Thermus promoters for gene expression

Abstract

The present invention relates to a system for identifying, isolating and utilizing promoter elements useful for expression of nucleotide sequences and the proteins encoded thereby in a thermophile. In one embodiment, a recombinant DNA molecule is provided, and comprises a reporter sequence, a putative thermophile promoter, a selectable marker sequence, and a 3′ and a 5′ DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile. Further, within the recombinant DNA, the reporter sequence is under the transcriptional control of a promoter which functions in a thermophile to form a promoter/reporter cassette, the promote/reporter cassette is flanked by said 3′ and said 5′ DNA targeting sequences, and the promoter/reporter cassette is positioned in the opposite orientation of the DNA targeting sequences.

We claim:  
     
         1 . An isolated, recombinant DNA molecule for identification of a regulatory region of a thermophile genome comprising: 
 a) a reporter sequence;    b) a putative thermophile promoter operably linked to said reporter sequence to form a promoter/reporter cassette;    c) a drug resistance marker; and,    d) a 3′ and a 5′ DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile;    wherein said promote/reporter cassette is flanked by said 3′ and said 5′ DNA targeting sequences; and, said promoter/reporter cassette is positioned in the opposite orientation of the DNA targeting sequences.    
     
     
         2 . A recombinant DNA of  claim 1  wherein said reporter sequence is tbg.  
     
     
         3 . A recombinant DNA of  claim 1  wherein said reporter sequence is lacZ.  
     
     
         4 . A recombinant DNA of  claim 1  wherein said drug resistantce marker confers upon a thermophile resistance to kanamycin.  
     
     
         5 . A recombinant DNA of  claim 1  wherein said putative promoter sequence is a fragment of the genome of a thermophile.  
     
     
         6 . A recombinant DNA of  claim 5  wherein said fragment was isolated following limited digestion of the genome with a restriction enzyme.  
     
     
         7 . A recombinant DNA of  claim 1  wherein said thermophile putative promoter sequence was isolated from a bacteria of the genus Thermus.  
     
     
         8 . A recombinant DNA of  claim 1  wherein said thermophile putative promoter sequence was isolated from the thermophile  Thermus flavus.    
     
     
         9 . A recombinant DNA of  claim 1  wherein said thermophile putative promoter sequence was isolated from the thermophile  Thermus thermophilus.    
     
     
         10 . A method of identifying a thermophile promoter comprising transforming a thermophile with a recombinant DNA molecule of  claim 1  and detecting expression of the reporter sequence.  
     
     
         11 . A method of  claim 10  wherein said thermophile is from the genus Thermus.  
     
     
         12 . A method of  claim 10  wherein said thermophile is  Thermus flavus.    
     
     
         13 . A method of  claim 10  wherein said thermophile is  Thermus thermophilus.    
     
     
         14 . An isolated DNA molecule comprising a promoter sequence identified by the method of  claim 10 .  
     
     
         15 . An isolated DNA molecule comprising a thermophile promoter sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.  
     
     
         16 . An isolated DNA molecule of  claim 15  wherein said thermophile promoter sequence is operably linked to a reporter sequence.  
     
     
         17 . An isolated DNA molecule of  claim 15  wherein said thermophile promoter sequence is operably linked to a nucleic acid sequence encoding a protein.  
     
     
         18 . An isolated DNA molecule comprising the thermophile terminator sequence of SEQ ID NO: 13.  
     
     
         19 . A method for expressing a protein in Thermus comprising transforming into Thermus a DNA molecule of  claim 17  wherein said DNA molecule is stably integrated into the Thermus genome.  
     
     
         20 . A host cell having a DNA molecule of  claim 17  stably integrated into its genome such that expression of said protein occurs within said host cell.  
     
     
         21 . An isolated, recombinant DNA molecule comprising: 
 a) a coding sequence comprising nucleic acid encoding a protein;    b) a putative thermophile promoter selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44 operatively linked to said coding sequence to form a promoter/coding sequence cassette;    c) a drug resistance marker; and,    d) a 3′ and a 5′ DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile;    wherein said promote/coding sequence cassette is flanked by said 3′ and said 5′ DNA targeting sequences; and, said promoter/coding sequence cassette is positioned in the opposite orientation of the DNA targeting sequences.