Abstract: A method of identifying nucleotide sequences coding for signal peptides in lactic acid bacteria, using a DNA molecule comprising a transposon including a promoterless reporter gene from which DNA molecule a region between the LR and the reporter gene is deleted and the DNA molecule comprises a DNA sequence coding for a secretion reporter molecule. By deleting the region between the LR and the reporter gene, stop codons in-frame with the secretion reporter molecule is removed which upon transposition permits translational fusions from upstream the LR.

Abstract: The present invention provides a genetically engineered microorganism belonging to the genus Gluconobacter or Acetobacter, which has an engineered gene for the biological activity of reducing L-sorbose which is more than 90% non-functional in developing the said biological activity. The engineered microorganism is derived from a microorganism belonging to the genus Gluconobacter or Acetobacter, and has the biological activity for reducing L-sorbose that is less than 10% of the amount of the activity of the wild type organism.

Abstract: The present invention relates to an automated method of transposon-mediated multiplex sequencing of DNA fragments inserted into a vector. It relates more particularity to an increased efficiency in such automated methods, where the increased efficiency is obtained by screening out before the sequencing those constructs in which the transposon inserted into the vector sequence. This prevents a waste of time and resources in performing reactions sequencing the vector instead of the DNA fragments of interest.

Abstract: The present method is useful for the identification of genes, ORF's and other nucleic acid molecules which are essential for the expression of a specific phenotype in microorganisms. The method employs In vitro transposition in conjunction with an chromosomal integration vector containing a specific gene or genetic element whose function is unknown. Subsequent transformation of a recombination proficient host with the vector and growth first under non-integrating conditions and then under integrating conditions, followed by a selection screen for either single or double crossover events results in transformants that may be subjected to phenotypic screens to determine gene function.

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: The invention relates to a method for determining if repetitive sequences from nucleic acid sequence databases are bona fide transposons.

Abstract: The invention relates to a method for generating a transgenic organism. The invention also relates to a method for detecting and characterizing a genetic mutation in a transgenic organism. The invention further relates to a method for isolating a gene which is correlated with a phenotypic characteristic in a transgenic animal. The invention further relates to a method for isolating an exon in a transgenic animal. The invention also relates to a method for modulating the expression of a gene in an organism.

Abstract: We disclose compositions and processes for transferring a nucleic acid into a mammalian cell utilizing a transposase to achieve nonviral integration of exogenous nucleic acid into the chromosomal DNA of the cell.

Abstract: Disclosed herein are methods for regulating fungal gene expression, and reagents for carrying out those methods.

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

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

Abstract: The present invention relates to an improved and efficient method for simultaneous monitoring of abundance of individual mutants of a microbe in mixed populations where insertion of a known transposon in the genome of a microbe such that each mutant caries a single transposon insertion, isolating the genomic DNA of the mixed population of mutants, fragmenting the same with a frequently cutting restriction enzyme, ligating a double standard adapter to the genomic DNA fragments, amplifying the DNA fragments adjoining transposon insertions thereby generating a set of DNA fragments corresponding only to mutated genes resolving the said amplified DNA fragments followed by comparing the intensity of DNA fragments obtained from population of mutants before selection with that obtained from the population subjected to selection and finally sequencing the DNA fragments that change in abundance to identify the mutated genes.

Abstract: The invention relates to methods, cells and nucleic acids for making transgenic animals. The methods generally comprise introducing into a genome of an animal a genetic construct comprising a transcriptional regulatory element operably linked to a heterologous marker gene encoding a marker, wherein the element drives expression of the marker across genera transgenic in the construct sufficient to visually detect the marker in photoreceptive cells or organs, and selecting for transgenesis by visually detecting the marker in a photoreceptive cell or organ of the animal.

Abstract: The present invention provides three insertion elements and transposases encoded by the insertion elements that are derived from the genome of Ralstonia solanacearum, which has been isolated with a transposon trap vector.

Abstract: The present invention provides three insertion elements and transposases encoded by the insertion elements that are derived from the genome of Ralstonia solanacearum, which has been isolated with a transposon trap vector.

Abstract: The present invention provides methods which greatly facilitate the rapidity in which cells and transgenic animals with targeted genes may be generated. The invention hastens the investigation of cells and transgenic animals bearing lowered expression of the targeted gene product, a truncated targeted gene product, a fusion protein of the targeted gene and exogenous DNA, or the expression of a different gene from the locus of the targeted gene whose product has reduced expression levels. Also disclosed is a transgenic animal having Cushing's disease. Also disclosed are diagnostic methods for detecting patients with endocrine disorders, and methods for treating or alleviating the symptoms of endocrine disorders.

Abstract: The invention is specifically directed to efficient, random, simple insertion of a transposon or derivative transposable element into DNA in vivo or in vitro. The invention is particularly directed to mutations in ATP-utilizing regulatory transposition proteins that permit insertion with less target-site specificity than wild-type. The invention encompasses gain-of-function mutations in TnsC, an ATP-utilizing regulatory transposition protein that activates the bacterial transposon Tn7. Such mutations enable the insertion of a Tn7 transposon or derivative transposable element in a non-specific manner into a given DNA segment. Insertion can be effected in plasmid and cosmid libraries, cDNA libraries, PCR products, bacterial artificial chromosomes, yeast artificial chromosomes, mammalian artificial chromosomes, genomic DNAs, and the like. Such insertion is useful in DNA sequencing methods, for genetic analysis by insertional mutagenesis, and alteration of gene expression by insertion of a given genetic sequence.

Abstract: The present invention provides three insertion elements and transposases encoded by the insertion elements that are derived from the genome of Ralstonia solanacearum, which has been isolated with a transposon trap vector.

Abstract: A method of efficiently sequencing multiple exons from complex genomic DNAs is disclosed. The methodology includes the use of bacterial and bacteriophage-derived artificial chromosomes (BBPACS) in novel gene trapping protocols. Targeted gene trapping by homologous recombination, and random gene trapping with the use of a transposon system are exemplified. Included in the invention are methods of preparing a gene map from BBPAC contigs, the resulting gene maps, methods of constructing a cDNA library from BBPAC contigs, and the resulting cDNA 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 cloning vector which comprises a plasmid or bacteriophage comprising the phage DNA of the invention.

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 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: 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: 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. The BAC system is used to provide to the prokaryotic host cell an additional copy of a known segment of DNA of the host cell (or of another prokaryotic cell whose genome is known) to construct merodiploid test cells wherein the chromosomal region of the host cell that is homologous to the DNA contained in the BAC becomes diploid. Alternatively, due to the high homology in essential genes of prokaryotes, the DNA contained in the BAC can be derived from a prokaryote other than the host cell. A transposon is then delivered randomly to the merodiploid cell.

Abstract: The invention provides a method for making a cell which does not contain a natural nisA gene but expresses a variant nisA gene, said method comprising the steps of providing a cell that contains a natural, chromosomal nisA gene and substituting the natural nisA gene or part thereof with a variant nisA gene at the chromosomal location of the natural nisA gene or part thereof. The invention further provided a process for producing variant nisin and cells for use in the same.

Abstract: The present invention provides plasmids for insertional mutagenesis in bacteria. Specifically, the invention provides plasmids comprising a transposition system including a transposon and a transposase, in which transposon insertion can be temporally regulated. The plasmids can be used to determine the relative importance of a particular gene in viability of the organism from which the gene is derived. The plasmids are particularly useful for large-scale screening of a multiplicity of genes.

Abstract: Methods for producing transposable elements with improved properties as vectors are provided. Directed evolution procedures are employed to improve characteristics of transposable elements, including transposons and insertion sequences as vectors. Methods for generating diversity in vivo and in vitro using transposable elements as vectors are provided.

Abstract: The invention is directed to improved methods for gene expression using vectors with multiple promoters. Multiple promoters are used in nucleic acid constructs to provide increased expression of a desired nucleic acid sequence. The sequence is introduced into a vector by conventional cloning or is expressed from an endogenous sequence in the genome that is activated by the vector containing the multiple promoters.

Abstract: The present invention provides three insertion elements and transposases encoded by the insertion elements that are derived from the genome of Ralstonia solanacearum, which has been isolated with a transposon trap vector.

Abstract: A novel insertion sequence, which has been found in the sMMO gene coding for methane monooxygenase of methane-assimilating bacterium Methylococcus capsulatus NCIMB 11132 strain, and has inverted repeat sequences consisting of a sequence of the nucleotide numbers 5-19 of SEQ ID NO: 1 at the both ends, can be utilized as effective means for genetic analysis including creation of insertion mutant strains, gene mapping, promoter searching, insertion of genetic information into chromosomal DNA, disruption of specific gene and the like, or utilized for improving methane-assimilating bacteria by chromosomal genetic engineering techniques.

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: The invention features a general system for the identification of essential genes in organisms. This system is applicable to the discovery of novel target genes for antimicrobial compounds, as well as to the discovery of genes that enhance cell growth or viability.

Abstract: The present invention relates to a process for integration of a chosen gene or of a specific DNA sequence in a DNA sequence such as the chromosome or episome of a bacterium, wherein: a) the said chosen gene or the chosen DNA sequence is cloned inside a defective transposon outside the essential parts of the transposon, b) the said transposon is integrated in the DNA sequence such as the chromosome or the episome of the said bacterium, and also the bacterium strains obtained by implementation of this process.

Abstract: A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

Abstract: A method of efficiently sequencing multiple exons from complex genomic DNAs is disclosed. The methodology includes the use of bacterial and bacteriophage-derived artificial chromosomes (BBPACs) in novel gene trapping protocols. Targeted gene trapping by homologous recombination, and random gene trapping with the use of a transposon system are exemplified. Included in the invention are methods of preparing a gene map from BBPAC contigs, the resulting gene maps, methods of constructing a cDNA library from BBPAC contigs, and the resulting cDNA libraries.

Abstract: A method of efficiently sequencing multiple exons from complex genomic DNAs is disclosed. The methodology includes the use of bacterial and bacteriophage-derived artificial chromosomes (BBPACs) in novel gene trapping protocols. Targeted gene trapping by homologous recombination, and random gene trapping with the use of a transposon system are exemplified. Included in the invention are methods of preparing a gene map from BBPAC contigs, the resulting gene maps, methods of constructing a cDNA library from BBPAC contigs, and the resulting cDNA libraries.

Abstract: A process for replacing a nucleotide sequence in the genome of a mycobacterium strain comprises the steps of:a) providing a vector containing SacB gene coding for levane saccharase enzyme and a nucleotide sequence of interest;b) transfecting the mycobacterium strain with the vector;c) selecting clones of the resulting transfected mycobacteria for replacement of the nucleotide sequence of interest by propagating the transfected clones in a culture medium supplemented with sucrose; andd) isolating the recombinant strain.The process is useful for positive selection of allelic exchange mutants, such as in Mycobacterium tuberculosis complex.

Abstract: This invention is directed to L5 shuttle phasmids capable of delivering foreign DNA into mycobacteria and to methods of producing L5 shuttle phasmids. In addition, this invention is directed to a method of generating mycobacterial mutations and to a method of producing mycobacterial vaccines.

Abstract: There are provided novel transposable elements isolated from Aspergillus. Also provided are novel fragments comprising the inverted repeat(s) of the transposable elements, such fragments being useful as probes to isolate transposable elements from other filamentous fungi.

Abstract: The present invention provides a conditional shuttle phasmid constructed by inserting a cosmid into a non-essential region of the TM4 mycobacteriophage that introduces DNA of interest into mycobacteria, especially M. tuberculosis complex organisms and other slow growing mycobacteria. The present invention provides a recombinant mycobacterium which expresses a DNA of interest incorporated into its chromosome by a TM4 conditional shuttle phasmid containing the DNA of interest. The present invention further provides a mycobacterial auxotrophic mutant and a method of generating auxotrophic mutants.

Abstract: A system for in vitro transposition includes a donor DNA that includes a transposable element flanked by a pair of bacterial transposon Tn5 outside end repeat sequences, a target DNA into which the transposable element can transpose, and a modified Tn5 transposase having higher binding avidity to the outside end repeat sequences and being less likely to assume an inactive multimer form than wild type Tn5 transposase.

Abstract: A vector containing a selected nucleic acid sequence interposed between the terminal ends of a Tn7 transposon and a method for site specific integration of genetic material into a chromosome using those vectors are provided.

Abstract: A system for in vitro transposition includes a donor DNA that includes a transposable element flanked by a pair of bacterial transposon Tn5 outside end repeat sequences, a target DNA into which the transposable element can transpose, and a modified Tn5 transposase having higher binding avidity to the outside end repeat sequences and being less likely to assume an inactive multimer form than wild type Tn5 transposase.

Abstract: The invention described here is a method whereby a molecular tag is put on a gene, transcript and protein in a single recombinational event. The protein tag takes the form of a unique peptide that can be recognized by an antibody or other specific reagent, the transcript tag takes the form of the sequence of nucleotides encoding the peptide that can be recognized by a specific polynucleotide probe, and the gene tag takes the form of a larger sequence of nucleotides that includes the peptide-encoding sequence and other associated nucleotide sequences. The central feature of the invention in its essential form is that the tag-creating DNA has a structure such that when it is inserted into an intron within a gene it creates two hybrid introns separated by a new exon encoding the protein tag. A major virtue of the method is that it allows one to identify new proteins or protein-containing structures, and, having done so, to readily identify and analyze the genes encoding those proteins.