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 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.

What is claimed is:  
     
         1 . A method for identifying a restriction endonuclease comprising the steps of: 
 (a) screening a target DNA sequence for the presence of known DNA 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 the open reading frames of step (b) for restriction endonuclease activity.    
     
     
         2 . The method of  claim 1 , wherein the target DNA sequence is selected from the group consisting of bacterial DNA sequences, archaeal DNA sequences and viral DNA sequences.  
     
     
         3 . The method of  claim 1 , wherein the screening of step(a) comprises searching DNA sequence databases.  
     
     
         4 . The method of  claim 1 , wherein the protein products of step(c) are produced by in vitro transcription and translation.  
     
     
         5 . The method of  claim 4 , wherein the restriction endonuclease is a thermophilic restriction endonuclease and the translation mix is selected from the group consisting of a Wheat Germ translation mix, a bacterial S30, or a rabbit reticulocyte system.  
     
     
         6 . The method of  claim 1 , wherein the protein products of step(c) are produced by recombinant DNA techniques.  
     
     
         7 . The method of  claim 1 , wherein step (c) further comprises the steps of: 
 (d) growing the original microorganism;    (e) preparing cell extracts; and    (f) testing the extracts of step(e) for restriction endonuclease activity.    
     
     
         8 . The method of  claim 1 , wherein the methylase sequence motifs of step(a) are selected from the group consisting of cytosine-5 methylase motifs, N4C-methylase motifs, and N6A-methylase motifs.  
     
     
         9 . A substantially pure restriction endonuclease MjaIV obtainable from  M. jannaschii , said endonuclease recognizing the following base sequence in double-stranded deoxyribonucleic acid molecules:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   5′- GTN↓NAC -3′ 
                 
                     
                     
                 
                     
                   3′- CAN↑NTG -5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
       and having a cleavage position defined by the arrows.  
     
     
         10 . Isolated DNA coding for the MjaIV restriction endonuclease, wherein the isolated DNA is obtainable from  Methanococcus jannaschii.    
     
     
         11 . A recombinant vector comprising a vector into which DNA coding for MjaIV restriction endonuclease has been inserted.  
     
     
         12 . The recombinant vector of  claim 11  wherein the DNA comprises residues 1748-2485 of GenBank Entry U67573.  
     
     
         13 . A host cell transformed with the recombinant vector of  claim 11  or  12 .  
     
     
         14 . A method of producing MjaIV restriction endonuclease comprising culturing a host cell transformed with the vector of  claim 11  or  12  under conditions suitable for expression of said endonuclease.  
     
     
         15 . Isolated DNA coding for the MjaII restriction endonuclease, wherein the isolated DNA is obtainable from  M. jannaschii.    
     
     
         16 . A recombinant vector comprising a vector into which DNA coding for MjaII restriction endonuclease has been inserted.  
     
     
         17 . The recombinant vector of  claim 16  wherein the DNA comprises residues 11380-12492 of GenBank Entry U67585.  
     
     
         18 . A host cell transformed with the recombinant vector of  claim 16  or  17 .  
     
     
         19 . A method of producing MjaII restriction endonuclease comprising culturing a host cell transformed with the vector of  claim 16  or  17  under conditions suitable for expression of said endonuclease.  
     
     
         20 . A substantially pure MjaIII restriction endonuclease obtainable from  M. jannaschii , which recognizes the following base sequence in double-stranded deoxyribonucleic acid molecules:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   5′- GATC -3′ 
                 
                     
                     
                 
                     
                   3′- CTAG -5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
       and wherein said endonuclease is an isoschizomer of Mbol.  
     
     
         21 . Isolated DNA coding for the MjaIII restriction endonuclease, wherein the isolated DNA is obtainable from  M. jannaschii.    
     
     
         22 . A recombinant vector comprising a vector into which DNA coding for MjaIII restriction endonuclease has been inserted.  
     
     
         23 . The recombinant vector of  claim 22 , wherein the DNA comprises residues 5632-6504 of GenBank Entry U67508.  
     
     
         24 . A host cell transformed with the recombinant vector of  claim 22  or  23 .  
     
     
         25 . A method of producing the MjaIII restriction endonuclease comprising culturing a host cell transformed with the vector of  claim 22  or  23  under conditions suitable for expression of said endonuclease.  
     
     
         26 . A method of identifying an isoschizomer of a known restriction endonuclease, said isoschizomer possessing a desired physical property, said method comprising the steps of: 
 (a) identifying any open reading frames in genomic DNA encoding said known restriction endonuclease;    (b) comparing said open reading frames of step(a) against known open reading frames in at least one organism possessing said desired physical property to identify potential sequence matches; and    (c) assaying the protein products of said candidate isoschizomer sequences of step(c) for restriction endonuclease activity under conditions selective for said desired physical property.    
     
     
         27 . The method of  claim 26 , wherein said desired physical property is selected from the group consisting of thermostability, halostability, acidostability, and cryostability.  
     
     
         28 . The method of  claim 26 , wherein said screening of step (b) comprises searching DNA sequence databases.  
     
     
         29 . The method of  claim 26 , wherein said protein products of step (c) are produced by in vitro transcription and translation.  
     
     
         30 . The method of  claim 29 , wherein said isoschizomer is thermostable and said translation mix is selected from the group consisting of a Wheat Germ translation mix, a rabbit reticulocyte system, or a bacterial S30.  
     
     
         31 . The method of  claim 26 , wherein said protein products of step (c) are produced by recombinant DNA techniques.  
     
     
         32 . A vector suitable for cloning a DNA sequence encoding a cytotoxic protein wherein, said vector comprises at least a first and a second transcription promotor and is adapted to accept said DNA sequence insert and wherein said first and said second transcription promoters are independently controllable.  
     
     
         33 . The vector of  claim 33 , wherein said first transcription promotor enables anti-sense strand transcription and said second transcription promotor enables sense strand transcription.  
     
     
         34 . The vector of  claim 33 , wherein said first transcription promotor comprises i phage promotor and said second transcription promotor comprises T7 RNA polymerase promotor.  
     
     
         35 . The vector of  claim 34 , wherein said vector comprises pLT7K.  
     
     
         36 . The vector of  claim 34 , wherein said independent control of said first and second transcription promotors comprises control by a member of the group consisting of temperature, IPTG addition, and inhibition of at least one RNA polymerase required for transcription of said vector.  
     
     
         37 . The vector of  claim 36  wherein said inhibition comprises inhibition by a member of the group consisting of bacteriophage T7 lysozyme expression, and utilization of a T7 RNA polymerase negative  E. coli  strain.  
     
     
         38 . An  E. coli  host cell transformed by the vector of any one of claims  32 ,  33 ,  34 ,  35 ,  36  and  37 .  
     
     
         39 . A method for producing a recombinant cytotoxic protein, said method comprising the steps of: 
 (1) inserting a DNA sequence encoding said cytotoxic protein into the vector of any one of claims  32 ,  33 ,  34 ,  35 ,  36 , and  37 ;    (2) transforming a host cell with the vector of step (1) under conditions which disallow the expression of said sense strand;    (3) culturing said transformed host cell of step (2) under conditions which disallow the expression of said sense strand;    (4) inducing the selective expression of said sense strand; and    (5) recovering said recombinant cytotoxic protein.    
     
     
         40 . The method of  claim 39 , wherein said induction of step(4) comprises induction by a member of the group consisting of temperature, IPTG addition, and inhibition of at-least one RNA polymerase required for transcription of said vector.  
     
     
         41 . A stable recombinant vector encoding R.NlaIII, said vector comprising pLT7-nlaIIIR, wherein said vector does not encode M.NlaIII.  
     
     
         42 . A host cell transformed by the vector of  claim 41 .  
     
     
         43 . Isolated DNA coding for the PacI restriction endonuclease, wherein the isolated DNA is obtainable from ATCC Accession No. 55044.  
     
     
         44 . A recombinant DNA vector comprising a vector into which a DNA segment coding for PacI endonuclease produced by  Pseudomonas alcaligenes  has been inserted.  
     
     
         45 . A cloning vector which comprises a vector into which the isolated DNA of  claim 43  has been inserted.  
     
     
         46 . A host cell transformed by the vector of  claim 44 .  
     
     
         47 . A method of producing PacI restriction endonuclease comprising culturing a host cell transformed with the vector of  claim 44  under conditions suitable for expression of said endonuclease.  
     
     
         48 . A substantially pure MjaV restriction endonuclease obtainable from  M. jannaschii , which recognizes the following base sequence in double-stranded deoxyribonucleic acid molecules:  
       
         
           
                 
               
                     
                 
                   5′-GTAC-3′ 
                 
                     
                 
                   3′-CATG-5′ 
                 
                     
                 
             
                
                
                
                
                
               
            
           
         
       
       and wherein said endonuclease is an isoschizomer of RsaI.  
     
     
         49 . Isolated DNA coding for the MjaV restriction endonuclease, wherein the isolated DNA is obtainable from  M. jannaschii.    
     
     
         50 . A recombinant vector comprising a vector into which DNA coding for MjaV restriction endonuclease has been inserted.  
     
     
         51 . The recombinant vector of claim  50 , wherein the DNA comprises residues 767-74 of GenBank Entry U67591.  
     
     
         52 . A host cell transformed with the recombinant vector of claim  50  or  51 .  
     
     
         53 . A method of producing the MjaV restriction endonuclease comprising culturing a host cell transformed with the vector of claim  50  or  51  under conditions suitable for expression of said endonuclease.