Multi-species polynucleotide control sequences

Abstract

The present invention relates to polynucleotide sequences which enable a polynucleotide control sequence, such as a promoter, to direct expression in a wide range of industrially relevant species, both prokaryotes and eukaryotes. When the polynucleotide sequences of the invention are applied in combination with selection marker genes it is possible to perform selectable cloning in a laboratory host and use the same construct in the final host

1 . A polynucleotide sequence of the general formula
   T-T-G-A-C-W-N( o )-Y1-A-Y2-A-A-T-H1-H2-N( p )-S1-S2-W-K1-K2-N( q )-H3-M1-M2-H4-A-T-G,  (formula I)
   wherein   N is any of the nucleotides A, C, G and T, and N(o) is 16, 17 or 18 nucleotides long;   N(p) is 21, 22 or 23 nucleotides long; N(q) is 2, 3, 4, 5 or 6 nucleotides long; and each individual N in N(o), N(p), and N(q) are the same or different;   W is any of the nucleotides A and T;   Y is any of the nucleotides C and T, and Y1 and Y2 are the same or different;   H is any of the nucleotides A, C, and T and H1, H2, H3 and H4 are the same or different;   S is any of the nucleotides G and C, and S1 and S2 are the same or different;   K is any of the nucleotides G and T, and K1 and K2 are the same or different;   M is any of the nucleotides A and C, and M1 and M2 are the same or different;   The AG-content of N(o) is below 50%   The A-content of N(o) is below 35%   The AG-content within each stretch of 6 nucleotides in N(o) is below 66%   The AG-content of N(p) is below 60%   The AG-content within each stretch of 6 nucleotides in N(p) is below 83%   N(o) and N(p) do not contain two consecutive G's   The G-content of N(q) is below 50%,   which sequence when present in a polynucleotide control sequence, allows the polynucleotide control sequence to direct expression of polypeptides   in a wide range of industrially relevant prokaryotes and eukaryotes.   
     
     
         2 . A polynucleotide sequence according to  claim 1 , wherein N(o) is 17 nucleotides long; N(p) is 23 nucleotides long; and N(q) is 3 or 4 nucleotides long. 
     
     
         3 . A polynucleotide sequence according to  claim 1 , selected from the group consisting of SEQ ID NO. 46 to 51. 
     
     
         4 . A polynucleotide construct comprising a polynucleotide sequence according to  claim 1 . 
     
     
         5 . A polynucleotide control sequence which directs the expression of polypeptides in at least three of the following groups: mammals, planta, algae, fungi, yeasts, gram-positive bacteria, gram-negative bacteria or archaebacteria. 
     
     
         6 . A polynucleotide control sequence which directs the expression of polypeptides in at least four of the following genera:  Escherichia, Streptomyces, Bacillus, Gluconobacter, Pseudomonas, Clostridium, Saccharomyces, Kluyveromyces, Pichia, Penicillium, Aspergillus, Mortierella, Chrysosporium, Acremonium, Trichoderma, Cricetulus, Homo.    
     
     
         7 . A polynucleotide control sequence which comprises a polynucleotide sequence according to  claim 1 . 
     
     
         8 . An expression cassette comprising a polynucleotide sequence according to  claim 1 , or a polynucleotide control sequence. 
     
     
         9 . A vector comprising a polynucleotide sequence according to  claim 1 . 
     
     
         10 . A host cell comprising a polynucleotide sequence according to  claim 1 . 
     
     
         11 . Use of a polynucleotide sequence according to  claim 1 , in restriction enzyme digestion, in recombination reactions, in molecular biology kits and reagents, in enzyme screening, in biocatalysis reactions, in biochemical reactions, or in fermentation processes. 
     
     
         12 . Use of a host cell according to  claim 10  for producing a polypeptide, a primary or secondary metabolite, an antibody or a pharmaceutical. 
     
     
         13 . Method for producing a polypeptide, a primary or secondary metabolite, an antibody or a pharmaceutical by cloning a gene of interest in a cloning host and producing the polypeptide, primary or secondary metabolite, antibody or pharmaceutical in a production host, which method comprises using the same polynucleotide control sequence in both cloning host and production host. 
     
     
         14 . Method according to  claim 13 , wherein the polynucleotide control sequence is a control sequence. 
     
     
         15 . Method according to  claim 13 , wherein in the production host is of a different species than the cloning host. 
     
     
         16 . Method according to  claim 13 , wherein in the gene of interest encodes the polypeptide, part of an antibody, one or more enzymes involved in the production of the primary or secondary metabolite or the pharmaceutical. 
     
     
         17 . A method for cloning a polynucleotide sequence according to  claim 1 , comprising:
 (a) cloning a polynucleotide sequence in front of a selectable marker gene on a vector according to the invention;   (b) transfecting a first species with the vector from step (a);   (c) obtaining clones after selection for active transcription of the selectable marker gene   (d) isolating DNA from these clones   (e) transfecting another species with the isolated DNA, whereby steps (c) to (e) are repeated minimally 2 times.