US2001047091A1PendingUtilityA1

Cryptic regulatory elements obtained from plants

Priority: Sep 9, 1998Filed: Dec 22, 2000Published: Nov 29, 2001

Est. expirySep 9, 2018(expired)· nominal 20-yr term from priority

Inventors:Brian Miki Therese Ouellet Jiro Hattori Elizabeth Foster Helene Labbe Teresa Martin-Heller Lining Tian Daniel Brown Peijun Zhang Keqiang Wu

C07K 14/415C12N 15/8234C12N 15/8216C12N 15/8222

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References

Claims

Abstract

T-DNA tagging with a promoterless β-glucuronidase (GUS) gene generated transgenic Nicotiana tabacum plant that expressed GUS activity either only in developing seed coats, or constitutively. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. Analysis of the region demonstrated that the seed coat-specificity of GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. This promoter is useful in controlling the expression of genes to the developing seed coat in plant seeds. Similarly, cloning and characterization of the cryptic constitutive promoter revealed the occurrence of several cryptic regulatory regions. These regions include promoter, negative regulatory elements, transcriptional enhancers, core promoter regions, and translational enhancers and other regulatory elements.

Claims

exact text as granted — not AI-modified

We claim:

1 . An isolated nucleic acid comprising a cryptic regulatory element obtained from a plant.

2 . The cryptic regulatory element of claim 1 , said cryptic regulatory element comprising a promoter.

3 . The cryptic regulatory element of claim 1 , said cryptic regulatory element comprising a core promoter.

4 . The cryptic regulatory element of claim 1 , said cryptic regulatory element comprising an enhancer.

5 . The cryptic regulatory element of claim 1 , said cryptic regulatory element comprising a negative regulatory element.

6 . The cryptic regulatory element of claim 1 , said cryptic regulatory element comprising a post-transcriptional regulatory element.

7 . The cryptic regulatory element of claim 4 , said enhancer comprising a transcriptional enhancer.

8 . The cryptic regulatory element of claim 1 , wherein said cryptic regulatory element is selected from the group consisting of a tissue-specific regulatory element and a constitutive regulatory element.

9 . The cryptic regulatory element of claim 8 , wherein said cryptic regulatory element is a seed-specific regulatory element.

10 . The cryptic regulatory element of claim 9 , wherein said cryptic regulatory element is a seed-coat specific regulatory element.

11 . The cryptic regulatory element of claim 8 , wherein said cryptic regulatory element is a constitutive regulatory element.

12 . The cryptic regulatory element of claim 1 , comprising a DNA fragment of about 2.5 kb and characterized by the restriction map of FIG. 2(B).

13 . The cryptic regulatory element of claim 10 comprising a DNA fragment that is substantially homologous to the nucleotide sequence of SEQ ID NO:1.

14 . The cryptic regulatory element of claim 10 , comprising a nucleotide sequence consisting of at least 19 contiguous nucleotides of nucleotides 1 to 993 of SEQ ID NO:1.

15 . The cryptic regulatory element of claim 10 , comprising a nucleotide sequence consisting of at least 19 contiguous nucleotides of nucleotides 1 to 467 of SEQ ID NO:1.

16 . A vector comprising the cryptic regulatory element as defined in claim 1 , operatively associated with a gene that encodes a protein, wherein the gene is under the control of said cryptic regulatory element.

17 . A plant cell which has been transformed with a vector as claimed in claim 16 .

18 . A transgenic plant containing a cryptic regulatory element as claimed in claim 1 , operatively linked to a gene encoding a protein.

19 . The cryptic regulatory element of claim 12 , comprising an XbaI—SmaI fragment of about 2 kb.

20 . The cryptic regulatory element of claim 12 , comprising an XbaI and SnaBI fragment of about 500 bp.

21 . The cryptic regulatory element of claim 12 , comprising an XbaI and SnaBI fragment of about 1.5 kb.

22 . The cryptic regulatory element of claim 12 , comprising an HindIII and SnaBI fragment of about 1.9 kb.

23 . The cryptic regulatory element of claim 12 , comprising an EcoRI and SnaBI fragment of about 2 kb.

24 . A seed obtained from a transgenic plant containing a cryptic regulatory element as claimed in claim 1 , operatively linked to a gene encoding a protein.

25 . The cryptic regulatory element of claim 11 comprising a DNA fragment that is substantially homologous to the nucleotide sequence of SEQ ID NO:2.

26 . The cryptic regulatory element of claim 11 , comprising a nucleotide sequence consisting of at least 18 contiguous nucleotides of SEQ ID NO:2.

27 . The cryptic regulatory element of claim 11 , comprising nucleotides 2055-2224 of SEQ ID NO:2.

28 . The cryptic regulatory element of claim 11 , comprising nucleotides 1875-1992 of SEQ ID NO:2.

29 . The cryptic regulatory element of claim 11 , comprising nucleotides 1-2224 of SEQ ID NO:2.

30 . The cryptic regulatory element of claim 29 , comprising nucleotides 415-2224 of SEQ ID NO:2.

31 . The cryptic regulatory element of claim 29 comprising nucleotides 750-2224 of SEQ ID NO:2.

32 . The cryptic regulatory element of claim 29 , comprising nucleotides 1370-2224 of SEQ ID NO:2.

33 . The cryptic regulatory element of claim 29 , comprising nucleotides 1660-2224 of SEQ ID NO:2.

34 . The cryptic regulatory element of claim 29 , comprising nucleotides 1875-2224 of SEQ ID NO:2.

35 . The cryptic regulatory element of claim 29 , comprising nucleotides 2086-2224 of SEQ ID NO:2

36 . The cryptic regulatory element of claim 29 , comprising nucleotides 1-1660 of SEQ ID NO:2

37 . The cryptic regulatory element of claim 29 , comprising nucleotides 1660-2086 of SEQ ID NO:2

38 . The cryptic regulatory element of claim 29 , comprising nucleotides 1-2086 of SEQ ID NO:2.

39 . The cryptic regulatory element of claim 29 , comprising nucleotides 1875-2086 of SEQ ID NO:2.

40 . A method of conferring expression of a gene in a host organism, comprising operatively linking an exogenous DNA of interest, for which expression is desired with the cryptic regulatory element of claim 1 , or a fragment thereof, to produce a chimeric gene construct, and introducing the chimeric gene construct into the host organism capable of expressing the chimeric gene construct.

41 . The method of claim 40 , wherein the host organism is selected from the group consisting of a plant, a tree, an insect, a fungi, a bacteria, a yeast and a non-human animal.

42 . The method of claim 41 , wherein the host organism is a plant, and the cryptic regulatory element is selected from the group consisting of a seed-coat specific regulatory element, and constitutive regulatory element.

43 . The method of claim 42 , wherein the seed-coat specific regulatory element comprises a nucleic acid that is substantially homologous with the sequence of SEQ ID NO:1.

44 . The method of claim 42 , wherein the constitutive regulatory element comprises a nucleic acid that is substantially homologous with the sequence of SEQ ID NO:2.

45 . The method of claim 43 , wherein the nucleic acid comprises at least a 19 bp contiguous sequence of SEQ ID NO:1.

46 . The method of claim 44 , wherein the nucleic acid comprises at least an 18 bp contiguous sequence of SEQ ID NO:2.

47 . The cryptic regulatory element of claim 6 , wherein the post-transcriptional regulatory element is a transcriptional enhancer.

48 . The cryptic regulatory element of claim 6 , wherein the post-transcriptional regulatory element is a translational enhancer.

49 . The cryptic regulatory element of claim 6 , wherein the post-transcriptional regulatory element is an mRNA stability determinant.

50 . A method of modulating expression of a gene in a host organism, comprising operatively linking an exogenous DNA of interest, for which expression is desired with a promoter of interest and the cryptic regulatory element of claim 1 , or a fragment thereof, to produce a chimeric gene construct, and introducing the chimeric gene construct into the host organism capable of expressing the chimeric gene construct.

51 . The method of claim 50 , wherein the host organism is selected from the group consisting of a plant, a tree, an insect, a fungi, a bacteria, a yeast and a non-human animal.

52 . The cryptic regulatory element of claim 6 , wherein the element comprises nucleotides 1-141 of SEQ ID NO:3.

53 . The cryptic regulatory element of claim 6 , wherein the element comprises nucleotides 1-188 of SEQ ID NO:3.

54 . The cryptic regulatory element of claim 6 , wherein the element comprises nucleotides 1-97 of SEQ ID NO:4.

55 . The cryptic regulatory element of claim 6 , wherein the element comprises nucleotides 1-129 of SEQ ID NO:4.

56 . The cryptic regulatory element of claim 6 , wherein the element comprises nucleotides 1-119 of SEQ ID NO:5.

57 . The cryptic regulatory element of claim 6 , wherein the element comprises nucleotides 1-86 of SEQ ID NO:5.

58 . The cryptic regulatory element of claim 3 , wherein the core promoter comprises nucleotides 1992-2042 of SEQ ID NO:2.

59 . The cryptic regulatory element of claim 28 , wherein nucleotides 1875-1992 of SEQ ID NO:2 are repeated.

60 . The cryptic regulatory element of claim 29 comprising nucleotides 1660-1992 of SEQ ID NO:2.

61 . The cryptic regulatory element of claim 60 , wherein nucleotides 1660-1992 of SEQ ID NO:2 are repeated.

62 . A method of modulating expression of a gene in a host organism, comprising operatively linking an exogenous DNA of interest, for which expression is desired with a promoter of interest and at least one fragment of the cryptic regulatory element of claim 1 to produce a chimeric gene construct, and introducing the chimeric gene construct into the host organism capable of expressing the chimeric gene construct.

63 . The method of claim 62 , wherein the at least one fragment of the cryptic regulatory element is repeated.

64 . The method of claim 63 , wherein the host organism is selected from the group consisting of a plant, a tree, an insect, a fungi, a bacteria, a yeast and a non-human animal.

65 . The method of claim 40 , wherein the exogenous DNA of interest encodes a protein selected from the group consisting of a pharmaceutically active protein, a growth factor, a growth regulator, an antibody, an antigen, an interleukin, insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF, an interferon, a blood clotting factor, an industrial enzyme, a protein supplement, a nutraceutical, a protease, an oxidases, a phytase, a chitinase, an invertase, a lipase, a cellulase, a xylanase, and an enzyme involved in oil metabolic and biosynthetic pathways.

66 . A transgenic host organism containing a cryptic regulatory element as claimed in claim 1 , operatively linked to a gene encoding a protein.

67 . The transgenic host organism of claim 64 , wherein the host organism is selected from the group consisting of a plant, a tree, an insect, a fungi, a bacteria, a yeast and a non-human animal.

68 . The vector of claim 16 , wherein the crytpic regulatory element is repeated.

69 . The vector of claim 16 , wherein the crytpic regulatory element is inverted.

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