmidic expression systems in hosts such as E. Coli and yeast, the polypeptide of formula I being also obtainable from psoriatic plaques.

Abstract: The present invention relates to cells for the production of helper dependent adenoviral vectors, including at least the following genic units: a first genic unit comprising an adenovirus defective genome having the inverted terminal repeats in head-to-tail configuration, the encapsidation signal inactivated, and at least one of the non-structural regions inactivated; a second genic unit comprising at least one inducible promoter and at least one of the regions inactivated in the first genic unit, said regions being under the control of said inducible promoter; whereby following the activation of the inducible promoter of the second genic unit and the infection of the cells with said helper dependent adenoviral vectors, the first genic unit and the second genic unit enable the production of said helper dependent adenoviral vectors in said cells in absence of helper vector.

Abstract: The present invention relates to a new plasmid originated from (Bifidobacterium) a recombinant expression vector and transformation method using the same. More particularly, the present invention relates to a plasmid pMG1 having nucleotide sequence represented by SEQ.ID.NO.1; (Bifidobacterium longum) MG1 including the plasmid pMG1; and a shuttle vector which can be replicated in both (Bifidobacterium) and (E. coli), and comprises (Mob) gene having nucleotide sequence represented by SEQ.ID.NO.2. (Rep) gene having nucleotide sequence represented by SEQ.ID.NO.3 and a selection marker. The shuttle vector and the promoter of the present invention can be used for expressing target gene without additional purification process.

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 nucleic 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 is directed to herbicide resistant N2 fixing bacteria. The bacteria are useful for effecting N2 fixation, nodulation, growth and yield of herbicide resistant or tolerant leguminous plants treated with herbicide. The bacteria are particularly useful for providing competitive advantage to superior N2 fixing rhizobial strains over non-resistant indigenous rhizobia for nodulation of herbicide resistant or tolerant leguminous plants.

Abstract: The production of a purified extracellular bacterial signal called autoinducer-2 is regulated by changes in environmental conditions associated with a shift from a free-living existence to a colonizing or pathogenic existence in a host organism. Autoinducer-2 stimulates LuxQ luminescence genes, and is believed also to stimulate a variety of pathogenesis related genes in the bacterial species that produce it. A new class of bacterial genes is involved in the biosynthesis of autoinducer-2.

Abstract: A single-copy BAC vector (containing or lacking an insert) is converted in a host cell into a conditional high-copy BAC vector by introducing a conditional origin of replication into the single-copy BAC vector. The conditional ori is introduced by site-specific recombination between the SC BAC vector and a vector that contains the conditional ori. The host cell comprises a recombinase that recognizes a site-specific recombination site on both the BAC vector and the conditional ori vector. In the presence of the recombinase, the conditional ori-containing vector recombines into the BAC vector to produce a high-copy BAC vector that can be conditionally amplified by activating the conditional origin of replication on command.

Abstract: A fast method of transforming competent cells is described. The competent cells are thawed at room temperature or in a water bath. Plasmid DNAs and competent cells are mixed together, then the mixture is subject to heat shock treatment. After plating the mixture on a low-temperature selective medium by a low-temperature plating tool, the competent cells are cultured on the selective medium.

Abstract: A method for introducing and stabilizing heterologous and recombinant genes in a thermophilic host in which a characteristic gene defining a detectable host characteristic is inactivated or deleted from the thermophilic host, resulting in a modified thermophilic host expressing an absence of the detectable host characteristic. A DNA fragment of interest is inserted into the modified thermophilic host together with an intact characteristic gene, whereby the detectable host characteristic is restored to the thermophilic host, thereby enabling detection and confirmation of successful transformation using plasmid vectors and integration of the DNA fragment into the chromosome of the thermophilic host.

Abstract: The invention relates to improved E. coli bacteria with enhanced viability at low temperatures, methods for producing improved bacterial strains capable of enhanced viability at low temperatures, and the isolation and use of genetic material capable of enhancing the viability of bacteria at low temperatures. In addition to the enhanced viability at low temperatures, the bacteria may exhibit enhanced transformation efficiencies after storage at low temperatures. As such, the invention may be used for the insertion of exogenous DNA sequences into the bacteria of the invention.

Abstract: Disclosed are methods for generating mycolic acid bacterial biosensors for particular analytes (especially industrial pollutants) by the use of innovative methods for isolating DNA encoding an inducible promoter which is induced in response to the specific analyte (and/or associated operon proteins), the methods generally comprising the steps of: (a) culturing a source of mycolic acid bacteria in a selective medium containing said specific analyte and being selective for oligotriphic bacteria; (b) identifying mycolic acid bacteria capable of subsisting on said medium, especially those which do not display catabolic repression; (c) extracting DNA from said mycolic acid bacteria; (d) incorporating said DNA into vectors, such as various shuttle vectors; (e) cloning said vector into a suitable host cell (which may be E.

Abstract: This invention is directed to Flavobacterium heparinum for use as a host cell organism for the expression of homologous and heterologous genes.

Abstract: The invention is directed to methods for purifying Troponin I, particularly recombinant Tropnin I produced in a bacterial expression system. Recombinant Tropnin I can be advantageously purified after reversibly protecting the free sulfhydryl groups, e.g., by forming sulfates. In a specific example, Tropnin I reacted with sodium tetrafhionate yielded sulfitolyzed Tropnin I, which was purified by chromatography on an anion exchanger, followed by hydrophobic interaction chromatography. Facile deprotection of the sulfhydryl groups yields a highly purified product ready for refolding.

Abstract: The present invention provides nucleic acid constructs, expression systems, and methods relating to the regulation of gene expression. The invention may be applied to regulate the expression of any coding sequence of interest, including those coding for viral components necessary for the packaging of viral particles.

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

Abstract: The invention concerns recombinant vectors replicated in mycobacteria, a set of sequences coding for exported polypeptides detected by fusion with alkaline phosphatase, in particular one polypeptide, called DP428, of about 12 kD corresponding to an exported protein found in mycobacteria belonging to the Mycobacterium tuberculosis complex. The invention also concerns methods and kits for detecting in vitro the presence of a mycobacterium and in particular a mycobacterium belonging to the Mycobacterium tuberculosis complex in a biological sample using said polypeptides, their fragments or polynucleotides coding for the latter. The invention also concerns immunogenic or vaccine compositions for preventing and/or treating infections caused by mycobacteria and in particular a mycobacterium belonging to said complex, particularly tuberculosis.

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: Methylation of DNA can be a critical step in the introduction of DNA into P. haemolytica. A methyltransferase has been isolated and molecularly cloned for this purpose. Use of the methyltransferase has allowed construction of defined, attenuated mutants for use as vaccines to protect cattle.

Abstract: A plasmid vector characterized by comprising a promoter sequence that can be recognized by an RNA polymerase which is not inherent in a host and that controls the expression of desired genes and a replication origin that increases the number of copies under the induction by exogenous factors; methods for expression and isolation of target genes by using the vector; a polypeptide having the activity of an AccIII restriction endonuclease; and a DNA encoding the polypeptide. The invention provides for the first time a plasmid vector which can introduce an exogenous desired gene encoding proteins which are lethal or harmful to hosts into the hosts, a method for efficiently expressing the proteins by using the vector, and also a method for permitting a restriction endonuclease-gene constituting a restriction-modification system to be isolated even in the absence of a modification enzyme gene, which has been difficult in the prior arts.

Abstract: Genes have been isolated from a Methylomonas sp encoding enzymes in the carbon flux pathway. The genes encode a 2-keto-3-deoxy-6-phosphogluconate (KDPGA) and a fructose bisphosphate aldolase (FFBPA), as well as numerous other genes. The genes will be useful in C1 metabolizing microorganisms for the manipulation of the carbon flux pathway.

Abstract: The present invention is directed to novel xylanases (referred to as XYL-IV) and to nucleic acid molecules encoding those xylanases. Also provided herein are vectors and host cells including those nucleic acid sequences, antibodies which bind to the xylanases of the present invention, methods for producing the xylanases of the present invention, and methods employing the xylanases of the present invention.

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: This invention relates to methods of screening molecules capable of inhibiting the survival of Helicobacter pylori in vivo by specifically inhibiting the activity of UreI, to the molecules identified by these methods, and to the use of these molecules to treat or prevent H. pylori infection.

Abstract: A novel rec-L-N-carbamoylase from Arthrobacter aurescens and its method of use for producing L-amino acids. The recombinantly produced L-carbamoylase is unexpectedly stable, so that an industrial method of producing L-amino acids can be established with it, in contrast to previously known L-carbamoylases.

Abstract: A protein capable of modulating or mediating the intracellular activity of RIP in inflammation, cell survival and cell death pathways is provided. DNA encoding it, a method for its production and its uses are also provided.

Abstract: This invention provides a purified extracellular bacterial autoinducer-2 signaling molecule, the production of which is regulated by changes in environmental conditions associated with a shift from a free-living existence to a colonizing or pathogenic existence in a host organism. The signaling molecule stimulates LuxQ luminescence genes, and is believed also to stimulate a variety of pathogenesis related genes in the bacterial species that produce it. This invention also provides is a new class of bacterial genes involved in the biosynthesis of the signaling molecule.

Abstract: The present invention relates to a method of obtaining altered plasmid contents in bacteria, bearing mutation in at least one of the chromosomal genes, nusG, rho, and dnaC, and the bacterial strains thereof, having the mutated chromosomal genes, individually or in various possible combinations, capable of altering the level of plasmids.

Abstract: A microorganism is described which is transformed with DNAs which encode a hydantoinnase, a racemase, and a carbamoylase. As a result, the microorganism is able to degrade hydantoins directly to amino acids. A process for the production of the microorganism and a process for producing amino acids with the microorganism is also described.

Abstract: NAIP and IAP polypeptides prevent neuronal cell death caused by ischemia, neurodegenerative conditions, and axotomy. The invention provides methods for neuroprotection by the prevention of cell death and kits and methods for the identification of neuroprotective therapeutic compounds.

Abstract: The instant invention is drawn towards transformed strains of Fusarium sporotrichioides effective for the production of lycopene. The transformed strains comprise an expression cassette having three genes encoding, respectively, geranylgeranyl-pyrophosphate synthase, phytoene synthase and phytoene desaturase (i.e. Tri5crtE, Tri5crtB and Tri5crtl). The transformed strains of Fusarium sporotrichioides of the instant invention produce lycopene at levels of up to 0.5 milligrams per gram culture dry weight.

Abstract: The present invention describes a mutant plasmid replication control region having the ability to convey temperature sensitivity to the plasmid on which it resides. The mutant replication control region is based on a similar region isolated from pBRH1. Plasmids containing this replication control region cannot be classed as belonging to any known incompatibility group and thus may co-exist with a broad range of other plasmids in a single host.

Abstract: Site-specific Listeria integration vectors and methods for their use are provided. The subject vectors include a bacteriophage integrase gene and a bacteriophage attachment site, where in many embodiments the bacteriophage that is the source of these elements is a listeriophage. In certain embodiments, the subject vectors further include a multiple cloning site, where the multiple cloning site may further include a polypeptide coding sequence, e.g., for a heterologous antigen. The subject vectors and methods find use in a variety of different applications, including the study of Listeria species and the preparation of Listeria vaccines.

Abstract: The invention relates to a process for the preparation of L-amino acids. The process involves fermenting an L-amino acid producing coryneform bacteria in a culture medium, concentrating L-amino acid in the culture medium or in the cells of the bacteria, and isolating the L-amino acid produced. The bacteria has an amplified gene encoding the Zwischenferment protein.