Nucleotide sequences for the control of the expression of DNA sequences in a cell host

Abstract

The present invention provides nucleotide sequences from Bacillus bacteria, which control the expression of other DNA sequences in a cell host.

1 . DNA sequence for the control of the expression of a sequence coding for a gene in a host cell, said sequence being characterized in that it comprises a promoter and a nucleotide sequence or downstream region under the control of said promoter, and in particular situated downstream from said promoter and upstream from said coding sequence, said nucleotide sequence or downstream region containing a region essentially complementary to the 3′ end of a bacterial ribosomal RNA, said DNA sequence being capable of intervening in order to increase the expression of said coding sequence, placed downstream in a cell host.  
     
     
         2 . DNA sequence according to  claim 1 , characterized by the following properties: 
 it is included in a DNA sequence about 1692 bp long, defined by the restriction sites HindIII-PstI (H 2 -P 1  fragment) such as obtained by partial digestion of the 6 kb BamHI fragment borne by the cryIIIA gene of  Bacillus thuringiensis  strain LM79;    it is capable of intervening in the control of the expression of a coding nucleotide sequence placed downstream in a bacterial cell host, in particular of the  Bacillus thuringiensis  and/or  Bacillus subtilis  type.    
     
     
         3 . DNA sequence according to  claim 2  characterized in that it corresponds to the HindIII-PstI sequence about 1692 bp long (H 2 -P 1  fragment) such as that obtained by partial digestion of the 6 kb BamHI fragment bearing the cryIIIA gene of  Bacillus thuringiensis  strain LM79.  
     
     
         4 . DNA sequence according to  claim 1 , characterized in that it corresponds to the following nucleotide sequence designated by the expression Seq. No.1 included between nucleotides 1 and 1692 of the sequence shown in FIG. 3.  
     
     
         5 . DNA sequence according to any one of the  claims 2  to  4 , characterized in that it corresponds to the sequence defined by the restriction site TaqI-TaqI.  
     
     
         6 . DNA sequence according to any one of the  claims 1  to  5 , characterized in that it corresponds to the following nucleotide sequence designated by the expression Seq No.2 included between nucleotides 907 and 1559 of the sequence shown in FIG. 3.  
     
     
         7 . DNA sequence according to any one of the  claims 2  to  4 , characterized in that it comprises the TaqI-PacI fragment corresponding to the sequence Seq No.3, in combination with the XmnI-TaqI fragment corresponding to sequence Seq No.4.  
     
     
         8 . DNA sequence according to any one of the  claims 2  to  7 , characterized in that it is a sequence intervening in the control of the transcription of the coding nucleotide sequence or a sequence intervening to increase the expression of a coding sequence.  
     
     
         9 . DNA sequence according to  claims 1  to  18 , characterized by: 
 its capacity to hybridize under non-stringent conditions with a nucleotide sequence selected from the sequences Seq No.1, Seq. No.2 and Seq. No.3 and Seq. No.4 and by 
 its capacity to intervene in the control of the expression in a cell host, in particular of the Bacillus type, of a coding nucleotide sequence placed downstream.  
 
 
     
     
         10 . Sequence according to  claim 1 , characterized in that said nucleotide sequence or downstream region is the sequence S2 isolated from a bacterium, in particular from a Gram +  bacterium, more particularly from a bacterium of the Bacillus type, or a sequence derived from said sequence S2, said S2 sequence being characterized in that it contains a region essentially complementary to the 3′ end of the 16S RNA of a bacterial ribosome, it is capable of intervening at the post-transcriptional level when said coding sequence is expressed, in particular without acting directly on the translation of the coding sequence to be expressed, in particular when said S2 sequence is situated between said promoter and said coding sequence for a gene so as to enhance the expression of said coding sequence in a bacterium, in particular a bacterium of the Bacillus type.  
     
     
         11 . DNA sequence according to  claim 10 , characterized in that said sequence S2 contains a first region sufficiently complementary to the 3′ end of the 16S RNA of a bacterial ribosome, in particular of a Gram +  bacterium, more particularly of a bacterium of the Bacillus type so that said first region is capable of hybridizing to a bacterial ribosome or to a part of said ribosome, and a second region downstream from the first region, said second region containing a sequence capable of having an additional positive effect at the level of the expression of the coding sequence.  
     
     
         12 . DNA sequence according to  claim 10 , characterized in that the nucleotide sequence or the downstream region exhibits the following properties: 
 it is contained in a nucleotide sequence hybridizing under non-stringent conditions with the DNA fragment included between the nucleotides 1413 and 1559 of the sequence shown in FIG. 3;    it is capable of intervening in the control of the expression of the coding sequence of a gene in a host cell, in particular of a sequence coding for Bacillus polypeptide toxic towards insects or a sequence coding for a polypeptide expressed during the vegetative phase, during the stationary phase or during the sporulation of the Bacillus.    
     
     
         13 . DNA sequence according to any one of the  claims 2  to  12 , characterized in that the nucleotide sequence or the downstream region corresponds to the sequence defined by the XmnI-TaqI restriction sites.  
     
     
         14 . DNA sequence according to  claim 13 , characterized in that the nucleotide sequence or downstream region corresponds to the sequence Seq. No.4 included between the nucleotides 1179 and 1559 of the sequence shown in FIG. 3, or in that it corresponds to any part of at least ten nucleotides of this sequence, naturally consecutive or not, capable of intervening in the control of the expression of a coding nucleotide sequence placed downstream in a cell host.  
     
     
         15 . DNA sequence according to  claim 13 , characterized in that the nucleotide sequence or the downstream region corresponds to the sequence Seq No.5 corresponding to the fragment comprising the nucleotides 1179 to 1556 of the sequence shown in FIG. 3.  
     
     
         16 . DNA sequence according to  claim 13 , characterized in that the nucleotide sequence or the downstream region corresponds to the sequence Seq No.11 corresponding to the fragment comprising the nucleotides 1413 to 1556 of the sequence shown in FIG. 3.  
     
     
         17 . DNA sequence according to  claim 13 , characterized in that the nucleotide sequence or the downstream region corresponds to the sequence Seq No.8 corresponding to the fragment comprising the nucleotides 1413 to 1461 of the sequence shown in FIG. 3.  
     
     
         18 . DNA sequence according to  claim 13 , characterized in that the nucleotide sequence or the downstream region corresponds to the sequence Seq No.9 corresponding to the following DNA fragment  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-AGCTTGAAAGGAGGGATGCCTAAAAACGAAGAACTGCA-3′ 
                     
                 
                     
                     
                 
                     
                   3′-ACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         19 . Sequence according to  claim 13 , characterized in that the nucleotide sequence or downstream region corresponds to the sequence Seq No.10 corresponding to the following DNA fragment  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-CTTGAAAGGAGGGATGCCTAAAAACGAAGAAC-3′ 
                     
                 
                     
                     
                 
                     
                   3′-GAACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         20 . Sequence according to  claim 13 , characterized in that the nucleotide sequence or downstream region corresponds to the following DNA fragment:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-GAAAGGAGG-3′ 
                     
                 
                     
                     
                 
                     
                   3′-CTTTCCTCC-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         21 . DNA sequence according to any one of the  claims 1  to  20 , characterized in that the promoter is included in the sequence defined by the TaqI-PacI restriction sites.  
     
     
         22 . DNA sequence according to  claim 21 , characterized in that the promoter corresponds to the sequence Seq. No.3 included between the nucleotides 907 and 990 of the sequence shown in FIG. 3, or a variant comprising the nucleotides 907 to 985, or characterized in that the promoter corresponds to any part of at least ten nucleotides of this sequence, naturally consecutive or not, capable of intervening in the control of the expression of a coding nucleotide sequence placed downstream in a bacterial cell host.  
     
     
         23 . Nucleotide sequence characterized in that it contains a region essentially complementary to the 3′ end of the 16S RNA of a bacterial ribosome, said sequence being capable of intervening at the post-transcriptional level when the sequencing coding for a gene is being expressed, in particular without acting directly on the translation of the coding sequence to be expressed, said nucleotide sequence being under the control of a promoter controlling said coding sequence.  
     
     
         24 . Nucleotide sequence according to  claim 23 , characterized in that said sequence is isolated from a bacterium, in particular from a Gram +  bacterium and more particularly from a bacterium of the Bacillus type.  
     
     
         25 . Nucleotide sequence according to  claim 23 , characterized in that said sequence is an S2 sequence containing a first region sufficiently complementary to the 3′ end of the 16S RNA of a bacterial ribosome, in particular of a Gram +  bacterium, more particularly of a bacterium of the Bacillus type so that said first region is capable of hybridizing with a bacterial ribosome or a part of a bacterial ribosome and a second region downstream from the first region, said second region containing a sequence capable of having an additional positive effect at the level of the expression of the coding sequence.  
     
     
         26 . Nucleotide sequence according to  claim 24 , characterized in that the nucleotide sequence corresponds to the sequence Seq No.5 corresponding to the fragment comprising the nucleotides 1179 to 1556 of the sequence shown in FIG. 3.  
     
     
         27 . Nucleotide sequence according to  claim 24 , characterized in that the nucleotide sequence corresponds to the sequence Seq No.11 corresponding to the fragment comprising the nucleotides 1413 to 1556 of the sequence shown in FIG. 3.  
     
     
         28 . Nucleotide sequence according to  claim 24 , characterized in that the nucleotide sequence corresponds to the sequence Seq No.8 corresponding to the fragment comprising the nucleotides 1413 to 1461 of the sequence shown in FIG. 3.  
     
     
         29 . Nucleotide sequence according to  claim 24 , characterized in that the nucleotide sequence or the downstream region corresponds to the sequence Seq No.9 corresponding to the following DNA fragment:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-AGCTTGAAAGGAGGGATGCCTAAAAACGAAGAACTGCA-3′ 
                     
                 
                     
                     
                 
                     
                   3′-ACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         30 . Nucleotide sequence according to  claim 24 , characterized in that the nucleotide sequence or the downstream region corresponds to the sequence Seq No.10 corresponding to the following DNA fragment:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-CTTGAAAGGAGGGATGCCTAAAAACGAAGAAC-3′ 
                     
                 
                     
                     
                 
                     
                   3′-GAACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         31 . Nucleotide sequence according to  claim 24 , characterized in that the nucleotide sequence corresponds to the following DNA fragment:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   5′-GAAAGGAGG-3′ 
                 
                     
                     
                 
                     
                   3′-CTTTCCTCC-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         32 . Recombinant DNA sequence, characterized in that it comprises a defined coding sequence under the control of a DNA sequence according to any one of the  claims 1  to  22 .  
     
     
         33 . Expression vector characterized in that it is modified at one of its sites by a DNA sequence according to any one of the  claims 1  to  22 , such that said DNA sequence intervenes in the control of the expression of defined nucleotide coding sequence.  
     
     
         34 . Expression vector according to  claim 32 , characterized in that it is a plasmid, for example an integrative plasmid or a replicative plasmid.  
     
     
         35 . Expression vector according to  claim 34 , characterized in that it is the plasmid pHT7902′lacZ deposited with the CNCM on Apr. 20, 1993 under the No. I-1301.  
     
     
         36 . Recombinant DNA sequence according to  claim 32  or expression vector according to  claim 33  or  34 , characterized in that the coding nucleotide sequence which it contains is a toxic sequence towards insects and in particular a sequence with larvicidal activity, for example the sequence coding for the cryIIIA gene of  B. thuringiensis.    
     
     
         37 . Recombinant DNA sequence according to  claim 32  or expression vector according to  claim 33  or  34 , characterized in that the nucleotide coding sequence which it contains is a sequence coding for an enzyme.  
     
     
         38 . Recombinant DNA sequence according to  claim 32  or expression vector according to  claim 33  or  34 , characterized in that the nucleotide coding sequence which it contains is a sequence coding for an antigen.  
     
     
         39 . Recombinant cell host, characterized in that it is modified by a DNA sequence according to any one of the  claims 1  to  32  or by an expression vector according to any one of the claims  33  or  34 .  
     
     
         40 . Cell host according to  claim 39 , characterized in that it is a Bacillus, for example  B. thuringiensis  or  B. subtilis.    
     
     
         41 . Cell host according to  claim 40 , characterized in that it is an asporogenic strain of Bacillus, for sample an asporogenic strain of  B. subtilis  in particular a strain capable of expressing the coding sequence of the DNA sequence according to any one of the  claims 14  to  18  during the stationary phase of growth.  
     
     
         42 . Cell host, characterized in that it is the strain 407-OA::Km R  (pHT305D) deposited with the CNCM on May 3, 1994 under No. I-1412.  
     
     
         43 . Production process for a recombinant protein encoded in a defined nucleotide sequence, said process being characterized in that it comprises: 
 the introduction into a cell host of a vector according to any one of the  claims 33  to  38 ,    the growth of said cell host under conditions permitting the expression of said defined nucleotide sequence, and    the recovery of the recombinant protein.