Control of gene expression in eukaryotes

• US 6,660,524 B1
• Filed: 12/22/1999
• Issued: 12/09/2003
• Est. Priority Date: 12/24/1998
• Status: Expired due to Fees

• Current Assignee: British American Tobacco Limited (British American Tobacco PLC)
• Original Assignee: Advanced Technologies Group Limited (British American Tobacco PLC)
• Inventors: Türck, Jutta Anna, Archer, John Anthony Charles
• Primary Examiner(s): MCKELVEY, TERRY ALAN
• Application Number:
  US09/469,211
• Time in Patent Office: 1,448 Days
• Field of Search: 435/419, 435/69.1, 435/243, 435/468, 435/325, 435/320.1, 435/375, 536/23.1, 536/24.1, 800/278, 800/295
• US Class Current: 435/419
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/8217 Gene switch
• CPC Class Codes: C12N 15/8238 chemically inducible, e.g. tetracycline

Abstract

A chemically inducible gene expression system is described. A chimeric gene having a first sequence comprising a promoter and a regulator polypeptide is linked with a second sequence comprising a promoter and a coding or non-coding sequence. Expression of the target gene of the second sequence is controlled by the regulator polypeptide which is acted upon by an inducer. The inducer is a chemical compound, such as OHP, which acts upon the OHP responsive regulator polypeptide, which can be obtained from Rhodococcus sp. V49. Various domain regions and complementary response elements are also described.

Claims

1. A system for modulating expression of a target nucleic acid sequence comprising:

(a) a first nucleic acid sequence operably linked to and regulates the expression of a second nucleic acid sequence, said second nucleic acid sequence encoding a regulator polypeptide; and

(b) a third nucleic acid sequence operably linked to and regulates the expression of a target nucleic acid sequence, whereby said regulator polypeptide binds to said third nucleic acid sequence and modulates the expression of said target nucleic acid sequence, and wherein said regulator polypeptide comprises (i) the amino acid sequence of SEQ ID NO;

2;

or (ii) an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complement of the nucleotide sequence from 295 to 1035 of SEQ ID NO;

1 under hybridization conditions comprising incubating at 37°

C. in 1×

SSC and 50% formamide, and/or, wherein said third nucleic acid sequence (i) comprises the nucleotide sequence from 1225 to 1260 of SEQ ID NO;

1;

or (ii) hybridizes to the complement of the nucleotide sequence from 1225 to 1260 of SEQ ID NO;

1 under hybridizing conditions comprising incubating at 37°

C. in 1×

SSC and 50% formamide.

2. The system according to claim 1, wherein said first and/or third nucleic acid comprises a promoter which regulates expression in eukaryotic cells and/or tissues.

3. The system according to claim 1, wherein said first nucleic acid comprises a promoter selected from the group consisting of constitutive, developmentally regulated, tissue-specific, cell-specific, and cell compartment-specific promoter.

4. The system according to claim 3, wherein said constitutive promoter is cauliflower mosaic virus 35S promoter or cauliflower mosaic virus 19S promoter.

5. The system according to claim 3, wherein said tissue-specific promoter is a patatin promoter or a petE promoter.

6. The system according to claim 3, wherein said cell compartment-specific promoter is a chloroplast gene promoter or a mitochondrial gene promoter.

7. The system according to claim 6, wherein said chloroplast gene promoter is from a gene encoding a large subunit of ribulose biphosphate carboxylase.

8. The system according to claim 6, wherein said mitochondrial gene promoter is from a 18S-5S rRNA gene.

9. The system according to claims 1, 2, 3, 4, 5, 6, 7, or 8, wherein said first and/or third nucleic acid sequence comprises one or more enhancer sequences.

10. The system according to claim 9, wherein said enhancer sequence is an intron, or said enhancer sequence is a transcriptional enhancer sequence and/or a translation enhancer sequence.

11. The system according to claim 10, wherein said enhancer sequence is a non-translated leader sequence.

12. The system according to claim 11, wherein said non-translated leader sequence is a viral non-translated leader sequence.

13. The system according to claim 12, wherein said viral non-translated leader sequence is from a virus selected from the group consisting of Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), Alfalfa Mosaic Virus (AMV), Picornavirus, Potyvirus, and AMV RNA4.

14. The system according to claim 11, wherein said enhancer sequence is a Heat Shock Protein 70 leader sequence.

15. The system according to claim 10, wherein said enhancer sequence is a transcriptional enhancer sequence.

16. The system according to claim 15, wherein said enhancer sequence is a petE enhancer sequence.

17. The system according to claim 10, wherein said enhancer sequence is an intron of the maize Adh1 gene or the Heat Shock Protein 70 intron from maize.

18. The system according claim 1, wherein said regulator polypeptide comprises one or more domains selected from the group consisting of a ligand binding domain, a nucleic acid binding domain, a transactivation domain, a silencing/repressing domain, a dimerization domain, and a targeting domain.

19. The system according to claim 18, wherein said ligand binding domain binds non-covalently to a ligand.

20. The system according to claim 19, wherein said ligand is an inducer, or a precursor of an inducer.

21. The system according to claim 18, wherein said nucleic acid binding domain comprises a sequence of amino acids which binds non-covalently to a response element.

22. The system according to claim 21, wherein said third nucleic acid sequence comprises said response element.

23. The system according to claim 21, wherein said response element is a combination of two or more response elements and responsive to one or more nucleic acid binding proteins.

24. The system according to claim 22, wherein said response element responds to a protein selected from the group consisting of LexA, Ga14, LacI, Tet, C1, and Ace1.

25. The system according to claim 18, wherein said transactivation domain is selected from the group consisting of herpes simplex virus Vp16 domain, maize C1 domain, rice Oshox1 silencing domain, rice Oshox1 repressing domain, and Kruppel Associated Box domain.

26. The system according to claim 18, wherein said targeting domain is selected from the group consisting of a plasma membrane targeting sequence, a golgi targeting sequence, an endoplasmatic reticulum targeting sequence, a nuclear targeting signal, a chloroplast targeting sequence, a mitochondrial targeting sequence, and an inner envelope targeting sequence.

27. The system according to any one of claims 18-26, wherein said second nucleic acid sequence which encodes said domain is modified for expression in eukaryotes.

28. The system according to claim 18, wherein said regulator polypeptide comprises a fusion protein.

29. The system according to claim 18, wherein said regulator polypeptide comprises a ligand binding domain and/or a DNA binding domain.

30. The system according to claim 1, wherein said third nucleic acid sequence comprises a promoter, and a response element that binds said regulator polypeptide.

32. The system according to claim 1, wherein the expression of said target nucleic acid sequence is increased by the binding of an inducer to said regulator polypeptide.

33. The system according to claim 32, wherein the inducer is 3-(2-hydroxyphenyl)propionic acid orthohydroxyphenylpropionic acid.

34. The system according to claim 32, wherein said regulator polypeptide is OhpR protein.

35. The system according to claim 32, wherein said third nucleotide sequence comprises a cauliflower mosaic virus 35S promoter.

31. A plasmid deposited under NCIMB Accession No. 40997.