Abstract: A composition and method for controlled in vitro fragmentation of nucleic acids. A transposase forms catalytically active complexes with a modified transposon end that contains within its end sequence degenerate, apurinic/apyrimidinic sites, nicks, or nucleotide gaps, to fragment or shear a target nucleic acid sample in a controlled process. This method yields desired average nucleic acid fragment sizes. The inventive composition and method may be applied for generation of DNA fragments containing shortened transposon end sequences to facilitate subsequent reactions, for production of asymmetrically tailed DNA fragments, etc.

Abstract: Wheat having a reduced level of SBEIIa activity, that may have a relative high amylose content. Wheat having a mutant SBEIIa gene in the A genome. The wheat might additionally have reduced levels of SBEIIb activity. The wheat grain of this invention can be of a non-shrunken phenotype despite a lesion in the amylopectin synthesis pathway, and may also have a high relative amylose content.

Abstract: An isolated polynucleotide is disclosed which comprises a nucleic acid sequence of a Brucella phage, the nucleic acid sequence being specific to the Brucella phage and comprising a sequence selected from the group consisting of SEQ ID NOs: 387-393. An exemplary polynucleotide sequence is one which comprises at least 100 consecutive nucleotides of a nucleic acid sequence as set forth in SEQ ID NO: 396. Uses of such sequences are further disclosed.

Abstract: An object is to provide a Tol1 element transposase and a use thereof. Provided is a Tol1 element transposase containing (a) a protein having the amino acid sequence of SEQ ID No: 1 or (b) a protein having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 1 and having an enzymatic activity for transferring Tol1 element. Further, provided are a polynucleotide encoding the transposase and an expression construct containing the polynucleotide therein. The present invention also provides a DNA introduction system including (a) a donor factor having such a structure that a desired DNA is inserted in a transposase gene-defected Tol1 element and (b) a helper factor containing the transposase or the polynucleotide.

Abstract: The present invention provides live, attenuated Mycoplasma gallisepticum bacteria that exhibit reduced expression of a protein identified as MGA_0621. In certain embodiments, the attenuated bacteria may additionally exhibit reduced expression of one or more proteins selected from the group consisting of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35, relative to a wild-type M. gallisepticum bacterium. Also provided are vaccines and vaccination methods involving the use of the live, attenuated M. gallisepticum bacteria, and methods for making live attenuated M. gallisepticum bacteria. An exemplary live, attenuated strain of M. gallisepticum is provided, designated MGx+47, which was shown by proteomics analysis to exhibit significantly reduced expression of MGA_0621, and was shown to be safe and effective when administered as a vaccine against M. gallisepticum infection in chickens.

Abstract: Protoporphyrinogen oxidase having an activity of imparting acifluorfen resistance and gene thereof are provided. Cyanobacterium protoporphyrinogen oxidase gene is identified by introducing a protoporphyrinogen oxidase gene of Arabidopsis into cyanobacterium, disrupting a cyanobacterium gene with a transposon, selecting a mutant strain in which protoporphyrinogen oxidase gene is disrupted, identifying the disrupted protoporphyrinogen oxidase gene, and isolating the disrupted protoporphyrinogen oxidase gene. This procedure is effective as a gene isolation technique when a protein derived from other organism species that is homologous to a known protein (e.g., protoporphyrinogen oxidase from cyanobacterium) can not be found in a gene database of the other species.

Abstract: The present invention relates to the generation of replication-competent viruses having therapeutic utility. The replication-competent viruses of the invention can express proteins useful in the treatment of disease.

Abstract: A high throughput bioluminescence mutant screening procedure is disclosed. This procedure utilizes robotics, and bacterial luciferase to allow real-time monitoring of mutant viability. The procedure was used to develop a live attenuated vaccine for a catfish against E. ictaluri, which is further claimed herein. Additionally, genes from other bacterial species are disclosed which may also be used to create vaccines.

Abstract: The present invention relates to genetic engineering and especially to the use of DNA transposition complex of bacteriophage Mu. In particular, the invention provides a gene transfer system for eukaryotic cells, wherein in vitro assembled Mu transposition complexes are introduced into a target cell and subsequently transposition into a cellular nucleic acid occurs. The invention further provides a kit for producing insertional mutations into the genomes of eukaryotic cells. The kit can be used, e.g., to generate insertional mutant libraries.

Abstract: There is provided a transposable element comprising at least four inverted repeats, at least two of which are each inverted in relation to another, the element comprising DNA for insertion into a host genome, the DNA being located between two pairs of opposing repeats excisable by a transposase in situ to leave said DNA without flanking transposon-derived repeats in the host genome. Also provided is a transposable element comprising at least three inverted repeats, at least one of which is inverted in relation to the others, wherein at least one non-terminal repeat is a minimal repeat. Both these elements allow for greater efficiency of insertion of nucleotide sequences into the genome.

Abstract: This document provides live non-pathogenic M. bovis bacteria and compositions containing live non-pathogenic M. bovis bacteria. This document also provides methods of using live non-pathogenic M. bovis bacteria to immunize cattle against infectious diseases (e.g., diseases caused by M. bovis bacteria). In addition, methods and materials that can be used to generate live non-pathogenic M. bovis bacteria are provided.

Abstract: The present invention provides a method and components thereof of performing genetic modification under a drug-free environment. The method comprises the steps of generating a trapped mammalian cell library by trapper constructs (including the element of piggyBac terminal inverted repeats (TIRs)), reporter constructs, and helper constructs (including a sequence of an internal ribosomal entry site (IRES)). The present art allows: (1) to target & identify the silenced loci; (2) to separate genes with low-level expression at certain differentiation stages; (3) to evaluate the efficiency of gene targeting in the silent or repressed loci. The present invention avoids the biased gene targeting observed in the prior arts, and eliminates the needs of introducing antibiotic genes into the host genome which may lead to a potential threat of drifting antibiotic resistant genes into environment.

Abstract: The present invention provides molecular methods for efficiently transforming the genome of common disease-transmitting parasites, such as Plasmodium falciparum. The transformation efficiencies are improved up to 100 times over those conventionally known. The methods provide high saturation of the target parasite genome, of 50% or greater, and target non-specifically TTAA-rich sites in the parasite genome. The invention also discloses a model that may be used to functionally annotate the genome of the Plasmodium falciparum, thus permitting the design and screening of compounds that may be useful in the control and inhibiting of diseases caused and transmitted by these parasites, including malaria. Highly efficient and multi-site integrating transposons, particularly piggyBac transposons, which provide for random and multi-site integration into parasite genomes in the presence of a helper plasmid, are also presented.

Abstract: Disclosed herein is a method for marking bio-information into the genome of an organism, an organism marked with inherent bio-information by the method and a method for reading the bio-information. The method is characterized in that inherent bio-information encoded to a DNA sequence or RNA sequence is inserted into genome of organisms through a gene delivery system. Inherent bio-information is inserted into genome of organisms, thus avoiding loss by cell culture or artificial manipulation and being widely used even for organisms whose host-vector system is not provided. Based on these features, the method has the following advantages. First, inherent bio-informtion can be clearly obtained from the organisms themselves, rather than an additional means such as catalogs. Second, when organisms developed through desperate efforts are stolen, they can be tracked down and identified. Third, when serious problems occur by overuse or misuse of organisms, the origin thereof can be clearly determined.

Abstract: Disclosed is a method for developing novel strains deleted specific chromosome sites, using transposon and Cre/loxP site-specific recombination by Cre expression vector, wherein the transposon comprises a selectable marker and loxP site. The method comprises the steps of: (1) preparing a transposon comprising a selectable marker and loxP site; (2) inserting the transposon into an optional position of microbial chromosome, and determining the inserted site; (3) integrating two transposons comprising a different selectable marker to one chromosome; (4) deleting a chromosomal site between the two lox sites by introducing a Cre expression vector into the chromosome of step (3); and (5) repeating steps (3 and 4) for the mutant deleted a part of chromosome, to shorten the chromosome of mutant gradually.

Abstract: The invention relates to hyperactive, non-phosphorylated, mutant transposases of mariner mobile genetic elements. The invention also relates to recombinant nucleotide sequences encoding such transposases. The invention further relates to a method of producing said transposases and to the use thereof for in vitro or in vivo transposition.

Abstract: A novel plant transposon that induces bud mutation, transposase thereof, and a method for efficiently inducing bud mutation by improvement in the transposition efficiency are provided. A novel transposon having terminal inverted repeat sequences (SEQ ID NO: 1 and SEQ ID NO: 2) shown below has been isolated for the first time from carnation (Dianthus caryophyllus) and the nucleotide sequence thereof has been determined. The transposon has contained a gene encoding the transposase in its sequence. The transposon induces bud mutation when it is inserted into a gene. The transposition frequency is elevated due to environmental stress. TAGAGCTGGCAAA---TTTGCCAGCTCTA (SEQ ID NOS 1 & 2, respectively in order of appearance).

Abstract: The disclosed invention relates to an isolated hydrogen gas producing microorganism, termed Enterobacter sp. SGT-T4™ and derivatives thereof. Compositions and methods comprising the disclosed microorganisms are also provided.

Abstract: The present invention provides engineered enzymes generated from protein scaffolds combined with Specificity Determining Regions, the production thereof and the use of said engineered enzymes for research, nutritional care, personal care and industrial purposes.

Abstract: The present invention relates to the generation of replication-competent viruses having therapeutic utility. The replication-competent viruses of the invention can express proteins useful in the treatment of disease.

Abstract: The disclosed invention relates to an isolated hydrogen gas producing microorganism, termed Enterobacter sp. SGT 06-1™.

Abstract: The present invention provides a simple method for splitting and loss of a chromosome in yeast. The method for modifying a chromosome in yeast includes preparing a linear chromosome splitting vector (1) having a target sequence (a), a marker gene sequence and (C4A2)n sequence in this order; preparing a linear chromosome splitting vector (2) having a target sequence (b), a centromere sequence of a yeast chromosome and (C4A2)n sequence in this order; and introducing the chromosome splitting vectors (1) and (2) into yeast. Herein, n is each independently an integer of 6 to 10. Although this chromosome splitting vector has a repetitive sequence of 5?-CCCCAA-3?, it can be amplified specifically with PCR, so that a chromosome splitting vector can be prepared significantly simply and easily, compared with the conventional DNA splitting method.

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 characterised Fusarium oxysporum Impala transposon in the genome of said fungi. The invention also concerns the resulting mutants.

Abstract: Methods are provided for manipulating nucleic acid to produce gene fusions, to delete or clone a portion of a chromosome, or to insert a sequence into a chromosome. The methods employ sequential transposition processes using two or more pairs of inverted repeat transposase-interacting sequences on a transposable polynucleotide wherein each pair of transposase-interacting sequences interacts with a distinct transposase enzyme.

Abstract: We disclose compositions and processes for enhancing transposon mediated integration of a nucleic acid molecule into another target nucleic acid molecule. Integration by an integrator complex is enhanced by cationic reagents.

Abstract: An isolated peptide fragment of the VapA protein that binds antibodies specific for Rhodococcus equi and the VapA protein. In a preferred form the peptide contains an amino acid sequence of 5 or more amino acid residues that is identical to or homologous to the amino acid sequence of at least one region of the VapA protein that is responsible for immunological recognition. Methods of diagnosing a vertebrate for the presence of R. equi using the peptide and methods of vaccinating a vertebrate against R. equi using the peptide are also claimed.

Abstract: A transposon-based mutagenesis method for altering DNA in Sorangium and other Myxococcales host cells is provided, along with vectors and transposases for use in the method.

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: 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: 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 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 an inclusion complex of Rifampicin and cyclodextrin (CD) that can be used as an anti-tubercular drug. The present invention also relates to a process for synthesizing inclusion complexes of the anti-tubercular drug, Rifampicin, with ?-CD (?-cyclodextrin) and HP-?-CD (2-hydroxy propyl cyclodextrin) and characterization of these inclusion complexes.

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: More efficient transfer of genes into host cells or embryos to transform the cells or embryos is facilitated by transposition vectors using the minimal amount of nucleotide sequences in the transposon piggyBac required for gene transfer. The transformed cells or embryos may be developed into transgenic organisms.

Abstract: A helper dependent adenoviral vector system is provided. The subject helper dependent adenoviral vector system is made up of: (1) a “gutless” adenoviral vector that include cis-acting human stuffer DNA that provides for in vivo long term, high level expression of a coding sequence present on the vector; (2) an adenoviral helper vector that is characterized by having an adenoviral genome region flanked by recombinase recognition sites, where the helper vectors further include a non-mammalian endonuclease recognition site positioned outside of the adenoviral genome region; and (3) a mammalian cell that expresses the corresponding recombinase and endonuclease, as well as the adenoviral preterminal and polymerase proteins. Also provided are methods of using the subject systems to produce virions having the subject helper dependent adenoviral vectors encapsulated in an adenoviral capsid. In addition, kits for use in practicing the subject methods are provided.

Abstract: The present invention relates to the production of proteins in host cells, and, more particularly to host cells containing multiple integrated copies of an integrating vector. Suitable integrating vectors for use in the present invention include retrovirus vectors, lentivirus vectors, transposon vectors, and adeno-associated virus vectors. Methods are provided in which the host cells are prepared by using the integrating vectors at a high multiplicity of infection. The host cells are useful for producing pharmaceutical proteins, variants of proteins for use in screening assays, and for direct use in high throughput screening.

Abstract: Recombinant hepatitis C virus (HCV) capsid proteins that self-assemble into large spherical virus-like particles structures and viral capsids that include conformational antigenic epitopes are provided. The large spherical virus-like particles structures and viral capsids, including capsid proteins that are expression products of a viral particle coding sequence protein, may be prepared as vaccines to induce a cellular or humoral immune response. The self assembling capsid proteins may also be used as elements of diagnostic immunoassay procedures for HCV infection.

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

Abstract: Methods of identifying a gene whose product modulates a control phenotype of interest are provided. The methods comprise introducing a promoter insertion construct of the present invention into the genomes of a collection of host cells having the control phenotype of interest; selecting mutagenized cells exhibiting a mutant phenotype to provide a pool of mutant cells; treating the mutant cells with the disrupting agent having recombinase activity; detecting changes or the lack thereof in the linkage between the promoter element and downstream host genomic DNA sequences in treated mutant cells, or in both treated and untreated mutant cells; and correlating the changes or lack thereof in the linkage between the promoter element and the downstream host genomic DNA sequences in treated mutant cells, or in both untreated and treated mutant cells, with the phenotypes of said cells. Also provided are compositions that are used in the present methods.

Abstract: Tn5 transposase (Tnp) mutants that have higher transposase activities than the wild-type Tnp are disclosed. The Tn5 Tnp mutants differ from the wild-type Tnp at amino acid positions 54, 242, and 372 and have greater avidity than the wild-type Tnp for at least one of a wild-type Tn5 outside end sequence as defined by SEQ ID NO:3 and a modified Tn5 outside end sequence as defined by SEQ ID NO:5. Also disclosed are various systems and methods of using the Tnp mutants for in vitro or in vivo transposition.

Abstract: A transposon-based mutagenesis method for altering DNA in Sorangium and other Myxococcales host cells is provided, along with vectors and transposases for use in the method.

Abstract: Certain embodiments are directed to using an insulator element in a transposon having at least one transcriptional unit and at least one insulator element. The transcriptional unit(s) may be flanked by at least one insulator element on each side. The transcriptional unit may include an exogenous nucleic acid for introduction into a cell, e.g., DNA encoding a marker molecule. The insulator element may include a binding site for a CTCF protein. And, for example, a transcriptional unit may be disposed between a first insulator element and a second insulator element, and the first insulator element and the second insulator element may be disposed between inverted repeats of a transposon. The exogenous nucleic acid may be, e.g., DNA encoding an antisense RNA or siRNA.

Abstract: The present invention relates to methods of creating genetic diversity. The invention also relates to methods of making nucleic acid libraries and their uses to screen for biological activities. The invention can be used to create, improve or modify nucleic acid sequences or repertoire, particularly from non-cultivable microorganisms and finds various applications in the medical, food, agriculture or veterinary industries, for instance. The invention particularly uses and discloses in situ gene shuffling.

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: We disclose compositions and processes for enhancing transposon mediated integration of a nucleic acid molecule into another target nucleic acid molecule. Integration by an integrator complex is enhanced by cationic reagents.

Abstract: A nucleic acid sequence required for regulating the autolytic activity of bacteria is provided. Also provided are polypeptides encoded by the gene or mutant gene as well as vector and host cells for expressing these polypeptides. Methods for identifying and using agents which interact with the gene or mutant gene or polypeptides encoded thereby to inhibit bacterial growth and infectivity are also provided.

Abstract: The present invention provides materials and methods for introducing genetic disruptions in bacterial genetic material, especially in that of Streptomyces spp.. A novel transposon is provided, which has an origin of transfer between inverted repeat sequences. The transposon may also include a genetic marker. The transposon introduces a disruption into DNA of interest, which disruption may then be conjugated into host bacteria, including bacteria of other species or strains. The host bacteria is incubated at conditions suitable for homologous recombination between the conjugated DNA and the host DNA. The effect of the disruption in different genetic backgrounds can therefore be investigated. The disruption may be stored as a mobile genetic element ready for transfer to a test host.

Abstract: The present invention is directed to improved transposons and transposases. The present invention also includes gene transfer systems, methods of using the transposons and transposases, and compositions including the transposons and transposases.

Abstract: The present invention relates to cells and cell strains that are resistant to the killing effects of one or more toxic genes, particularly those that kill hosts in the absence of a suppressing function, e.g., kicB or ccdB. The host cells may comprise one or more suppression mutations, such as deletional or insertional mutations in gyrA, endA, or recA, or combinations thereof (particularly gyrA/endA or gyrA/recA), which allow cell strains carrying the one or more suppression mutations to survive the presence and/or expression of one or more toxic genes within their genome or in extrachromosomal genetic elements within the host cell. Preferred host cell strains include prokaryotic host cells, particularly specified strains of E. coli containing the gyrA462 mutation and/or one or more additional mutations, such as DB3, DB3.1, DB4 and DB5.

Abstract: Methods for homologously recombining an exogenous nucleic acid into a target cell genome of a target cell are provided. In the subject methods, a targeting vector that includes a linearizing endonuclease site, e.g., a recombinase recognition site, and a homologous recombination integrating element, is contacted with the target cell(s) such that the target cell(s) take up the targeting vector. The targeting vector is originally a circular targeting vector that is linearized by a linearizing endonuclease, e.g., a recombinase, either prior to administration (e.g., in vitro treatment with an endonuclease) or upon entry into the multicellular organism by endonucleases in the extracellular or intracellular fluids that can be introduced to or already present in the muticellular organism. The integrating element homologously recombines into the target cell genome from the linearized targeting vector. Also provided are targeting vectors, systems and kits for use in practicing the subject methods.