Abstract: A composition for external use, comprising 2-O-(?-D-glucopyranosyl)ascorbic acid represented by the formula (I): or a salt or ester thereof which is safe to the human body, and a koji mold or a processed koji. The composition for external use is excellent in skin permeability, containing an ascorbic acid derivative which is excellent in stability, utilized persistently in the living body, and strong in antioxidant activity, and has little skin irritation.

Abstract: A process for producing an optical active &bgr;-amino alcohol, the method comprising the step of allowing at least one microorganism selected from the group consisting of microorganisms belonging to the genus Morganella and others, to act on an enantiomeric mixture of an &agr;-aminoketone or a salt thereof having the general formula (I): to produce an optical active &bgr;-amino alcohol with the desired optical activity having the general formula (II) described below in a high yield as well as in a highly selective manner:

Abstract: The invention relates to DNA constructs encoding a safe, selectable marker, other than an antibiotic resistance marker, vectors and/or cells including said constructs; and vaccines based on said constructs for use in animals and particularly humans. The safe, selectable marker being a marker that confers resistance to an agent, other than an antibiotic, which would otherwise deleteriously affect the growth of a cell in which said construct was placed.

Abstract: A method for generating adenoviral vectors from polynucleotides such as plasmids wherein there occurs recombinase-mediated transfer of an adenoviral ITR and terminal proteins bound to the ITR to a plasmid, thus enabling the plasmid to replicate as an adenoviral vector. Such method enables the rapid generation of adenoviral vectors at high titers from plasmids without the use of selectable markers and screening procedures. Such method enables the rapid generation of adenoviral vectors devoid of adenovirus backbone genes. The method also may be employed to generate hybrid adenoviral-retroviral vectors that convert transduced cells into producer cells that produce retroviral vectors to effect high level, permanent genetic modification of cells in vivo.

Abstract: The present invention makes available methods and reagents for novel manipulation of nucleic acids. As described herein, the present invention makes use of the ability of intronic sequences, such as derived from group I, group II, or nuclear pre-mRNA introns, to mediate specific cleavage and ligation of discontinuous nucleic acid molecules. For example, novel genes and gene products can be generated by admixing nucleic acid constructs which comprise exon nucleic acid sequences flanked by intron sequences that can direct trans-splicing of the exon sequences to each other. The flanking intronic sequences can, by intermolecular complementation, form a reactive complex which promotes the transesterification reactions necessary to cause the ligation of discontinuous nucleic acid sequences to one another, and thereby generate a recombinant gene comprising the ligated exons.

Abstract: The invention includes a method for recombining and, optionally, cloning nucleic acids without the use of restriction enzymes or DNA ligase. The method involves recombining an insert and a recipient nucleic acid using custom designed complementary regions to anneal strands from the nucleic acids. The complementary regions on double stranded nucleic acids are exposed by limited digestion of the ends of the nucleic acids. After annealing the digested ends, single stranded gaps on the hybridized nucleic acid are closed to yield a double stranded nucleic acid which may, optionally, be cloned into a microorganism. The invention also includes kits for recombining and, optionally, cloning nucleic acids using the methods of the invention. The invention further includes recombinant nucleic acids prepared using the methods of the invention.

Abstract: The present invention is directed to methods for the expansion of non-terminally differentiated cells ("precursor cells") using agonists of Notch function, by inhibiting the differentiation of the cells without inhibiting proliferation (mitotic activity) such that an expanded population of non-terminally differentiated cells is obtained. The cells are preferably stem or progenitor cells. These expanded cells can be used in cell replacement therapy to provide desired cell populations and help in the regeneration of diseased and/or injured tissues. The expanded cell populations can also be made recombinant and used for gene therapy, or can be used to supply functions associated with a particular precursor cell or its progeny cell.

Abstract: Double-stranded DNA molecules characterised in that they are circular and in that they essentially include one or more genes of interest.

Abstract: Chain reaction cloning methods and reagents and kits for performing such methods are provided. Chain reaction cloning allows ligation of double-stranded DNA molecules by DNA ligases and bridging oligonucleotides. Double-stranded nucleic acid molecules are denatured into single-stranded molecules. The ends of the molecules are brought together by hybridization to a template. The template ensures that the two single-stranded nucleic acid molecules are aligned correctly. DNA ligase joins the two nucleic acid molecules into a single, larger, composite nucleic acid molecule. The nucleic acid molecules are subsequently denatured so that the composite molecule formed by the ligated nucleic acid molecules and the template cease to hybridize to each. Each composite molecule then serves as a template for orienting unligated, single-stranded nucleic acid molecules. After several cycles, composite nucleic acid molecules are generated from smaller nucleic acid molecules.

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 invention comprises a series of adenovirus-based vectors having deletions in the E1 and/or E3 regions, and also insertions of pBR322 sequences, which can be used to deliver nucleic acid inserts into host cells, tissues or organisms that then can express the insert. The invention includes the use of the vectors in introducing genes into cells, in making vaccines and in gene therapy.

Abstract: A recombinant cell stably transformed with a cDNA of a silent, inducible replicon encoding a recombinant protein is disclosed. Transcription of the replicon cDNA is under the control of a silent promoter inducible by a DNA virus and expression of the recombinant protein is dependent upon the presence of the DNA virus in the cell. The cell can be engineered to package the replicon upon infection by the DNA virus, leading to intercellular amplification of expression of the recombinant protein. Where the recombinant protein is a reporter gene product, the recombinant cell may be used in an assay for detecting DNA viruses. A kit for performing such an assay is also provided.

Abstract: Disclosed are methods, DNA sequences, vectors and cell lines useful for the rapid generation of stable mammalian cell lines expressing high levels of recombinant proteins.

Abstract: The invention provides a method and materials for analyzing the frequency of sequences in a population of polynucleotides, such as a cDNA library. A population of restriction fragments is formed which is inserted into vectors which allow segments to be removed from each end of the inserted fragments. The segments from each restriction fragment are ligated together to form a pair of segments which serves as a tag for the restriction fragment, and the polynucleotide from which the fragment is derived. Pairs of segments are excised from the vectors and ligated to form concatemers which are cloned and sequenced. A tabulation of the sequences of pairs provides a frequency distribution of sequences in the population.

Abstract: A noninfectious insect virus is described having an altered genetic element whose function is restored by genetic complementation, thereby again producing the insect infectious form of the virus. Also described is the insertion of a heterologous gene into the viral genome, such that an insect controlling or modifying substance is also produced by the virus for an improved bioinsecticidal effect and genetic stability of desired traits.

Abstract: The invention provides a method of preventing or reducing the severity of a cancer in a subject by stimulating the subject's immune response against the cancer. The invention provides, for example, a method of stimulating an immune response in a subject by administering to the subject tumor cells that are substantially similar to the subject's cancer cells and that are genetically modified to reduce or inhibit the expression of one or more immunosuppressive agents. The invention also provides a method of preventing or reducing the severity of cancer in a subject by stimulating the subject's immune response against the cancer by administering to the subject tumor cells that are substantially similar to the subject's cancer cells and that are genetically modified to prevent the expression of an immunosuppressive agents and, in combination with the genetically modified tumor cells, an immunostimulatory agent. The invention further provides compositions useful for practicing the claimed methods.

Abstract: The invention features a synthetic gene encoding a protein normally expressed in a mammalian cell wherein at least one non-preferred or less preferred codon in the natural gene encoding the protein has been replaced by a preferred codon encoding the same amino acid.

Abstract: A cytotoxin associated immunodominant antigen and the nucleic acid encoding the antigen from Helicobacter pylori are described. This antigen was identified from the cytotoxin positive CCUG 17874 Helicobacter pylori strain, and both the antigen and the DNA encoding it have been sequenced. The antigen is a hydrophilic, surface-exposed protein having a molecular weight of 120-132 kDa. The nucleic acid encoding the antigen may be incorporated into a vector for transformation of host cells for expression of the antigen. Both the DNA and the antigen can be used in assays for detection of disease or infection by Helicobacter pylori, and may find use in treating and preventing infection by Helicobacter pylori and the diseases associated with such infection.

Abstract: The subject invention finds utility in the area of gene therapy of diseases. More specifically, the invention concerns the making of a novel non-viral vector which can bind to desired DNA to form a combination useful to transfect diseased mitochondria of human or animal cells. The non-viral vector comprises a dequalinium salt subjected to standard liposome production procedures to obtain the vector named DQAsomes.

Abstract: An baculovirus artificial chromosome is disclosed which has the lef-8 gene inactivated. The baculovirus artificial chromosome allows the cloning and expression of heterologous genes in insect and mammalian cells without killing the host cells. Also disclosed is an infectious baculovirus comprising the artificial chromosome and a cell containing the chromosome. Also disclosed is an insect cell which expresses the LEF-8 gene in the absence of a baculovirus chromosome. Also disclosed is a method to express heterologous proteins in eukaryotic cells using the baculovirus artificial chromosome.

Abstract: The invention relates to the specific expression of heterologous sequences, particularly genes encoding cytotoxic products, in tumor cells under the control of regulatory transcriptional sequences. Particularly preferred promoters include H19 regulatory sequences, the IGF-1 promoter and the IGF-2 P3 and P4 promoters. The invention provides expression constructs and methods of administering such expression constructs. The compositions and methods of the invention are useful in the treatment of cancer.

Abstract: The Thermus aquaticus gene encoding a thermostable DNA polymerase (Taq Pol) is altered in the N-terminus-encoding region to provide mutant genes with improved expression in E. coli.

Abstract: Non-replicating vectors containing a nucleotide sequence coding for an F protein of respiratory syncytial virus (RSV) and a promoter for such sequence, preferably a cytomegalovirus promoter, are described for in vivo immunization. The nucleotide sequence encloding the RSV F protein may lack a sequence encoding the homologous signal peptide but possessing a heterologous signal peptide enhancing RSV F protein expression. Such non-replicating vectors, including plasmids, also may contain a further nucleotide sequence located adjacent to the RSV F protein encoding sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo. Such non-replicating vectors may be used to immunize a host against disease caused by infection with RSV, including a human host, by administration thereto, and may be formulated as immunogenic compositions with pharmaceutically-acceptable carriers for such purpose. Such vectors also may be used to produce antibodies for detection of RSV infection in a sample.

Abstract: A composition for the transfection of higher eucaryotic cells, comprising complexes of nucleic acid, a substance having an affinity for nucleic acid and optionally an internalizing factor, contains an endosomolytic agent, e.g. a virus or virus component, which may be conjugated. The endosomolytic agent, which is optionally part of the nucleic acid complex, is internalized into the cells together with the complex and releases the contents of the endosomes into the cytoplasm, thereby increasing the gene transfer capacity. Pharmaceutical preparations, transfection kits and methods for introducing nucleic acid into higher eucaryotic cells by treating the cells with the composition are also disclosed.

Abstract: This invention provides an isolated, vertebrate nucleic acid molecule encoding the normal protein that prevents development of Wilson's disease. This invention also provides a nucleic acid molecule comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of the above-described nucleic acid molecule. Finally, this invention provides various uses of the isolated Wilson's disease gene.

Abstract: Novel helper vectors are provided for complementing defective recombinant viral vectors, characterized in that they are provided with recombination sequences recognized by a recombinase. A complementation cell expressing the recombinase, and a method for preparing recombinant viral vectors as infectious viral particles for transferring and expressing genes of interest in a host organism or cell, are also provided. The invention is particularly suitable for use in gene therapy, especially in humans.

Abstract: The invention is directed to inter alia two related but self-sufficient improvements in conventional display methods. The first improvement provides methods of enriching conventional display libraries for members displaying more than one copy of a polypeptide prior to affinity screening of such libraries with a target of interest. These methods can achieve diverse populations in which the vast majority of members retaining full-length coding sequences encode polypeptides having specific affinity for the target. In a second aspect, the invention provides methods of subcloning nucleic acids encoding displayed polypeptides of enriched libraries from a display vector to an expression vector without the need for clonal isolation of individual members. These methods result in polyclonal libraries of antibodies and other polypeptides for use, e.g., as diagnostic or therapeutic reagents.

Abstract: The present invention features biarsenical molecules and target sequences that specifically react with the biarsenical molecules. Methods of using the biarsenical molecules, tetraarsenical molecules and the target sequences are included.

Abstract: Methods and compositions for the isolation and specific targeting of any single stranded DNA sequence to be acted upon by any desired double stranded DNA genetic element or recognition sequence in a cis-oriented fashion. The invention involves construction of a vector comprised of single stranded sequences that form "stem-loop" structures wherein the "loop" comprises the single stranded target sequence and the "stem" comprises the double stranded "functional" cis-acting genetic elements or recognition sequences. The in vivo formation of this single stranded intermediate (prior to stem-loop folding) is performed by genetic elements which direct the normal DNA replication of any one of a number of prokaryotic and eukaryotic viruses during their life cycles. Constructs containing these replicative functions are used to produce the desired single stranded intermediates. Also included in the stem-loop structure is an appropriate inverted tandem repeat which, forms the "stem" of the stem-loop structure.

Abstract: The invention involves a method of identifying nucleic acid sequences encoding signal peptide-containing proteins. The method features chimeric constructs containing a KRE9 gene that lacks a signal sequence. Yeast containing chimeric KRE9 plasmid constructs that encode signal sequences are selected based on their ability to grow on media in which sucrose is the sole carbon source.

Abstract: A method of synthesis of new and useful single-stranded DNAs which have a stem-loop configuration (ss-slDNA). The method is an in vivo or an in vitro synthesis. Replicating vehicles which produce these ss-slDNAs. The ss-slDNAs are described. Uses for these slDNAs are disclosed. They can be used for introducing random mutations, they lend themselves for replication by a variant of the PCR method. They can also be used for regulating gene function. Other uses are disclosed.

Abstract: The present invention is directed to methods, compositions, kits and apparatus to identify and detect the presence or absence of target analytes. The embodiments of the present invention have utility in identification of protein component of human telomerase and measurement of its levels in specimens and samples, as well as the design of test kits and apparatus for implementing such methods.

Abstract: A modified Aspergillus oryzae cell having an area gene which does not express a functional AreA activator, DNA sequences encoding the area gene, and method for using the cell of the invention for production of a heterologous polypeptide.

Abstract: Selectable Herpesvirus saimiri vectors which have a selection gene inserted into a junction region of the L- and H-DNA are described. Vectors of this type are able persistently to infect human T cells and thus are suitable for the expression of foreign genes in human T cells. An additional advantage is that no infectious virus particles are produced during this.

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: Vectors containing a nucleotide sequence coding for an F protein of respiratory syncytial virus (RSV) and a promoter for such sequence, preferably a cytomegalovirus promoter, are described. Such vectors also may contain a further nucleotide sequence located adjacent to the RSV F protein enclosing sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo. Such vectors may be used to immunize a host, including a human host, by administration thereto. Such vectors also may be used to produce antibodies for detection of RSV infection in a sample.

Abstract: The present invention provides a method of preparing a plasmid having both the ability of expressing retroviral genes and the ability of effecting the after-translational processing of the encoded products, and the resultant plasmid and the expression products thereof. The method of the present invention is to prepare a plasmid by insertion-linking a cDNA fragment containing at least the protease gene from among retroviral genes with a highly expressing gene or a gene direct expressing vector, thereby causing expression of the retroviral genes, and at the same time, to process said expression product itself with the protease in that expression product, thereby mass-producing three kinds of core protein encoded by vaq gene, and three kinds of enzymes encoded by pol gene, in the form of individual single mature or active proteins.

Abstract: Methods and compositions are provided herein for the use of a novel mutant form of E. coli heat-labile enterotoxin which has lost its toxicity but has retained its immunologic activity. This enterotoxin is used in combination with an unrelated antigen to achieve an increased immune response to said antigen when administered as part of an oral vaccine preparation.

Abstract: Provided are hydrophobic targeting sequences, which may serve to target heterologous proteins to a variety of cellular membranes. In particular, the structural components of the nuclear envelope, or those components which become nucleus-associated, may be targeted with the sequences provided. Also provided are methods of targeting heterologous proteins to particular membranes, and the use of these targeted proteins in therapeutic, diagnostic and insecticidal applications.

Abstract: Chimeric cDNA for the expression of immunogenic polypeptides include the genetic epitopic determinants for a base infectious bursal disease virus strain and at least one other infectious bursal disease virus strain. The genetic epitopic determinants encode amino acids or amino acid sequences which define epitopes bound to by previously established monoclonal antibodies. The immunogens expressed by the cDNA may be employed to provide a vaccine against a plurality of IBDV strains. The epitopic determinant of IBDV lethal strains has been detected, and an immunogen for conferring immunity with respect thereto is disclosed. Similarly, a monoclonal antibody specific for IBDV lethal strains is identified, and a vaccine for passive immunization therewith is also disclosed. Immunogens exhibiting conformational epitopes, in the form of virus-like particles, are effective in the preparation of vaccines.

Abstract: The present invention provides a protein regulating the sensitivity of fungus to an antimycotic aureobasidin, a gene coding for this protein, the use thereof, an antibody for the protein and the use thereof. The invention is useful in the diagnosis and treatment for diseases including mycoses.The invention also provides a novel chromosome integration vector capable of imparting a novel selective marker of a drug resistance to a fungal transformant, a transformant transformed with this vector and a process for producing the same. In particular, it provides a protein capable of imparting the resistance to aureobasidin and acting as a selective marker and a DNA coding for the same.

Abstract: A human mutY polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for preventing and/or treating diseases associated with a mutation in this gene. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention for detecting diseases, for example, cancer, are also disclosed.

Abstract: The invention provides isolated nucleic acids molecules, designated Siva nucleic acid molecules, which encode proteins involved in immune cell apoptosis. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing Siva nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a Siva gene has been introduced or disrupted. The invention still further provides isolated Siva proteins, fusion proteins, antigenic peptides and anti-Siva antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

Abstract: A plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus (BVDV), chimeric derivatives of the plasmid and a method of producing an infectious bovine viral diarrhea virus using the plasmid are disclosed. The invention relates to a plasmid DNA molecule that replicates easily in E. coli and contains a sufficient portion of the genome of BVDV, cloned as cDNA, to be a suitable template to produce RNA in vitro which, upon transfection into bovine cells, gives rise to infectious BVDV. The BVDV created by the process of the invention can be engineered for use as a vector in many advantageous applications.

Abstract: A recombinant adenovirus and a method for producing the virus are provided which utilize a recombinant shuttle vector comprising adenovirus DNA sequence for the 5' and 3' cis-elements necessary for replication and virion encapsidation in the absence of sequence encoding viral genes and a selected minigene linked thereto, and a helper adenovirus comprising sufficient adenovirus gene sequences necessary for a productive viral infection. Desirably, the helper gene is crippled by modifications to its 5' packaging sequences, which facilitates purification of the viral particle from the helper virus.

Abstract: The present invention provides a method for preparing HSV vectors. The method comprises co-transfecting a source vector and a mutating cassette together into a population of appropriate host cells, such that homologous recombination occurs between the mutating cassette and the source vector whereby the mutating cassette replaces a region of the HSV genome. The mutating cassette has a unique restriction site not present in the sequence of the vector. The method further comprises plaquing the co-transfected host cells, selecting plaques in which recombination has occurred between the source vector and the mutating cassette, and isolating the viral DNA from the plaques. The isolated viral DNA is digested with a restriction endonuclease appropriate for cleaving the viral DNA at the unique restriction site within the mutating cassette to produce two viral polynucleotides. Following purification, the two viral polynucleotides can be ligated to form an HSV vector comprising the two viral polynucleotides.

Abstract: An expression control polynucleotide capable of affecting the expression of a second polynucleotide, and derived from a spliceosomal protein gene promoter. Isolation of plant spliceosomal protein gene promoters from potato and maize is described. Partial sequences of the promoters of two potato spliceosomal protein gene promoters are disclosed.

Abstract: An isolated and purified DNA molecule comprising a sugarcane bacilliform virus promoter and expression cassettes comprising said promoter are provided. Also provided is a method of using a sugarcane bacilliform virus promoter to express proteins, RNA transcripts, or mixtures thereof, in transgenic plants.

Abstract: The present invention provides multiply deficient adenoviral vectors and complementing cell lines. Also provided are recombinants of the multiply deficient adenoviral vectors and a therapeutic method, particularly relating to gene therapy, vaccination, and the like, involving the use of such recombinants.

Abstract: Methods and compositions are provided for transferring DNA sequence information from a first vector to a second vector. In the subject methods, a first vector comprising a region of DNA having a sequence of interest is contacted with a set of three pairs of oligonucleotide primers under conditions sufficient to produce three different PCR products, where each product corresponds to a different reading frame. The oligonucleotide primers comprise a first region of sequence identity with the first vector and a second region permissive of site specific recombination with the second vector. The resultant PCR products are combined with the second vector under conditions sufficient for site specific recombination to occur. Also provided are kits for use in performing the subject methods.