Method for screening restriction endonucleases

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 promoters 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 Pacl and NlaIII.

1 . Isolated DNA coding for the Pacl restriction endonuclease, wherein the isolated DNA is obtainable from ATCC Accession No. 55044.  
     
     
         2 . A recombinant DNA vector comprising a vector into which a DNA segment coding for Pacl endonuclease produced by  Pseudomonas alcaligenes  has been inserted.  
     
     
         3 . A cloning vector which comprises a vector into which the isolated DNA of  claim 1  has been inserted.  
     
     
         4 . A host cell transformed by the vector of  claim 2 .  
     
     
         5 . A method of producing Pacl restriction endonuclease comprising culturing a host cell transformed with the vector of  claim 2  under conditions suitable for expression of said endonuclease.