Abstract: Compositions and methods are provided for preparing an hsiRNA mixture and for silencing of gene expression in vivo. The composition relates to a mutant RnaseIII. The methods are directed to reacting a preparation of dsRNA with an effective amount of a mutant RNAse III to produce the hsiRNA mixture.

Abstract: Methods and compositions are provided for selecting drug targets in silico, that include the following steps performed in the order presented or an alternative order of partially or entirely at the same time: (a) identifying one or more essential or functionally important sequences from a model organism using pre-existing genomic and phenotypic data; (b) comparing sequences from (a) with a DNA or peptide sequence from a pathogen to store homologous sequences; (c) comparing sequences from the pathogen with the DNA or peptide sequence from a host organism to store those sequences absent in the host organism; and (d) comparing sequences from (b) and (c) to identify shared sequences, the shared sequences being a drug target. Additionally, identified drug targets are provided.

Abstract: Methods and kits are provided for detecting chitin in biological samples using chitin-binding domain (CBD).

Abstract: Compositions and methods are provided for creating and identifying mutant carbohydrate-binding proteins that reversibly bind to carbohydrate substrates under conditions where the native protein remains bound. Examples of modified chitin-binding domains are provided which can be eluted from chitin in the presence of a reducing agent or at a pH within the range of 5-10.

Abstract: In accordance with the present invention, there is provided a novel type II restriction endonuclease, obtainable from Corynebacterium striatum M82B, hereinafter referred to as “CstMI”, which endonuclease: (1) recognizes the nucleotide sequence 5?-AAGGAG-3? in a double-stranded DNA molecule as shown below, 5?-AAGGAGN20?-3? 3?-TTCCTCN18?-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 at the phosphodiester bonds between the 20th and the 21th nucleotides 3? to the recognition sequence in the 5?-AAGGAG-3 strand of the DNA, and between the 18th and 19th nucleotides 5? to the recognition sequence in the complement stand, 5?-CTCCTT-3?, to produce a 2 base 3?extension; and (3) possesses a second enzymatic activity that recognizes the same DNA sequence, 5?-AAGGAG-3?, but modifies this sequence by the addition of a methyl group to prevent cleavage by the CstMI endon

Abstract: Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with T7 Endo I. The modified enzyme includes two catalytic centers separated by a ?-bridge where the ?-bridge contains at least one mutation having an effect of altering enzyme cleavage activity compared to the unmodified enzyme. Activities associated with the modified DNA cleaving enzyme that can be modulated in different reaction conditions include at least one of: (a) non-sequence specific nicking activity; (b) cleaving the second strand of a duplex DNA at a preexisting nick site to produce a linear duplex with a single strand overhang; (c) non-sequence specific DNA cleavage; (d) cleaving DNA flanking a mismatch; and (e) cleavage at a cruciform structure in a DNA duplex.

Abstract: In accordance with the present invention, there are provided selection systems and methods for screening for agents that control splicing of inteins in their native host protein (extein) or in homologous exteins. Specifically, there are provided positive genetic selection systems for the screening of agents which inhibit or activate protein splicing which comprise: a host cell containing a chromosomal gene encoding either a drug-resistant form of a target enzyme or a wild-type target enzyme, and a plasmid-borne gene encoding either a drug-sensitive form of the target enzyme, which is dominantly cytotoxic upon interaction with the drug, or a dominantly cytotoxic form of the target enzyme. In these systems the plasmid-borne gene contains an intein, and the inhibition or activation of splicing of the dominant cytotoxic form of the target enzyme by a given reagent results in the survival or death of the host cell. More specifically, positive genetic selection systems which utilize the M. xenopi GyrA intein or M.

Abstract: Methods and compositions are provided that achieve depletion of a nucleotide pool by means of a phosphate-transferring enzyme such as a nucleoside phosphate or a polyphosphate glucokinase. Depletion of a nucleotide pool using a nucleoside kinase may additionally utilize a phosphotransferase in a second phosphate-transferring reaction.

Abstract: The naturally-occurring amino acid selenocysteine (SEC) is incorporated uniquely and specifically in the context of a polypeptide displayed on the surface of an amplifiable genetic particle (phage, cell or spore) in response to incorporation signals engineered in the encoding DNA. In addition to conferring the unique activities of the selenol group to the chemistry of the displayed peptide, Sec also provides a unique handle for specific chemical modification of the display peptide. In addition to increasing the palette of available residues in a random peptide library to 21 possibilities, present invention also provides a means of tethering virtually any desired chemical functionality to the incorporated Sec.

Abstract: In accordance with the present invention, there is provided a DNA (deoxyribonucleic acid) fragment which encodes the MmeI type II restriction endonuclease enzyme. This one polypeptide possesses two related enzymatic functions; namely an endonuclease activity which recognizes the DNA sequence 5?-TCC(Pu)AC-3? and cleaves as indicated by the arrows: 5?-TCCRAC(N20)?-3? 3?-AGGYTG(N18)?-5? and a second enzymatic activity that recognizes the same DNA sequence, 5?-TCC(Pu)AC-3?, but modifies this sequence by the addition of a methyl group to prevent cleavage by the MmeI endonuclease activity.

Abstract: Compositions and methods are provided for reversibly binding chitin-binding domain (CBD) to a chitin or equivalent substrate under non-denaturing conditions. CBD from either prokaryotes or eukaryotes are modified for example, by random mutation, and screened to identify mutants that achieve this change in properties. Creating a modified CBD with an altered binding affinity for substrate provides new uses for CBD not previously possible with unmodified CBD that binds irreversibly to chitin.

Abstract: Substantially pure glycosidases capable for cleaving selected glycosidic bonds have been described including glycosidases isolated from Xanthomonas and recombinant glycosidases. Substrate specificity of isolated enzymes have been identified for GlcNac?1-x, Gal?1-3R, Gal?1-6R, Gal?1-3R, Fuc?-2R, Fuc?1-3R, Fuc?1-4R, Man?1-2R, Man?1-3R, Man?1-6R, Man?1-4R, Xyl?1-2R, Glc?1-4R, and Gal?1-4R providing improved capability for selectively cleaving a glycosidic linkage in a carbohydrate substrate and for forming modified carbohydrates.

Abstract: Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/or yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI. The reaction mixture may further contain an AP endonuclease and a polymerase. If used, these enzymes may be selected according to their ability to withstand high temperatures. For example, the reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture in as much as the repair is effected rapidly and prolonged incubation is not generally adverse.

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: Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a single strand helicase preparation or a thermostable helicase in the absence of a single strand binding protein and a DNA polymerase such that the amplification can be performed isothermally.

Abstract: Compositions and methods are provided for reversibly binding chitin binding domain (CBD) to a chitin or equivalent substrate under non denaturing conditions. CBD is modified preferably by a mutation to achieve this change in properties. In one embodiment, an aromatic amino acid residue located in the binding cleft of the CBD was altered resulting in reversible binding affinity for substrate in select conditions. Creating a modified CBD with an altered binding affinity for substrate provides new uses for CBD not previously possible with unmodified CBD which binds irreversibly to chitin.

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: Present embodiments of the invention describe computational methods for performing a systematic, genome-wide search for novel drug targets in pathogenic organisms for example, the human filarial parasites. Cofactor independent phosphoglycerate mutase (iPGM) was identified by this search as a candidate target for identifying therapeutic agents for use in treating animal or plant subjects infected with parasitic nematodes, microbial pathogens including microsporidia, fungi etc. A consensus amino acid or nucleotide sequence that characterizes iPGM is further provided.

Abstract: Methods and compositions are provided for repairing a polynucleotide so that it can be synthesized efficiently with improved fidelity and yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI. The reaction mixture may further contain an AP endonuclease and a polymerase. These enzymes are optionally selected according to their ability to withstand high temperatures so they can be included in an amplification mixture. The reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture.

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.