Chimaeric gene coding for a transit peptide and a heterologous peptide

• US 5,717,084 A
• Filed: 06/06/1995
• Issued: 02/10/1998
• Est. Priority Date: 12/28/1984
• Status: Expired due to Term

• Current Assignee: Bayer Cropscience NV (Bayer AG)
• Original Assignee: Bayer AG, Plant Genetic Systems NV (Bayer AG)
• Inventors: Van Den Broeck, Guido, Bohnert, Hans J., Schreier, Peter, Kausch, Albert P., Schell, Jeff, Van Montagu, Marc, Timko, Michael P., Herrera-Estrella, Luis, Cash more, Anthony R.
• Primary Examiner(s): Fox, David T.
• Application Number:
  US08/470,719
• Time in Patent Office: 980 Days
• Field of Search: 435/69.7, 435/69.8, 435/70.1, 435/172.3, 435/240.4, 536/23.4, 536/24.1, 536/23.6, 536/23.7
• US Class Current: 536/23.4
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8221 Transit peptides

Abstract

A chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence:

1) a transit peptide of a cytoplasmatic precursor of a chloroplast protein or polypeptide of a plant species, and,

2) a protein or polypeptide of interest which is different from a mature chloroplast protein or polypeptide contained in a natural cytoplasmatic precursor thereof, and,

b) a promoter upstream of the nucleic acid sequence recognized by polymerases endogenous to a plant, whereby the chimaeric DNA sequence can be expressed in cells of the plant under the control of the promoter and the protein or polypeptide of interest can be translocated in the chloroplasts of the cells of the plant.

Claims

1. A chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a protein or polypeptide of interest which is different from a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to a plant, wherein the chimaeric DNA sequence can be expressed in cells of said plant under the control of said promoter and said protein or polypeptide of interest can be translocated into the chloroplasts of said cells of said plant.

2. The chimaeric DNA sequence of claim 1 in which said protein or polypeptide of interest is of bacterial origin.

3. The chimeric DNA sequence of claim 1 in which said protein or polypeptide of interest is of plant origin.

4. The chimaeric DNA sequence of claim 1 in which said protein or polypeptide of interest confers resistance to a herbicide.

5. The chimaeric DNA sequence of claim 1 in which the first amino acid of said protein or polypeptide of interest is a methionine.

6. The chimaeric DNA sequence of claim 1 in which no more than the first seven amino acids of said protein or polypeptide of interest are encoded by a synthetic nucleotide linker.

7. The chimaeric DNA sequence of claim 1 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is the small subunit of ribulose-1,5-bisphosphate carboxylase or chlorophyll a/b binding proteins.

8. The chimaeric DNA sequence of claim 7 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of soybean, pea, duckweed or wheat.

9. The chimaeric DNA sequence of claim 7 in which the transit peptide has the sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C or M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

10. The chimaeric DNA sequence of claim 1 in which said promoter is normally associated with the DNA encoding said transit peptide.

11. The chimaeric DNA sequence of claim 10 in which said promoter is of a plastocyanin gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-bisphosphate carboxylase gene, or a chlorophyll a/b binding protein gene.

12. The chimaeric DNA sequence of claim 11 in which said promoter is foreign to the DNA encoding said transit peptide.

13. The chimaeric DNA sequence of claim 12 in which said promoter is a promoter of the nopaline synthase gene.

14. A chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a fusion protein comprising in sequence;

2.1) the N-terminal part of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and

2.2) a protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to a plant,

wherein the chimaeric DNA sequence can be expressed in cells of said plant under control of said promoter and said fusion protein can be translocated into the chloroplasts of said cells of said plant.

15. The chimaeric DNA sequence of claim 14 in which said protein or polypeptide of interest is of bacterial origin.

16. The chimaeric DNA sequence of claim 14 in which said protein or polypeptide of interest is of plant origin.

17. The chimaeric DNA sequence of claim 14 in which said protein or polypeptide of interest confers resistance to an herbicide.

18. The chimaeric DNA sequence of claim 14 in which said N-terminal part is encoded by a DNA sequence that comprises an intron.

19. The chimaeric DNA sequence of claim 14 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is the small subunit of ribulose-1,5-bisphosphate carboxylase or chlorophyll a/b binding proteins.

20. The chimaeric DNA sequence of claim 19 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of soybean, pea, duckweed or wheat.

21. The chimaeric DNA sequence of claim 20 in which the transit peptide has the sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C or M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

22. The chimaeric DNA sequence of claim 14 in which said promoter is normally associated with the DNA encoding said transit peptide.

23. The chimaeric DNA sequence of claim 22 in which said promoter is of a plastocyanin gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-bisphosphate carboxylase gene, or a chlorophyll a/b binding protein gene.

24. The chimaeric DNA sequence of claim 14 in which said promoter is foreign to the DNA encoding said transit peptide.

25. The chimaeric DNA sequence of claim 24 in which said promoter is a promoter of the nopaline synthase gene.

26. A chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a fusion protein comprising in sequence;

2.1) a N-terminal part comprising no more than the first 22 N-terminal amino acids of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and

2.2) a protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to a plant,

whereby the chimaeric DNA sequence can be expressed in cells of said plant under control of said promoter and said fusion protein can be translocated into the chloroplasts of said cells of said plant.

27. The chimaeric DNA sequence of claim 26 in which said protein or polypeptide of interest is of bacterial origin.

28. The chimaeric DNA sequence of claim 26 in which said protein or polypeptide of interest is of plant origin.

29. The chimaeric DNA sequence of claim 26 in which said protein or polypeptide of interest confers resistance to an herbicide.

30. The chimaeric DNA sequence of claim 26 in which said N-terminal part is encoded by a DNA sequence that comprises an intron.

31. The chimaeric DNA sequence of claim 26 in which said N-terminal part is from the small subunit of the ribulose-1,5-bisphosphate carboxylase of Pisium sativum.

32. The chimaeric DNA sequence of claim 26 in which said N-terminal part comprises no more than the first five N-terminal amino acids of said mature chloroplast protein or polypeptide.

33. The chimaeric DNA sequence of claim 32 in which said N-terminal part comprises the sequence M-Q-V-W-P.

34. The chimaeric DNA sequence of claim 26 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is the small subunit of ribulose-1,5-bisphosphate carboxylase or chlorophyll a/b binding proteins.

35. The chimaeric DNA sequence of claim 34 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of soybean, pea, duckweed or wheat.

36. The chimaeric DNA sequence of claim 35 in which the transit peptide has the sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C, or M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

37. The chimaeric DNA sequence of claim 26 in which said promoter is normally associated with the DNA encoding said transit peptide.

38. The chimaeric DNA sequence of claim 37 in which said promoter is a promoter of a plastocyanin gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-bisphosphate carboxylase gene, or a chlorophyll a/b binding protein gene.

39. The chimaeric DNA sequence of claim 26 in which said promoter is foreign to the DNA encoding said transit peptide.

40. The chimaeric DNA sequence of claim 39 in which said promoter is a promoter of the nopaline synthase gene.

41. A chimaeric DNA sequence comprising a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence:

1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a protein or polypeptide of interest which is different from a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof.

42. The chimaeric DNA sequence of claim 41 in which said protein or polypeptide of interest is of bacterial origin.

43. The chimaeric DNA sequence or claim 41 in which said protein or polypeptide of interest is of plant origin.

44. The chimaeric DNA sequence of claim 41 in which said protein or polypeptide of interest confers resistance to a herbicide.

45. The chimaeric DNA sequence of claim 41 in which the first amino add of said protein or polypeptide of interest is a methionine.

46. The chimaeric DNA sequence of claim 41 in which no more than the first seven amino adds of said protein or polypeptide of interest are encoded by a synthetic nucleotide linker.

47. The chimaeric DNA sequence of claim 41 in which the transit peptide is from a cytoplasmatic precursor of a chloroplast protein which is the small subunit of ribulose-1,5-bisphosphate carboxylase or chlorophyll a/b binding proteins.

48. The chimaeric DNA sequence of claim 47 in which the transit peptide is from a cytoplasmatic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of soybean, pea, duckweed or wheat.

49. The chimaeric DNA sequence of claim 47 in which the transit peptide has the sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S -R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C or M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N -P-S-S-Q-E-I-G-A-A-R-P-T.