Abstract: The present invention is directed to methods and compositions for DNA subcloning using bacterial recombinase-mediated homologous recombination. The invention relates to methods for cloning, compositions comprising polynucleotides usefall as cloning vectors, cells comprising such polynucleotide compositions, and kits useful for cloning mediated by bacterial recombinases, such as RecE/T and Red&agr;/&bgr;.

Abstract: Disclosed is a method for obtaining glucose-regulated expression of active insulin in the cells of a mammalian subject. The method involves delivering into the subject a genetic construct comprising a coding sequence for a human proinsulin operably connected a promoter functional in the host cells. The construct includes a glucose responsive regulatory module having at least one glucose inducible regulatory element comprising a pair of CACGTG motifs linked by a five base nucleotide sequence, which confers glucose inducible expression of the proinsulin coding sequence. To ensure proper processing of the proinsulin to active insulin, the coding sequence was modified to direct the synthesis of a mutant proinsulin polypeptide having amino acid sequences that can be cleaved to mature insulin in suitable host cells, such as hepatocytes.

Abstract: The invention provides pskG polypeptides and polynucleotides encoding pskG polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing pskG polypeptides to screen for antibacterial compounds.

Abstract: A method of quantitating the activity of a selected protein kinase on a peptide substrate is provided. The peptide substrate is conjugated to a binding compound. The modified peptide substrate is then added to a solution containing the selected protein kinase. The protein kinase and the peptide are incubated along with a label for sufficient time to form a modified peptide product having the binding compound and the label. The modified peptide product is then bound to a matrix having a high binding compound affinity. The bound peptide is then washed and the activity of the protein kinase is measured. Also provided is a kit for the stated method.

Abstract: There is disclosed novel peptides, fragments or analogues thereof and polynucleotides encoding the same, obtained from streptokinase suitable for use in the amelioration of cell death and methods related thereto.

Abstract: The present invention relates to a whole cell assay for cathepsin K. The present invention is useful for determining cathepsin K activity in a mammalian cell systems and for identifying and evaluating inhibitors of cathepsin K.

Abstract: Methods are provided for locally administering an agent to a host. Specifically, the subject methods provide for the local administration of an agent to an interstitial space of a host. In the subject methods, an agent is retroinfused into a vessel of a host, typically a vein, under conditions sufficient for the agent to enter an interstitial space of the host proximal to the vessel location into which the agent is retroinfused. In practicing the subject methods, the agent is administered to the host in combination with the production of vascular stress at the site of administration, where the vascular tissue stress is sufficient to provide for transport of the agent from the vascular site of deposition into the target interstitial space. In a preferred embodiment, the agent is retroinfused at a pressure sufficient to provide for mechanical stress on the vessel proximal to the target interstitial space. Also provided are kits for use in practicing the subject methods.

Abstract: This invention relates to recombinant-DNA-technology. Specifically this invention relates to new recombinant yeast cells transformed with SEB1 gene. Yeast cells transformed with several copies of SEB1 gene, or overexpressing the Seb1 protein by some other means, have an increased capacity to produce secreted foreign or endogenous proteins. Further, said new recombinant cells, when transformed with genes expressing suitable hydrolytic enzymes can utilize appropriate macromolecular compounds more efficiently, which results in increased cell mass production and/or more versatile utilization of the compounds in relevant biotechnical applications.

Abstract: A method for isolating DNA contained in a biological sample, including: lysing a DNA-containing biological sample and forming a DNA-bound carrier by placing a lysing solution, including the DNA-containing biological sample, a salt, and a cationic surfactant, and having a salt concentration higher than the DNA precipitation inhibition-initiating concentration, into contact with a DNA-binding carrier to bind DNA to the DNA-binding carrier to form the DNA-bound carrier; separating the DNA-bound carrier from other components; dissociating the bound DNA from the DNA-binding carrier; and recovering dissociated DNA. By the method, DNA purified with no preliminary treatment of a biological sample can be recovered at a high yield.

Abstract: A method for identifying a microorganism having a reduced adaptation to a particular environment comprising the steps of: (1) providing a plurality of microorganisms each of which is independently mutated by the insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence so that each mutant contains a different marker sequence, or clones of the said microorganism; (2) providing individually a stored sample of each mutant produced by step (1) and providing individually stored nucleic acid comprising the unique marker sequence from each individual mutant; (3) introducing a plurality of mutants produced by step (1) into the said particular environment and allowing those microorganisms which are able to do so to grow in the said environment; (4) retrieving microorganisms from the said environment or a selected part thereof and isolating the nucleic acid from the retrieved microorganisms; (5) comparing any marker sequences in the nucleic acid isolated in step (4) to the unique m

Abstract: A cell culture medium which is low in dissolved carbon dioxide is disclosed. The medium contains less than about 1 g/L added sodium bicarbonate and includes an organic buffer and a metal complexing agent. The medium is preferably essentially free of added sodium bicarbonate. Methods of use of the medium in culturing mammalian cells, particularly cells engineered to produce recombinant factor VIII, are also disclosed.

Abstract: Exposure of colorectal cancer (CRC) cells to ionizing radiation results in a growth arrest, with cells blocked in both the G1 and G2 phases of the cell cycle. The G1 block has been shown to be due to the p53-mediated induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/SDI1, but the basis for the G2 arrest is unknown. Through a quantitative analysis of gene expression patterns in CRC cell lines, we have discovered that 14-3-3&sgr; is strongly induced by &ggr;-irradiation and other DNA-damaging agents. The induction of 14-3-3&sgr; is mediated by a p53-responsive element located 1.815 kb upstream of its transcription start site. Exogenous introduction of 14-3-3&sgr; into cycling cells results in a G2 block similar to that observed following irradiation. These results document a molecular mechanism for G2/M control that is regulated in human cells by p53.

Abstract: The present invention relates to an automated format for the yeast two hybrid assay for protein-protein interactions.

Abstract: The present invention relates generally to the field of human genetics. Specifically, the present invention relates to human CDC14A gene which has been found to be mutated in certain tumor cell lines. More specifically, the invention relates to a novel sequence for the human CDC14A gene. The present invention further relates to somatic mutations in the CDC14A gene in human cancer and their use in the diagnosis and prognosis of human cancer. The invention also relates to the therapy of human cancers which have a mutation in the CDC14A gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the invention relates to the screening of the CDC14A gene for mutations, which are useful for diagnosing the predisposition to cancer.

Abstract: Nucleic acid molecule are provided comprising a nucleic acid sequence which encodes, in order, an alphavirus capsid, a signal peptide, and an alphavirus E1 or E2 glycoprotein. Also provided are vectors encoding such nucleic acid molecules, and use of such vectors or expression cassettes to generate recombinant alphavirus particles and alphavirus packaging cell lines. In addition, modified alphavirus vector constructs are provided that permit reduced transgene expression during vector packaging, as well as methods of using such vector constructs for the production of alphavirus vector particles.

Abstract: The present invention provides a dual selection cassette (DSC) comprising first and second DNA segments having homology to a eukaryotic viral vector, positive and negative selection genes, each operably linked to their own promoter, and one or more unique restriction enzyme sites (URES) or site-directed homologous recombination sites. The present invention also provides a plasmid, pN/P, comprising an independent positive selection marker gene, an origin of replication, and a dual selection cassette. The dual selection cassette and pN/P plasmid can be used to produce eukaryotic gene transfer vectors without requiring temporally-linked double recombination events or the use of specialized bacterial strains that allow the replication of plasmids comprising defective origins of replication. This method usefully increases the ratio of desired to undesired plasmid and vector constructs. Additionally, this invention provides a method for the creation of eukaryotic viral vector libraries.

Abstract: Pharmaceutical compositions are disclosed which comprise recombinant viruses containing a DNA fragment encoding a polypeptide recognized by a particular antibody H23, which recognizes a particular tumor antigen expressed on breast cancer cells. This antibody specifically binds to an epitope comprising a tandem repeat sequence of 20 amino acids comprised in a transmembrane form as well as a secreted form of the polypeptide specifically bound by antibody H23.

Abstract: The present invention discloses deoxyribonucleic acid enzymes—catalytic or enzymatic DNA molecules—capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

Abstract: The present invention relates to a substantially pure population of viable pancreatic progenitor cells, and methods for isolating such cells. The present invention further concerns certain therapeutic uses for such progenitor cells, and their progeny.

Abstract: The present invention relates to nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, antagonists and agonists vectors and host cells expressing, and immunoadhesions and antibodies to PRO201, PRO308 or PRO309 polypeptides. The invention further relates to compositions and method for the diagnosis and treatment of neoplasfic cell growth and proliferation in mammals, including humans. The invention is based in part on the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumorigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain tumors (e.g. cancer) and may act as predictors of the prognosis of tumor treatment.