The patent badge is an abbreviated version of the USPTO patent document. The patent badge does contain a link to the full patent document.
The patent badge is an abbreviated version of the USPTO patent document. The patent badge covers the following: Patent number, Date patent was issued, Date patent was filed, Title of the patent, Applicant, Inventor, Assignee, Attorney firm, Primary examiner, Assistant examiner, CPCs, and Abstract. The patent badge does contain a link to the full patent document (in Adobe Acrobat format, aka pdf). To download or print any patent click here.
Patent No.:
Date of Patent:
Feb. 02, 1999
Filed:
Oct. 17, 1997
Shuang-yong Xu, Lexington, MA (US);
Jian-ping Xiao, Wenham, MA (US);
New England Biolabs, Inc., Beverly, MA (US);
Abstract
The methylase selection method was used to clone the BslI methylase gene (bslIM) from Bacillus species. A partially active BslI methylase lacking the 17 amino acid residues at the N-terminus was cloned in E. coli using expression vector pRRS. Inverse PCR was used to clone the missing portion of the BslI methylase. After cloning the complete BslI methylase gene and its upstream DNA sequences, a RadC homolog was found upstream of the BslI methylase. Because methylase gene and restriction endonuclease gene are located in proximity to each other in a particular restriction-modification system, efforts were made to clone the downstream DNA by inverse PCR. After two round of inverse PCR reactions two open reading frames (ORF) were found downstream of the BslI methylase gene. Expression of the first ORF (ORF1) in a T7 expression vector did not yield any active BslI endonuclease. Expression of the second ORF (ORF2) in E. coli and assay of the crude cell extract indicated that this gene product has DNA nicking activity. The gene product of ORF2 alone does not constitute BslI endonuclease activity. Expression of ORF1 and ORF2 in the same E. coli cell produces BslI endonuclease activity. BslI endonuclease activity can be reconstituted in vitro by mixing gene product of ORF1 and ORF2 together.