New England Biolabs, Inc.

Elisabeth A Raleigh

Romualdas Vaisvila

Yu Brian Zheng

Shuang-Yong Xu

Richard D Morgan

Geoffrey G Wilson

Theodore B Davis

Richard J Roberts

Rebecca B Kucera

James C Samuelson

Sriharsa Pradhan

Christopher J Noren

Hang-Gyeong Chin

Jurate Bitinaite

Maurice W Southworth

Brian P Anton

Toby E Claus

Devora Cohen-Karni

Ellen S Hauck

Deborah K Dila

Aleksei Fomenkov

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

Compositions and methods are provided for expression of a toxic protein in a host cell preferably a bacterial host cell where at least one T7 RNA polymerase gene Is contained on the host cell chromosome and one or more genes encoding a T7 RNA polymerase inhibitor is located on an F' plasmid or on the chromosome.

Expression of toxic genes in vivo in a non-natural host

Compositions and methods are provided in which the composition is a protein with at least 50% but less than 100% amino acid sequence identity with McrA or is a variant McrA protein with at least one amino acid sequence modification. The variant or protein has the property of cleaving DNA with methylated cytosine and not hydroxymethylated cytosine in a target DNA sequence, or substantially lacks catalytic activity while maintaining binding activity. Methods are provided in which the protein or McrA variant are used to identify methylation sites either by cleavage or by binding to the methylation site in the presence of a marker or by binding to an immobilized protein or McrA variant.

McrA variants and uses thereof

A method for cloning restriction-modification system is provided whereby the target modification methylase is produced and confers full protection during all growth phases in which the cognate restriction enzyme is present. The method is employed in the cloning of the MseI restriction-modification system.

Method for cloning and producing the Msel restriction endonuclease

The present invention relates to host cells suitable for expressing genes under the direction of foreign RNA polymerases and to providing very low levels of expression of such genes and RNA polymerases in the absence of induction.

Host strain for low uninduced expression of foreign RNA polymerase genes

The present invention discloses a novel method for the direct cloning of nuclease genes such as restriction endonuclease genes in E. coli. In addition, there is provided a novel strain which facilitates application of the method. This method has been successfully employed to clone a number of genes coding for endonuclease including restriction endonuclease genes.

Method for direct cloning of nuclease genes in E. coli

The present invention relates to a recombinant McrBC endonuclease obtainable from Escherichia coli, two components of which, McrB.sub.L and McrC, have been purified in active form. McrBC is active in the presence of GTP and at a low pH. The McrBC endonuclease is also substantially free of a third component, McrB.sub.S, which is believed to inhibit or otherwise interfere with the activity of the enzyme. McrBC has various desirable properties, including the ability to recognize a methylated DNA sequence and also its ability to cleave such a sequence in the presence of GTP. Also provided is a process for the production of recombinant McrBC endonuclease, a process for the determination of the modification state of DNA a process for the determination of an epigenetic alteration or defect (including 'imprinting'), as well as a process for identifying and isolating additional enzymes that cleave modified DNA.

Process for producing recombinant McrBC endonuclease and cleavage of

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Somerville, MA, United States of America

Elisabeth A Raleigh