Abstract: A restriction endonuclease having one DNA binding site is proposed, synthesized from a restriction endonuclease that has one C-terminal domain and one N-terminal domain and two DNA binding sites, by proteolytic cleavage into the two domains or by cloning the gene segment that codes for the domains and expression of the domains and selection of the endonucleolytic domains having one DNA binding site. In addition, a method of synthesis of the restriction endonuclease and its use are claimed.

Abstract: An enzyme is re-engineered to be a zymogen, an enzyme precursor which is converted into an enzyme by protease cleavage. In the example described here, an RNase A enzyme is converted into a zymogen by adding to the enzyme a bridge of amino acids linking the amino and carboxyl termini of the enzyme. The bridge has built in it a protease cleavage site for a specific protease, for example the protease plasmepsin II, produced by the malaria parasite. Since RNase A can be made cytotoxic, this permits a cytotoxic enzyme to be made in the form of a zymogen that becomes active only when it is acted on by a protease only present in a particular target cell such as a pathogen.

Abstract: The invention provides T4 endonuclease V compositions that exhibit enhanced stability, including stability at non-refrigerated temperatures, through reduced activity of cryptic proteases. Methods for detecting cryptic protease activity and methods for reducing such activity are also provided.

Abstract: The present invention provides artificial endonucleases and methods to prepare and use those endonucleases.

Abstract: Methods are provided for converting into a sequence specific strand specific and location specific DNA nicking endonuclease, a restriction endonuclease that recognizes an asymmetric DNA sequence, the endonuclease having two catalytic sites and one or more single sequence specific DNA-binding domains. In one embodiment the method requires inactivating one of the catalytic sites of the restriction endonuclease. In another embodiment, the restriction endonuclease is a dimer having a first and second subunit each comprising a sequence specific DNA binding domain, a catalytic site and a dimerization domain. The nicking endonuclease is formed from combining one subunit having an inactivated catalytic site and a second subunit having an inactivated DNA binding domain. The nicking endonuclease may be converted into a restriction endonuclease by the addition of manganese cations in the digestion buffer.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3? to 5? exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: Compositions and method for making and using a synthetic bovine DNase I are disclosed. More particularly, the sbDNase I of the present invention is a versatile enzyme that cleaves DNA nonspecifically to release 5?-phosphorylated nucleotides. The sbDNase I molecules of the present invention find particular use in a wide range of molecular biology applications, including: degradation of contaminating DNA after RNA isolation; RNA clean-up prior to, or in conjunction with, RT-PCR after in vitro transcription; identification of protein binding sequences on DNA (DNase I footprinting); prevention of clumping when handling cultured cells; tissue dissociation and creation of fragmented DNA for in vitro recombination reactions.

Abstract: Methods and compositions are provided for altering the DNA recognition and cleavage characteristics of an endonuclease without prior knowledge of the endonuclease's three-dimensional structure and/or amino acid residues responsible for activity and/or specificity. Methods include subjecting a mutagenized endonuclease gene library to a genetic selection in prokaryotic cells which tolerate the expression of mutated endonuclease and where the endonuclease is active and determining the altered recognition-site specificity for the endonuclease.

Abstract: This invention provides a novel acid DNase (DLAD) which is an endonuclease capable of cleaving DNA independently from divalent cations, under acidic conditions, which retains its activity in acidic to even neutral pH range, and which is not inhibited by G-actin. This invention also provides a DNA encoding the enzyme, an expression vector containing the DNA, and a host cell transformed with the expression vector. Furthermore, a pharmaceutical composition containing DLAD, DLAD expression vector or a host cell transformed with the expression vector as an active ingredient is provided. The pharmaceutical composition is useful as a therapeutic agent replacing DNase I for cystic fibrosis, and can provide a new approach for the prophylaxis and treatment of infectious diseases.

Abstract: In accordance with the present invention, there is provided a novel restriction endonuclease obtainable from Bacillus thermoglucosidasius 36A (NEB#1384), hereinafter referred to as “BtgZI”, which endonuclease: (1) recognizes the nucleotide sequence 5?-GCGATG-3? in a double-stranded DNA molecule as shown below, 5?-GCGATGNNNNNNNNNN?-3? (SEQ ID NO:9) 3?-CGCTACNNNNNNNNNNNNNN?-5? ?(wherein G represents guanine, C represents cytosine, A represents adenine, T represents thymine and N represents either G, C, A, or T); (2) cleaves said sequence in the phosphodiester bonds between the 10th and the 11th nucleotides 3? to the recognition sequence in the 5?-GCGATG-3 strand of the DNA, and between the 14th and 15th nucleotides 5? to the recognition sequence in the complement stand, 5?-CATCGC-3?, to produce a 4 base 5? extension; and (3) cleaves double-stranded pBR322 DNA to produce 3 fragments of 2892, 1181 and 288 base pairs.

Abstract: This invention provides RNase A superfamily polypeptides with modified amino terminal which can be used to selectively kill target Kaposi's sarcoma cells, neoplastic endothelial cells, and non-neoplastic endothelial cells. In certain embodiments of the invention, the amino terminal modification consists of an addition of 4 amino acid sequence consisting of the SLHV sequence at position ?4 to ?1 to the eosinophil derived neurotoxin protein. The amino terminal addition is capable of directing the claimed RNase A superfamily polypeptides to proliferating endothelial cells, such as Kaposi's sarcoma cells, and selectively killing these cells.

Abstract: The present invention relates to the use of calcium ion and/or sugars to minimize thermal aggregation of DNase and to the use of calcium ion to stabilize liquid solutions of DNase, the solutions having a pH of less than neutral. DNase is the active pharmaceutical principle and the solutions may contain other pharmaceutically acceptable excipients making them suitable for pharmaceutical administration. In the first instance, calcium ion/sugar minimizes the effects of thermal aggregation in the solution. In the second aspect, calcium ion stabilizes the lower pH solutions from protein precipitation.

Abstract: The present invention relates to recombinant DNA encoding the AcuI restriction endonuclease as well as AcuI methylase, and expression of AcuI restriction endonuclease and AcuI methylase in E. coli cells containing the recombinant DNA.

Abstract: The present invention provides a transgenic plant characterized by suppressed flowering. The transgenic plant contains a nucleic acid molecule including a floral organ selective regulatory element operatively linked to a nucleotide sequence encoding a cytotoxic gene product, wherein the nucleic acid molecule is heritable by progeny thereof.

Abstract: A process for purifying a phosphodiesterase 1 (PDE-1) from a cell including heating an extract of a cell formed from a solution including at least one divalent cation, to increase the specific activity of PDE-1 in the extract.

Abstract: The present invention relates to: recombinant DNA encoding the SbfI restriction endonuclease as well as the SbfI methylase, and expression of the SbfI restriction endonuclease and SbfI methylase in E. coli cells containing the recombinant DNA; and methods for cloning the SbfI restriction gene (sbfIR) from Streptomyces species Bf-61 into E. coli by PCR. The method relied on primers based on DNA sequences predicted from amino acid sequences of the purified SbfI restriction endonuclease.

Abstract: The invention features novel RNase P molecules and nucleic acids encoding the same. Methods for discovery of antimicrobial compounds are also featured.

Abstract: The present invention provides the identification and characterization of two components of a recombinant preparation of DNase. These components are the purified deamidated and non-deamidated human DNases. Taught herein are the separation of these components and the use of the non-deamidated species as a pharmaceutical per se, and in particular in compositions wherein the species is disclosed within a plastic vial, for use in administering to patients suffering from pulmonary distress.

Abstract: The present invention relates to recombinant DNA which encodes the Tth111II restriction endonuclease-methylase fusion protein (Tth111IIRM), expression of Tth111II restriction endonuclease-methylase fusion protein in E. coli cells containing the recombinant DNA, and purification of Tth111II endonuclease-methylase fusion protein to near homogeneity.

Abstract: A method is provided for identifying a restriction endonuclease, which includes the steps of (a) screening a target DNA sequence for the presence of known methylase sequence motifs, (b) identifying any open reading frames which lie close to the methylase sequence motifs screened in step (a), and (c) assaying the protein products of these open reading frames for restriction endonuclease activity. Methods for identifying isoschizomers of known restriction endonucleases, which isoschizomers possess a desired physical property, such as thermostability, are also provided by the present invention, as are several novel restriction endonucleases isolated from M. jannaschii, MjaIII and MjaIV. Additionally, a gene was identified that encoded a previously observed endonuclease activity, designated MjaII. Also provided by the present invention are vectors suitable for cloning a DNA sequence encoding a cytotoxic protein, via independent transcription promotors which may be selectively controlled by several conditions.

Abstract: A process for producing a polynucleotide encoding a restriction endonuclease with an altered specificity, which process comprises: (a) mutagenising a polynucleotide encoding a restriction endonuclease with specificity for a recognition sequence so as to produce one or more mutated polynucleotides; and (b) isolating therefrom a polynucleotide encoding a mutated restriction endonuclease with specificity for an altered recognition sequence by selecting a polynucleotide which expresses a restriction endonuclease with methylase specificity for the altered recognition sequence.

Abstract: The present invention relates to a novel endonuclease enzyme which is secreted from immune cell and recognizes bacterial DNA as foreign agent and processes it to produce about 10 bp single-stranded oligonucleotide including CpG motif which is involved in immune response. Also, the present invention relates to a process for producing the endonuclease which comprises culturing human B-lymphoblastic IM9 cell line or TPA-treated myelogenous U937 cell line on an appropriate medium to produce the said endonuclease and isolating the said endonuclease from the cell lysate or the culture medium. In addition, the present invention relates to an immune adjuvant comprising about 10 bp single-stranded oligonucleotide having CpG motif produced by treatment of bacterial DNA by endonuclease.

Abstract: The present invention relates to thermostable mutants of B-type DNA polymerases comprising a Y-GG/A amino acid motif between the N-terminal 3?-5?-exonuclease domain and the C-terminal polymerase domain whereas the tyrosine of the Y-GG/A amino acid motif is mutated and whereas these mutant DNA polymerases are suitable for PCR.

Abstract: This invention provides for new recombinant ribonuclease proteins which are active when expressed by bacteria. This allows the recombinant ribonucleases of this invention to be fused in-frame with ligand binding moieties to form cytotoxic fusion proteins. Furthermore, these proteins are more active than ribonucleases currently available even though the proteins of this invention lack an N-terminal pyroglutamic acid, which has been found to be necessary for ribonucleolytic activity. Because these proteins are recombinant proteins, mutations which increase cytotoxicity can be engineered.

Abstract: The present invention relates to recombinant DNA encoding the BsrGI restriction endonuclease as well as BsrGI methyltransferase, expression of BsrGI restriction endonuclease and BsrGI methyltransferase in E. coli cells containing the recombinant DNA.

Abstract: This invention relates to plants having modified reproductive capacity. In particular, it relates to a plant reproductive tissue specific promoter, the PrAG1 promoter isolated from Pinus radiata, and its use in promoting transcription/expression of associated sequences in plant reproductive tissue, including for the purpose of producing plants which have diminished reproductive capacity or which are sterile.

Abstract: A human RNase H polypeptide and methods of use are provided.

Abstract: The invention is related to a method and a DNA construct for controlling transgene segregation in a sexually reproducing multicellular organism (SRMO), especially in such SRMOs which are susceptible of interbreeding with their cultured or wild-type relatives. The method and construct allows the farmer to reuse the transgenic crop without risk for escape of the transgene into the environment. The DNA construct is a recoverable block of function (RBF) system comprising at least one blocking construct (BC), and at least one means for recovering the blocked functions. The BC has a capacity of blocking at least one molecular or physiological function essential for the development and/or reproductive cycle of the SRMO. The BC is closely linked to the transgene of intereset (TGI). The blocked function is recoverable by an external treatment or intervention, which is optionally combined with one or more recovering constructs (RC).

Abstract: The present invention provides a process for economically producing N-acetylneuraminic acid without using expensive materials such as pyruvic acid and phosphoenolpyruvic acid. The process comprises: allowing (i) a culture of a microorganism having N-acetylneuraminic acid aldolase activity or N-acetylneuraminic acid synthetase activity, or a treated matter of the culture, (ii) a culture of a microorganism capable of producing pyruvic acid or a treated matter of the culture, or a culture of a microorganism capable of producing phosphoenolpyruvic acid or a treated matter of the culture, (iii) N-acetylmannosamine, and (iv) an energy source which is necessary for the formation of pyruvic acid or phosphoenolpyruvic acid to be present in an aqueous medium to form and accumulate N-acetylneuraminic acid in the aqueous medium; and recovering N-acetylneuraminic acid from the aqueous medium.

Abstract: Genes involved in double-stranded RNA interference (RNAi pathway genes) are identified and used to investigate the RNAi pathway. The genes and their products are also useful for modulating RNAi pathway activity.

Abstract: The present invention provides compositions and methods for recombinational cloning. The compositions include vectors having multiple recombination sites and/or multiple topoisomerase recognition sites. The methods permit the simultaneous cloning of two or more different nucleic acid molecules. In some embodiments the molecules are fused together while in other embodiments the molecules are inserted into distinct sites in a vector. The invention also generally provides for linking or joining through recombination a number of molecules and/or compounds (e.g., chemical compounds, drugs, proteins or peptides, lipids, nucleic acids, carbohydrates, etc.) which may be the same or different.

Abstract: Methods and compositions for subcellular fractionation of biological samples before proteomic analysis are given. Of particular interest is the separation of DNA binding nuclear proteins.

Abstract: It is intended to provide novel mutant RNA polymerases enabling a transcriptional sequencing method whereby a high SN ratio can be achieved in sequence analysis with the use of capillary and longer and more accurate base sequencial data can be obtained by a single reaction. More specifically, a mutant RNA polymerase derived from a wild type RNA polymerase by substitution of at least one amino acid and deletion of at least one amino acid, or a mutant RNA polymerase derived from a wild type RNA polymerase by deletion of at least one amino acid, wherein the above-described substitution and/or deletion of amino acid(s) have been performed so that the resultant mutant RNA polymerase has an enhanced ability to incorporate 3′-deoxynucleotide as a substrate compared with the wild type RNA polymerase corresponding thereto.

Abstract: The present invention provides compounds and methods for the detection of protein-protein interactions wherein said interactions are dependent on the presence or absence of post-translational modifications (PTMs) of at least one of the proteins.

Abstract: The RecQ4 helicase gene, belonging to the RecQ helicase gene family, is revealed herein to be the causative gene of Rothmund-Thomson syndrome. The present inventors found out that it is possible to diagnose Rothmund-Thomson syndrome by detecting mutation of this gene. Further, they uncovered that it is possible to treat patients of Rothmund-Thomson syndrome by utilizing normal RecQ4 helicase gene or proteins thereof.

Abstract: A heat-resistant ligase isolated from Aeropyrum pernix, and homologues thereof, the activity of which is not substantially decreased by heat treatment at 100° C., is disclosed.

Abstract: This invention provides DNA polymerases with mutations in the charge-switch nucleotide interaction region that increase activity for charge-switch nucleotides. Such polymerases can be generated by introducing mutations in specific residues which are identified as being in the appropriate region through structural models, by homology to polymerases with known structures, or experimental analysis. In some embodiments, the mutant DNA polymerases have additional mutations that decrease activity for non-charge-switch nucleotides and mutations that decrease exonuclease activity. In another aspect, the invention provides methods of sequencing a target nucleic acid with the above described mutated DNA polymerases.

Abstract: A method is provided for the production of a bovine pancreatic protein with a specific desoxyribonuclease activity of at least 6,000 units per mg of protein. Methylotrophic yeast is used as a heterologous host organism. The bovine pancreatic protein is secreted into the growth medium from which it is purified.

Abstract: A DNA sequence encoding a human adenylyl cyclase is described. The amino acid sequence of the adenylyl cyclase is also described.

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: This invention provides fragments of HCV NS3 helicase, and crystalline compositions thereof, based on subdomains of HCV helicase protein. The protein fragments are stable, soluble, and structurally sound. They can be expressed at high levels in conventional expressions systems, such as E. coli, to permit efficient, large-scale production for NMR-based screening applications and production of [2H,13C,15N]- and [13C,15N]-labeled polypeptides for structural NMR studies. Helicase fragments of the present invention are useful in the most advanced NMR techniques available, e.g., NMR-based drug discovery techniques such as SAR-by-NMR, in biological assays to discover inhibitors of HCV NS3 helicase, and to evaluate the mechanism of action and substrates for HCV NS3 helicase. Crystals of the present invention are useful for structure-based drug design studies using x-ray crystallographic techniques.

Abstract: The invention provides isolated Rad50 nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering Rad50 levels in plants. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions.

Abstract: Provided are a protein complex having the F0F1-ATPase activity; a DNA encoding the protein complex; a method for producing the protein complex, using the DNA; and a method for producing nucleoside 5′-triphosphate using the protein. The present invention further provides a recombinant DNA with the DNA inserted therein; a transformant carrying the recombinant DNA; and a method for producing a protein complex, using the transformant.

Abstract: A polypeptide factor derived from the thermophilic eubacterial species Thermus thermophilus has universal protein expression-assisting activity. The polypeptide factor has been named the CzrB protein active in full length or truncated form has the potential to act as a universal protein expression-assisting molecule which can increase the yields of all heterologous proteins produced in E. coli by a mechanism that is independent of the protein being expressed.

Abstract: This invention provides for murine telomerase reverse transcriptase (mTERT) enzyme proteins and nucleic acids, including methods for isolating and expressing these nucleic acids and proteins, which have application to the control of cell proliferation and aging, including the control of age-related diseases, such as cancer.

Abstract: According to the invention, the desoxyribonuclease with increased thermolability is a variant, by way of amino acid substitution, of bovine pancreatic desoxyribonuclease I. The variant protein retains desoxyribonuclease activity. Moreover, the specific desoxyribonuclease activity of the variant of bovine pancreatic desoxyribonuclease I is approximately zero units per mg of protein following heating of the variant of bovine pancreatic desoxyribonuclease I for about 5 min at a temperature below 95° C. In addition, the variant of bovine pancreatic desoxyribonuclease I has no measurable ribonuclease activity.

Abstract: The invention provides TBX5 protein fragments that regulate normal and malignant cell growth and nucleic acids encoding these fragments. Such protein fragments include a translated 5′ T-box sequence capable of binding to the major groove of target DNA and lack a translated 3′ T-box sequence that binds to target DNA's minor groove. In particular, the invention protein fragments that inhibit cell proliferation, this activity requiring T-box interaction with target DNA's major groove, but not target DNA's minor groove.

Abstract: The present invention describes methods of using terminal-phosphate-labeled nucleotides in the presence of a manganese salt to enhance their substrate properties towards various enzymes. Particularly described are methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases, in the presence of a manganese salt. Further provided are manganese complexes of terminal-phosphate-labeled nucleotides as well as terminal-phosphate-labeled nucleotides with new linkers with enhanced substrate properties.

Abstract: Compositions and methods are provided for producing adenine nucleotide translocator (ANT) polypeptides and fusion proteins, including the production and use of recombinant expression constructs having a regulated promoter. ANT ligands and compositions and methods for identifying ANT ligands, agents that bind ANT and agents that interact with ANT are also disclosed.

Abstract: Tn5 transposase (Tnp) mutants that have higher transposase activities than the wild-type Tnp are disclosed. The Tn5 Tnp mutants differ from the wild-type Tnp at amino acid positions 54, 242, and 372 and have greater avidity than the wild-type Tnp for at least one of a wild-type Tn5 outside end sequence as defined by SEQ ID NO:3 and a modified Tn5 outside end sequence as defined by SEQ ID NO:5. Also disclosed are various systems and methods of using the Tnp mutants for in vitro or in vivo transposition.