Control of gene expression in eukaryotic cells

• US 20030224483A1
• Filed: 02/06/2003
• Published: 12/04/2003
• Est. Priority Date: 07/01/1999
• Status: Active Grant

• Current Assignee: Monsanto Company (Bayer AG)
• Original Assignee: Calgene LLC (Bayer AG)
• Inventors: Adams, Thomas H., Oulmassov, Tim N., McBride, Kevin E., Miller, Paula C., Anderson, John C., Crossland, Lyle D., Gavrias, Victoria, Qurollo, Barbara A.
• Granted Patent: US 6,943,243 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: C07D 307/33 in position 2, the oxygen atom being in its keto or unsubstituted enol form
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/8214 Plastid transformation
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8217 Gene switch
• CPC Class Codes: C12N 15/8238 chemically inducible, e.g. tetracycline
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/001 controllable enhancer/promoter combination
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/30 being an enhancer not forming part of the promoter region
• CPC Class Codes: C12N 2830/36 being a transcription termination element
• CPC Class Codes: C12N 2840/105 enhancing translation
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

DNA constructs and other compositions and methods for controlling gene expression in eukaryotic cells and organisms are derived from bacterial quorum sensing systems. One or more cis elements from the luxI promoter (“lux box”) or a functionally similar sequence are incorporated in a eukaryotic promoter. A receptor protein from the LuxR family of transcriptional regulators, upon binding an acylated homoserine lactone (AHL) compound, interacts with the lux box, modulating the activity of the promoter.

Claims

1. A non-naturally occurring polynucleotide selected from the group consisting of:

a promoter that is functional in a eukaryotic cell comprising an AHL-response element;

a promoter that is functional in a eukaryotic cell operably linked to a sequence encoding an AHL synthase; and

a promoter that is functional in a eukaryotic cell operably linked to a sequence encoding a polypeptide that binds specifically to an AHL-response element.

2. The polynucleotide of claim 1 comprising a promoter that is functional in a eukaryotic cell comprising an AHL-response element.

3. The polynucleotide of claim 1 comprising a promoter that is functional in a eukaryotic cell operably linked to a sequence encoding an AHL synthase.

4. The polynucleotide of claim 3 comprising a promoter that is functional in a eukaryotic cell comprising an AHL-response element operably linked to the sequence encoding an AHL synthase.

5. The polynucleotide of claim 1 comprising a promoter that is functional in a eukaryotic cell operably linked to a sequence encoding a DNA-binding polypeptide that binds specifically to an AHL-response element.

6. The polynucleotide of claim 5 wherein the DNA-binding polypeptide comprises an AHL-binding motif and an AHL response element-binding domain.

7. The polynucleotide of claim 6 wherein the DNA-binding polypeptide further comprises a member of the group consisting of a eukaryotic transciptional activation domain and a eukaryotic transcriptional repression domain.

8. The polynucleotide of claim 6 wherein binding of an AHL by the AHL-binding motif causes the DNA-binding polypeptide to bind specifically to an AHL-response element.

9. The polynucleotide of claim 6 wherein the DNA-binding polypeptide binds to an AHL-response element unless an AHL is bound by the AHL-binding motif.

10. The polynucleotide of claim 1 comprising a promoter that is functional in a eukaryotic cell operably linked to a sequence encoding an AHL receptor.

11. The polynucleotide of claim 1 comprising:

a first sequence comprising a first promoter that is functional in a eukaryotic cell comprising an AHL-response element, and

a second sequence comprising a second promoter that is functional in the eukaryotic cell operably linked to a sequence encoding an AHL receptor, wherein the AHL receptor binds to the AHL-response element and modulates transcription of the first promoter.

12. The polynucleotide of claim 1 comprising:

a first sequence comprising a first promoter that is functional in a eukaryotic cell comprising an AHL-response element,

a second sequence comprising a second promoter that is functional in a eukaryotic cell comprising an AHL-response element operably linked to a sequence encoding an AHL synthase that, when expressed in the eukaryotic cell, synthesizes an AHL, and

a third sequence comprising a third promoter that is functional in the eukaryotic cell operably linked to a sequence encoding an AHL receptor, wherein binding of the AHL by the AHL receptor causes the AHL receptor to bind to the AHL-response element and modulate transcription of the first promoter.

13. An organism comprising the polynucleotide of claim 1 selected from the group consisting of a yeast, a mammal, and a plant.

14. A eukaryotic cell comprising

a first sequence comprising a first promoter that is functional in the eukaryotic cell comprising an AHL-response element operably linked to a gene of interest;

and

a second sequence comprising a second promoter that is functional in the eukaryotic cell operably linked to a sequence encoding an AHL receptor, wherein binding of an AHL by the AHL receptor causes the AHL receptor to bind to the AHL-response element and modulate transcription of the first promoter.

15. The eukaryotic cell of claim 14 wherein at least one of the first or second promoters is a non-constitutive promoter.

16. The eukaryotic cell of claim 14 wherein the gene of interest is a selectable or screenable marker.

17. A eukaryotic cell comprising

a nucleus;

an organelle;

a first sequence comprising a promoter that is functional in the organelle comprising an AHL-response element operably linked to a gene of interest; and

a second sequence comprising a second promoter that is functional in the nucleus operably linked to a sequence that encodes a polypeptide comprising (1) an organelle-transport peptide and (ii) an AHL receptor, wherein the AHL receptor binds to the AHL-response element and modulates transcription of the first promoter upon binding of an AHL.

18. The eukaryotic cell of claim 17 wherein the organelle is a plastid and the organelle-transport peptide is a plastid-transport peptide.

19. The eukaryotic cell of claim 18 wherein the plastid is a chloroplast and the plastid-transport peptide is a chloroplast-transport peptide.

20. A eukaryotic cell comprising an organelle, the organelle comprising:

a first sequence comprising a promoter that is functional in the organelle comprising an AHL-response element operably linked to a gene of interest; and

a second sequence comprising a promoter that is functional in the organelle operably linked to a sequence that encodes a polypeptide comprising an AHL receptor, wherein the AHL receptor binds to the AHL-response element and modulates transcription of the first promoter upon binding of an AHL.

21. The eukaryotic cell of claim 20 wherein the organelle is a plastid.

22. The eukaryotic cell of claim 21 wherein the plastid is a chloroplast.

23. A composition comprising (a) an amount of an AHL or AHL analog that is effective, when applied to a cell comprising an AHL receptor and a promoter comprising an AHL-response element, to cause the AHL receptor to bind the AHL-response element and modulate transcription of the promoter, and (b) a carrier that is substantially nontoxic to the eukaryotic cell.

24. The composition of claim 23 comprising (a) an amount of an AHL or AHL analog that is effective, when applied to a plant comprising the cell, to cause the AHL receptor to bind the AHL-response element and modulate transcription of the promoter, and (b) an agronomically carrier that is substantially non-phytotoxic.

25. The composition of claim 24 further comprising an agronomically acceptable additive or adjuvants.

26. The composition according to claim 23, wherein said AHL analog is selected from the group consisting of N-(Beta-ketooctanoyl)-2-methyl-homoserine lactone, N-(Beta-ketooctanoyl)-3-methyl-homoserine lactone, N-(Beta-ketooctanoyl)-4-methylhomoserine lactone, N-(Beta-ketooctanoyl)-4-ethyl-homoserine lactone, N-(Beta-ketooctanoyl)-4-butyl-homoserine lactone, N-(Beta-ketooctanoyl)-4-phenyl-homoserine lactone, N-(alpha-methyl-Beta-ketooctanoyl)-homoserine, N-(alpha, alpha-dimethyl-Beta-ketooctanoyl)-homoserine, N-(alpha-ethyl-Beta-ketooctanoyl)-homoserine, N-(alpha-butyl-Beta-ketooctanoyl)-homoserine, and N-(alpha-hexyl-Beta-ketobutanoyl)-homoserine.

27. A method of modulating the expression of a polynucleotide that comprises a first promoter that is functional in a eukaryotic cell comprising an AHL-response element, the method comprising

expressing in the eukaryotic cell an AHL receptor, and

applying to the cell a composition comprising an AHL that is bound by the AHL receptor, causing the AHL receptor to modulate transcription of the polynucleotide.

28. The method of claim 27 wherein the step of applying to a cell comprises applying the composition to an organism comprising the cell.

29. The method of claim 28 wherein the organism is a mammal and the step of applying to a cell comprises administering the composition to the mammal.

30. The method of claim 28 wherein the cell is a plant cell and the step of applying to a cell comprises applying the composition to a seed or a plant comprising the cell or to soil.

31. A method of identifying an AHL that is bound by an AHL receptor, the method comprising the steps of:

(a) providing a eukaryotic cell comprising (i) a first promoter that is functional in the eukaryotic cell comprising an AHL-response element operably linked to a sequence encoding a selectable or screenable marker, and (ii) a second promoter that is functional in the eukaryotic cell operably linked to a sequence encoding an AHL receptor, wherein binding of an AHL by the AHL receptor causes expression of the marker;

(b) applying to the cell a composition comprising the AHL, and

(c) detecting the expression of the marker.