Abstract: The present invention provides a method for producing nested deletions in vitro in desired DNA, comprising: 1) preparing a vector containing a DNA fragment in which nested deletions are to be generated and the terminal repeat of a transposon; 2) incubating the vector in vitro with transposase and a DNA replication system; 3) obtaining the vector incorporating the DNA fragment with nested deletions as a reaction product; and optionally, 4) transforming a host cell with the reaction product and growing it; and 5) recovering a vector incorporating said DNA fragment with nested deletions from the grown host cell.

Abstract: The present invention provides an expression vector comprising an RK2 minimum replicon together with an expression cassette comprising the regulatory functions of a TOL plasmid, and, in particular, an expression vector comprising a RK2 minimum replicon together with a promoter Pm and/or Pu and a corresponding regulatory gene xylS and/or xylR as derived from a TOL plasmid. Such expression vectors may be used to express desired genes in a wide range of gram negative and gram positive bacterial hosts.

Abstract: The present invention relates to the utilization of conditionally replicating recombinant nucleic acid molecules rescued from the integrated state for the expression of foreign proteins. The usefulness of the system is illustrated with a conditionally replicating recombinant nucleic acid molecule encoding the adeno-associated virus (AAV) capsid proteins. The present invention also relates to methods employing and conditionally replicating recombinant nucleic acid molecules for the packaging of recombinant AAV nucleic acid molecule into AAV capsids. The present invention also relates to packaging cell lines for recombinant AAV, expressing both the AAV rep and cap-genes.

Abstract: The promoter of the human p27Kip1 gene is provided. The promoter region is useful to screen a compound that regulates the promoter of the human p27Kip1 gene or regulates the activity of the promoter. It enables the gene therapy utilizing the promoter.

Abstract: The present invention provides nucleic acid molecules capable of binding phospholipid membranes, and more particularly RNA molecules capable of binding and forming channels in biological membranes. In this regard the present invention provides methods for screening nucleic acid molecules that bind phospholipid membranes. The present invention also provides compositions of nucleic acid molecules capable of binding phospholipid membranes, as well as methods employing these compositions to alter the permeability or detectably label phospholipid membranes.

Abstract: The coding information for three putative chicken anemia virus proteins (VP1, VP2, VP3) was inserted into a baculovirus vector and expressed in insect cells. The immunogenic properties of the chicken anemia virus (CAV) proteins produced separately or together in insect-cell cultures were analyzed by inoculating them into chickens. Only lysates of insect cells which have synthesized equivalent amounts of all three recombinant CAV proteins or cells which synthesized mainly VP1 plus VP2 induced neutralizing antibodies directed against CAV in inoculated chickens. Progeny of those chickens were protected against clinical disease after CAV challenge. Inoculation of a mixture of lysates of cells that were separately infected with VP1-, VP2- and VP3-recombinant baculovirus did not induce significant levels of neutralizing antibody directed against CAV and their progeny were not protected against CAV challenge.

Abstract: The current invention concerns the use of a receptor from the RZR/ROR receptor family or of a functional fragment thereof in a test of a compound for anti-autoimmune, anti-arthritic, anti-tumor, melatonin-like and/or melatonin-antagonistic activity and the production of a receptor ligand complex comprising said receptor or a functional fragment thereof and a ligand of said receptor. Described is also a method for testing compounds for said activity (screening for ligands) and the active compounds identified therewith.

Abstract: The present invention relates to nuclease resistant nucleic acids in general and ribonuclease resistant RNAs in particular. Methods of making and using such nucleic acids are disclosed.

Abstract: The present invention relates to synthetic leader peptide sequences for secreting polypeptides in yeast.

Abstract: Methods and vectors (both DNA and retroviral) are provided for the construction of a Library of mutated cells. The Library will preferably contain mutations in essentially all genes present in the genome of the cells. The nature of the Library and the vectors allow for methods of screening for mutations in specific genes, and for gathering nucleotide sequence data from each mutated gene to provide a database of tagged gene sequences. Such a database provides a means to access the individual mutant cell clones contained in the Library. The invention includes the described Library, methods of making the same, and vectors used to construct the Library. Methods are also provided for accessing individual parts of the Library either by sequence or by pooling and screening. The invention also provides for the generation of non-human transgenic animals which are mutant for specific genes as isolated and generated from the cells of the Library.

Abstract: The present invention relates to cloned DNA containing origin of DNA replication and to cloned DNA encoding repliation protein, RepT.

Abstract: Proteins such as human &bgr;-interferon or human erythropoietin are prepared by culturing mammalian cells which harbour a nucleic acid sequence comprising: (i) a coding sequence which encodes the desired protein and which is operably linked to a promoter capable of directing expression of the coding sequence in a mammalian cell in the presence of a heavy metal ion; and (ii) a first selectable marker sequence comprises a metallothionein gene and which is operably linked to a promoter capable of directing expression of the metallothionein gene in a mammalian cell in the presence of a heavy metal ion; and optionally (iii) a second selectable marker sequence which comprises a neo gene and which is operably linked to a promoter capable of directing expression of the neo gene in a mammalian cell.

Abstract: A plasmid was constructed, in which the hiNOS structural gene was replaced with the luciferase structural gene as a reporter gene, with retaining functions of the hiNOS gene 5′-promoter region and 3′-untranslated region. This plasmid was stably transfected into human cell lines. The above transformed cells selectively expressed the reporter gene in the presence of inducers. It has become possible, by examining the reporter gene expression in these transformed cells, to simply and easily screen, with high sensitivity, a compound which is expected to be useful for treating inflammations and sepsis by suppressing the hiNOS expression, or a compound which is expected to be useful for antitumor, antiviral, and vascular restenosis prevention treatments by the hiNOS induction.

Abstract: The present method provides a method for inhibiting restenosis associated with mechanical injury of a blood vessel. Human heme oxygenase I (HO1) is directly administered at the site of injury. The present inventors have discovered that carbon monoxide generated by HO1 is involved in the molecular pathogenesis of vascular proliferative disorders. By using adenoviral-mediated expression of inducible heme oxygenase 1 in primary vascular smooth muscle cells (vsmc) in vivo, the present inventors demonstrate that in vivo expression of HO1 can be used to treat restenosis.

Abstract: The promoter of the EPCR gene has been isolated from both murine (SEQ. ID No. 1) and human (SEQ. ID No. 2) genomic libraries. The promoter includes a region (nucleotides 3130 to 3350 of SEQ. ID No. 1 which affects selective gene expression in endothelial cells), and a region (nucleotides 2270 to 2840 of SEQ. ID No. 1) which affects selective gene expression in large vessel endothelial cells, as compared to expression in all endothelial cells. The EPCR promoter contains a thrombin responsive element, CCCACCCC (SEQ. ID No. 3), (murine, nucleotides 3007 to 3014 SEQ. ID No. 1 and human, nucleotides 2722 to 2729 SEQ. ID No. 2). The EPCR also contains a serum response element (nucleotides 2990 to 3061 of SEQ. ID No. 1). The regulatory sequences present in the EPCR promoter can be used for thrombin or serum controlled recombinant gene expression specific to either all endothelial cells or primarily endothelial cells of large vessels.

Abstract: A new method for the identification of useful promoters is disclosed. The method is capable of identifying bacterial promoters sensitive to a particular cellular insult and may be modified to identify promoters sensitive to herbicides and crop protection chemicals. Constructs comprising promoters upstream of a luminescent reporter genes are placed in transformed hosts. Transformants grown in liquid media to a predetermined growth stage and contacted with a cellular insult are assessed for regulatory region activity by measurement of the resulting change in bioluminescence. The method is able to identify promoters undetectable by standard methods.

Abstract: The present invention is directed to the isolation and use of super-infective, tumor-specific vectors that are strains of parasites including, but not limited to bacteria, fungi and protists. In certain embodiments the parasites include, but are not limited to, the bacterium Salmonella spp., such as Salmonella typhimurium, the bacterium Mycobacterium avium and the protozoan Leishmania amazonensis. In other embodiments, the present invention is concerned with the isolation of super-infective, tumor-specific, suicide gene-containing strains of parasites for use in treatment of solid tumors.

Abstract: The present invention is directed to a method of vascular proliferative disease by administering in vivo a gene encoding p27.

Abstract: Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

Abstract: The present invention provides a method of expressing an exogenous nucleic acid in a mammal. The method comprises non-systemically administering to a non-neuronal tissue of said mammal an exogenous nucleic acid operatively linked to a promoter. The exogenous nucleic acid is proximal to at least one native parvoviral inverted terminal repeat and does not require encapsidation. The expression of the exogenous nucleic acid in the tissue is not substantially diminished at 28 days after administration of the exogenous nucleic acid.