Abstract: On the basis of a first repertoire of genes coding for a population of one of two kinds of polypeptides capable of being combined, particularly an antibody light chain variable region, and at least one gene, and preferably a second repertoire coding for the other kind of polypeptide, particularly an antibody heavy chain variable region, the genes from the first repertoire are inserted into a first vector to form a vector population and the genes from the second repertoire are inserted into a second vector, at least one of said vectors being a recipient for the expression of both genes as polypeptides irreversibly combined with the outer surface of the product of said vector.

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 process for the preparation of L-amino acids, in which the following steps are carried out, a) fermenting the desired L-amino acid-producing bacteria in which at least the glyA gene is attenuated, in particular by removal of the natural promoter, and optionally b) concentrating the desired product in the medium or in the cells of the bacteria and c) isolating the L-amino acid, and optionally bacteria in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally amplified are employed, or bacteria in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated are employed, and nucleotide sequences of the lacI-tac-5′glyA or lacI-tac-glyA unit.

Abstract: The present invention is directed to isolated polynucleotides coding for phosphoglucose isomerase (pgi) from coryneform bacteria. In addition, the invention includes methods for increasing the metabolic flux through pentose phosphate cycle of bacteria by reducing or eliminating the activity of pgi. These methods may be used to increase the fermentative production of nucleotides, vitamins and amino acids.

Abstract: A recombinant vaccinia virus derived from the vaccinia virus Ankara (MVA) encoding and capable of expressing the E2 gene of Bovine papillomavirus. Also, the use of the virus in the treatment of lesions caused by papillomavirus.

Abstract: The present invention relates to a class of microbial coding sequences the transcription or cotranscription of which is specifically induced during microbial infection of a host. These particular coding sequences or defined regions thereof may be used as probes to identify and isolate microbial virulence genes. The products of these virulence genes will provide potential targets for the development of vaccines or antimicrobial agents.

Abstract: The present invention relates to a DNA cassette intended for the transformation of yeast, leaving no useless exogenous DNA but the gene(s) of interest comprising at least one negative dominant marker, two direct repeat sequences (DRS) which are non exogenous and non recombinogenic with the genome of the host strain, these two direct repeat sequences flanking the negative dominant marker and optionally at least one gene of interest containing, if necessary, the elements necessary for its expression in the host cell. The invention relates as well to a method of integration of gene(s) of interest or inactivation of a gene in yeast, and of transformation of yeast with the DNA cassette, and to yeast strains thus obtained.

Abstract: The present invention relates to a recombinant organism, and a bacterial strain such as Staphylococcus, and S. aureus, in particular. The organism has a regulatable gene for encoding an RNA polymerase specificity factor required for expression of at least one gene essential for growth of the organism. The regulatable gene is one which is responsive to an exogenous effector molecule, and may in particular be an inducer-responsive gene including an operator site, such as a lac operator, to which a repressor, such as a lacl-encoded repressor, is capable of binding. The regulatable gene may in particular be an IPTG responsive plaC allele.

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 invention relates to a process for the selection from a gene library of a gene encoding an enzyme that is capable of catalyzing the conversion of a prodrug to its active drug form. The method comprises contacting a library of lysogenic bacteria with a prodrug that causes activation of bacterial RecA when converted to its active drug form. Activation of RecA causes lysis of the bacteria, so allowing separation of bacteriophage particles released into the medium, and their subsequent genotypic analysis to isolate nucleic acid molecules in the library that encode a desired prodrug-activating enzyme.

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

Abstract: A process for site-directed integration of multiple copies of a gene in a mould is provided, which comprises transforming a mould cell containing in its chromosomal DNA a restriction site for a rare-cutting endonuclease, e.g., I-Scel, preferably introduced at a desired locus, e.g., within a selectable marker gene or in the neighborhood thereof, with a piece of DNA comprising multiple copies of at least one expressible gene comprising at least one structural gene encoding a desired protein, surrounded by two DNA fragments homologous to part of the DNA upstream and downstream, and in the neighborhood, of said restriction site, while during the transformation of the mould the presence of the rare-cutting endonuclease is provided, followed by selecting or screening for a mould cell in which the multiple gene copies of said expressible gene are inserted into the chromosomal DNA of the mould.

Abstract: The present invention relates to filamentous fungi that comprise in their genomes at least two substantially homologous DNA domains which are suitable for integration of one or more copies of a recombinant DNA molecule and wherein at least two of these DNA domains comprise an integrated copy of a recombinant DNA molecule. The invention also relates to methods for preparing such filamentous fungi and for further multiplying the DNA domains with integrated recombinant DNA molecules through gene conversion or amplification.

Abstract: The present invention relates to a method for recovering recombinant HBsAg, wherein recombinant methylotrophic yeast cells which are capable of expressing HBsAg are disrupted using a high pressure homogenizer, and HBsAg is recovered from the cell debris obtained. The inventive method is characterized by a high product yield per g of cell dry weight and thus constitutes a considerable improvement over the former methods for recovering HBsAg.

Abstract: Disclosed is a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, where an “unmarked mutation” is a mutated nucleotide sequence introduced into a mycobacterium where the introduced mutated nucleotide sequence does not contain a selectable marker, such as a gene conferring antibiotic resistance to the recombinant mycobacterium incorporating the mutated nucleotide sequence. Also disclosed is a method for preparing a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, as well as a vaccine comprising a recombinant slow-growing mycobacterium having at least one mycobacterial gene containing an unmarked mutation dispersed in a physiologically acceptable carrier. Further disclosed is a method of treating or preventing tuberculosis in a subject comprising administering the vaccine of the present invention in an amount effective to treat or prevent tuberculosis in the subject.

Abstract: The present invention relates to a method of improving the strain used for the production of erythromycin through the disruption of the melA gene.

Abstract: The invention provides shuttle vectors, and methods of using shuttle vectors, capable of expression in, at least, a mammalian cell. Furthermore, the shuttle vectors are capable of replication in at least yeast, and optionally, bacterial cells. Also provided is a method wherein yeast are transformed with a shuttle vector as provided herein. Heterologous nucleic acids flanked by 5′ and 3′ ends identical to a homologous recombination site within the shuttle vector are introduced to the transformed yeast and allowed to homologously recombine with the shuttle vector such that they are inserted into the vector by the yeast organism. The shuttle vector is then recovered and transferred to a mammalian cell for expression.

Abstract: A method of DNA library screening includes homologous recombination in E. coli utilizing lambda phage recombination functions. Inserting a positive selection marker such as antibiotic resistance into the target sequence by homologous recombination facilitates isolation of target sequences and requires only about 58-100 base pairs of total homology, thus allowing the use of synthetic fragments of DNA for targeting. DNA vector is designed for genomic library construction that features a novel genetic selection for inserts, automatic subcloning of isolated genomic clones and the presence of a negative selection marker adjacent to the genomic inserts to facilitate later gene targeting.

Abstract: The invention provides novel yeast cells comprising genes whose expression can be modulated by growth in the presence or absence of metal ions, methods for making such yeast cells, and methods of using such yeast cells for determining the requirement for expression of particular genes for the growth or viability of the yeast cells.

Abstract: A method of transforming slow-growing mycobacteria, such as M. bovis BCG, M. leprae, M. tuberculosis, M. avium, M. intracellulare and M. africanum; a method of manipulating genomic DNA of slow-growing mycobacteria through homologous recombination; a method of producing homologously recombinant (HR) slow-growing mycobacteria in which heterologous DNA is integrated into the genomic DNA at a homologous locus; homologously recombinant (HR) slow-growing mycobacteria having heterologous DNA integrated into their genomic DNA at a homologous locus; and mycobacterial DNA useful as a genetic marker.

Abstract: We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kantr2). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu−Kmr transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

Abstract: A system for performing antiviral drug susceptibility and resistance testing is automated using software and robotics. The system includes a transfection apparatus, an infection apparatus and a plate reading apparatus. One or more of the apparatuses may be automated.

Abstract: The present invention relates to a system for identifying, isolating and utilizing promoter elements useful for expression of nucleotide sequences and the proteins encoded thereby in a thermophile. In one embodiment, a recombinant DNA molecule is provided, and comprises a reporter sequence, a putative thermophile promoter, a selectable marker sequence, and a 3′ and a 5′ DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile. Further, within the recombinant DNA, the reporter sequence is under the transcriptional control of a promoter which functions in a thermophile to form a promoter/reporter cassette, the promote/reporter cassette is flanked by said 3′ and said 5′ DNA targeting sequences, and the promoter/reporter cassette is positioned in the opposite orientation of the DNA targeting sequences.

Abstract: The present invention relates to a method for in vivo recombination of homologous DNA sequences. The method is a forced artificial evolution resulting in a DNA sequence encoding a polypeptide having an advantageous property.

Abstract: This invention relates to a method for enhancing the production of biologically active proteins and peptides in bacterial cells by infecting bacterial cells of the producer strain, which contain a plasmid with one or more targeted genes, with bacteriophage &lgr; with or without the targeted gene(s). The phage increases synthesis of the targeted protein and induces lysis of the producer strain cells. Super-production is achieved by cultivating the producer strain cells under culture conditions that delay lytic development of the phage. The biologically active proteins and peptides subsequently accumulate in a soluble form in the culture medium as the cells of the producer strain are lysed by the phage.

Abstract: The present invention provides the complete nucleotide sequence of a bovine adenovirus. The invention further provides bovine adenovirus vectors and expression systems which can be used, among other things, for insertion of foreign sequences, for provision of DNA control sequences including transcriptional and translational regulatory sequences, for diagnostic purposes to detect the presence of viral nucleic acids or proteins encoded by these regions in a subject or biological sample, for provision of immunogenic polypeptides or fragments thereof, for vaccines and for gene therapy. Cell lines comprising the vectors of the invention, and methods for making bovine adenovirus vectors are also provided.

Abstract: A coryneform bacterium in which a DNA fragment is incorporated into its chromosome is prepared by (a) obtaining a recombinant plasmid through ligation of a DNA fragment having a sequence homologous to a gene present on a chromosome of a coryneform bacterium to a plasmid that has a wild-type replication control region segment of a particular nucleotide sequence including a mutation and is autonomously replicable in a coryneform bacterium cell at a culture temperature lower than 31° C. but not autonomously replicable in the cell at a temperature of 31° C. or higher, (b) introducing the recombinant plasmid into the coryneform bacterium cell, (c) culturing the bacterium at a temperature of 31° C. or higher, (d) causing homologous recombination between the DNA fragment and the gene present on the chromosome of the coryneform bacterium and having a sequence homologous to the DNA fragment, and (e) selecting a coryneform bacterium in which the DNA fragment is incorporated into its chromosome.

Abstract: The invention relates to a method for producing an integrant(s) of Bacillus thuringiensis which produces a larger quantity of a crystal delta-endotoxin with greater pesticidal activity as compared to the crystal delta-endotoxin produced by the corresponding parental strain. The crystal delta-endotoxin produced by the integrant Bacillus thuringiensis will have an activity directed towards the same pest(s) as its parent Bacillus thuringiensis crystal delta-endotoxin. The invention further relates to such integrants, compositions comprising such integrants, as well as methods for controlling a pest(s) using these compositions.

Abstract: Provided is a genetic identification and characterization of a gene which encodes an essential yeast mitotic spindle protein. The protein functions in anaphase spindle elongation. The invention also provides an identification of a protein which interacts with this mitotic spindle protein. The proteins identified and characterized by the present invention are useful as development candidates for cancer chemotherapeutic agents, anti-fungal compounds, and other anti-mitotic agents.

Abstract: The present invention relates to a system for identifying, isolating and utilizing promoter elements useful for expression of nucleotide sequences and the proteins encoded thereby in a thermophile. In one embodiment, a recombinant DNA molecule is provided, and comprises a reporter sequence, a putative thermophile promoter, a selectable marker sequence, and a 3′ and a 5′ DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile. Further, within the recombinant DNA, the reporter sequence is under the transcriptional control of a promoter which functions in a thermophile to form a promoter/reporter cassette, the promote/reporter cassette is flanked by said 3′ and said 5′ DNA targeting sequences, and the promoter/reporter cassette is positioned in the opposite orientation of the DNA targeting sequences.

Abstract: The invention relates to a method for the preparation of DNA virus vectors capable of replication in eukaryotic as well as in prokaryotic cells as well as to DNA virus vectors prepared by this method. Preferably, the method is used to prepare Epstein-Barr virus vectors.

Abstract: The invention concerns a method for the introduction of a foreign DNA into the genome of a target cell by homologous recombination as well as for the homologous recombination of suitable DNA constructs.

Abstract: The invention provides shuttle vectors, and methods of using shuttle vectors, capable of expression in, at least, a mammalian cell. Furthermore, the shuttle vectors are capable of replication in at least yeast, and optionally, bacterial cells. Also provided is a method wherein yeast are transformed with a shuttle vector as provided herein. Heterologous nucleic acids flanked by 5′ and 3′ ends identical to a homologous recombination site within the shuttle vector are introduced to the transformed yeast and allowed to homologously recombine with the shuttle vector such that they are inserted into the vector by the yeast organism. The shuttle vector is then recovered and transferred to a mammalian cell for expression.

Abstract: The present invention provides a method for high frequency of allelic exchange in the slow-growing mycobacteria using in vitro generated specialized transducing mycobacteriophages, as well as the recombinant slow-growing mycobacteria generated using the disclosed method. A transducing mycobacteriophage of the present invention comprises a conditional mycobacteriophage containing an E. coli bacteriophage lambda cosmid inserted into a non-essential region of the mycobacteriophage, said cosmid containing a mutated DNA substrate which is homologous to a wildtype nucleic acid sequence of a slow-growing mycobacterium. When slow-growing mycobacteria infected with the conditional transducing phage are cultured under conditions wherein the conditional transducing phage does not replicate, the mutated DNA substrate is incorporated into the chromosomal DNA of the slow-growing mycobacteria by homologous recombination, thereby generating the recombinant slow-growing mycobacteria of the present invention.

Abstract: The invention relates to Agrobacterium mediated transformation of moulds comprising species of the fungal sub-divisions Ascomycotina, Basidiomycotina, Deuteromycotina, Mastigomycotina, and Zygomycotina. Examples demonstrate the transformation of Aspergillus awamori (both protoplasts and conidia), Aspergillus nidulans, Aspergillus niger, Colletotrichum gloeosporioides, Fusarium solani pisi, Neurospora crassa, Trichoderma reesei, Pleurotus ostreatus and Agaricus bisporus (all conidia), and Fusarium graminearum (both conidia and rehydrated freeze dried ATCC material). Especially for Aspergillus awamori the transformation frequency is much higher than with conventional mould transformation techniques. It has further been found that not only one expressable gene can be introduced into these moulds, but even multiple copies of such gene, which, moreover, can be targeted e.g. in the chromosomal pyrG locus, as exemplified for A. awamori.

Abstract: The present invention relates to a novel drug discovery system for generating molecular diversity. The system provides methods for subjecting the genetic materials from a plurality of species of organisms to homologous or homeologous recombination to create novel genes and metabolic pathways. The recombined genetic materials are cloned to form recombined combinatorial gene expression libraries. Methods for screening such gene expression libraries containing recombined genes and metabolic pathways for novel activities and compounds are also provided.

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

Abstract: A method for transforming Schizosaccharomyces pombe which comprises integrating a vector into a chromosome of Schizosaccharomyces pombe through homologous recombination, wherein the vector has an expression cassette containing a heterologous protein structural gene and a promoter and a gene segment which induces homologous recombination of the chromosome and has lost a replication origin which functions in cells of an organism other than Schizosaccharomyces pombe required for construction of the vector.

Abstract: Methods of identifying agents or compounds which are capable of inhibiting the replication and/or accumulation of DNA circles in cells are described. Also described are methods of assessing the ability of a compound to extend life span, as well as methods of extending life span, comprising administering to a cell a compound identified by the assays described herein which extends life span. The invention also pertains to isolated mWRN, or an active derivative or fragment thereof and to an isolated nucleic acid molecule which encodes mWRN, or an active derivative or fragment thereof.

Abstract: Homozygous gene replacement can be created in unicellular diploid organisms by individually targeting each allele of a gene with genetic constructs containing two different and independent selectable markers. Selection for both markers indicates replacement of both alleles of the gene, or portion thereof. The method can be used to study gene function in these organisms and to create mutant organisms such as attenuated strains of parasitic protozoans for use in live vaccines.

Abstract: The functional analysis of genes frequently requires the manipulation of large genomic regions. A yeast-bacteria shuttle vector is described, that can be used to clone large regions of DNA by homologous recombination. The important feature of present invention is the presence of the a bacterial replication origin, which allows large DNA insert capacity. The utility of this vector lies in its ability to isolate, manipulate and maintain large fragments in bacteria and yeast, allowing for mutagenesis by yeast genetics and simplified preparation of plasmid DNA in bacteria.

Abstract: Disclosed are nucleic acids from tomato encoding polypeptides having xyloglucan endo-transglycosylase (XET) activity, comprising residues 21-289 of the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or or comprising residues 19-287 of the amino acid sequence shown in FIG. 5 (SEQ ID NO:8), and transgenic plants comprising said nucleic acids.

Abstract: A vector having a gene for resistance to an antibiotic otherwise capable of killing a host yeast cell, the gene being transcribed from a yeast promoter sequence.

Abstract: The invention provides novel erythromycin derivatives in which methyl groups on the macrolactone ring have been substituted with —H, -Et, and/or —OH. The invention also provides reagents such as isolated polynucleotides, vectors comprising the polynucleotides and host cells transformed with the vectors for making the novel compounds. Methods for making the compounds utilizing genetic engineering techniques are also disclosed.

Abstract: A tryptophan producing strain of microorganism is selected from E. coli and Corynebacteria and is tryptophan feedback resistant and serine feedback resistant. The serine feedback resistance is by a mutation in a serA allele, where the mutated serA allele codes for a protein which has a Ki value for serine between 0.1 mM and 50 mM. The tryptophan feedback resistance is by a trpE allele which codes for a protein which has a Ki value for tryptophan between 0.1 mM and 20 mM. A process for preparing this microorganism and a process for using this microorganism are disclosed.

Abstract: On the basis of a first repertoire of genes coding for a population of one of two kinds of polypeptides capable of being optionally covalently combined, particularly variable regions of either the antibody light chain type or the antibody heavy chain type, and at least one gene coding for the other type of polypeptide, particularly a variable region of the other type, an antibody chain or preferably a second repertoire of genes coding for a population of said other type, the genes from the first repertoire are inserted into a first vector to form a population of vectors carrying the various genes of said first repertoire, and said gene of said other type or the genes from said second repertoire is/are inserted into a second vector. Both starting vectors have means enabling each to exchange one part by one or more irreversible recombinations to generate recombinant final vectors of which one contains a gene of one of said types and a gene of the other type.

Abstract: A process for increasing the amount of clavam produced by an organism having both a clavam pathway or a portion thereof and a cephalosporin pathway or a portion thereof by interfering with the conversion of L-lysine to L-&agr;-aminoadipic acid in the cephalosporin pathway. Plasmids containing a defective LAT (lysine amino transferase) gene and organisms containing such plasmids are also provided.

Abstract: A method of introducing exogenous cloned DNA into a bacterial chromosome of a bacteria in which the transposon Tn5 and the FLP recombinase are functional in vivo is disclosed. In one embodiment, the method comprises the steps of: (a) introducing FLP recombination target sites (FRTs) permanently at random locations in a bacterial chromosome using a plasmid vector that contains an FRT within a modified Tn5 transposon, two selectable markers, and a removable replication origin; (b) mapping the FRT introduced into the bacterial chromosome; (c) cloning exogenous DNA into a vector comprising two FRT sites, two selectable markers, and a removable replication origin; (d) removing the replication origin in the vector of step (c); (e) introducing the altered plasmid vector into bacterial cells, wherein the bacteria cells comprise a functional FLP recombinase; and (f) obtaining targeted integrants.

Abstract: The present invention is directed to recombinant nucleic acids encoding lactoferrin variants and portions thereof, having modified iron-binding capacity, and to vectors comprising same recombinant nucleic acids. The present invention is further directed to methods of producing such vectors, and to transfected cells harboring the same. Methods for the production of lactoferrin variants and portions thereof, in various eukaryotic or prokaryotic cells are also provided. Finally, the invention is directed to lactoferrin variants and portions thereof encoded by the nucleic acids of the invention and produced by the processes of the invention. Thus, the invention provides an efficient and economical means for the production of recombinant lactoferrin variants and portions thereof.

Abstract: Tools for genetically engineering Pasteurellaceae are provided. Replication-conditional plasmids which are useful for the Pasteurellaceae have been isolated and characterized. The plasmids can be utilized for delivery of DNA segments into the Pasteurellaceae in situations where control of extra-chromosomal replication desired, such as in achieving allelic exchange or site-directed mutagenesis. A restriction endonuclease, HsoI, was isolated from a bovine lung isolate of Haemophilus somnus. The enzyme was found to be a true isoschizomer of HinPI, a commercially available enzyme originally isolated from Haemophilus influenzae PI. Commercially available HhaI methyl transferase was found to protect against cleavage by both enzymes. Methylation of foreign plasmid DNA was found to enhance transformation of Haemophilus somnus in excess of four orders of magnitude.