New England Biolabs, Inc.

Christopher J Noren

Richard J Roberts

Richard D Morgan

Devon R Byrd

Jay Patti

Yu Brian Zheng

Geoffrey G Wilson

Yinhua Zhang

Romualdas Vaisvila

Ming-Qun Xu

Francine B Perler

Sriharsa Pradhan

Donald G Comb

Elisabeth A Raleigh

William E Jack

George Tzertzinis

Larry A McReynolds

George R Feehery

Jurate Bitinaite

Hang-Gyeong Chin

Eric E Adam

Robert A Hodges

Maurice W Southworth

Karen E Sandman

Devora Cohen-Karni

Paul D Evans

Shaorong S Chong

Corinna Tuckey

Methods and compositions are described for detecting hydroxymethylated nucleotides (hmNs) in a polynucleotide preparation with a view to mapping the location of hmNs in a genome, quantifying the occurrence of hmNs at selected loci and correlating the occurrence of hmNs with gene expression and phenotypic traits. Embodiments describe the use of modifying enzymes together with site-specific endonucleases to detect the hmNs.

Detection and quantification of hydroxymethylated nucleotides in a polynucleotide preparation

A method for obtaining a mixture of heterogenous short double-stranded RNA molecules suitable for use in gene silencing (hsiRNA) by subjecting large double-stranded RNA to enzymatic cleavage under specified conditions. The resulting mixture consistently includes enhanced representation of fragments having a size of 21-22 nucleotides absent any fractionation step. The fragments contain sequences that collectively span the entire length of the large double-stranded RNA from which they are derived. Double-stranded RNA with sequences that individually represent segments of a target mRNA may be analyzed using the methods described herein to identify the most active subset of hsiRNA fragments or individual siRNA fragments for achieving gene silencing for any gene or transcribed sequences. A method is additionally provided for preparing and cloning DNA encoding selected siRNA, hsiRNA mixtures or hairpin sequences to provide a continuous supply of a gene silencing reagent derived from any long double-stranded RNA.

Methods and compositions relating to gene silencing

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.

Surface display of selenocysteine-containing peptides

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, 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. A method for producing these cytotoxic proteins using such vectors is also provided, as are stable clones of PacI and NlaIII.

Method for screening restriction endonucleases

A method is provided for identifying a restriction endonuclease that includes: screening a target DNA sequence for the presence of known methylase sequence motifs, identifying any open reading frames which lie close to the screened methylase sequence motif and assaying the protein products of the open reading frames for restriction endonuclease activity.

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 , 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. A method for producing these cytotoxic proteins using such vectors is also provided, as are stable clones of PacI and NlaIII.

The present invention is directed to modified proteins and methods of their production. The modified proteins comprise a controllable intervening protein sequence (CIVPS) inserted into or adjacent a target protein, the CIVPS being capable of excision from or cleavage of the modified protein under predetermined conditions in cis or in trans, i.e., increase in temperature, exposure to light, unblocking of amino acid residues by dephosphorylation, treatment with chemical reagents or deglycosylation. If desired, the modified protein can be subjected to these conditions. The CIVPS may also be inserted into a region that substantially inactivates target protein activity. The CIVPS may be used in a number of applications including purification of the target protein in a one-step protocol.

Modified proteins comprising controllable intervening protein sequences

Multipurpose cloning in vitro phagemid vectors are disclosed, which vectors may be used to generate high specific-activity RNA probes. The multipurpose vectors also possess a bidirectional in vitro transcription system. Methods for constructing this system are also disclosed.

Bidirectional in vitro transcription vectors utilizing a single RNA

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Rockport, MA, United States of America

Christopher J Noren