U.S. patent application number 09/747368 was filed with the patent office on 2001-11-29 for cryptic regulatory elements obtained from plants.
Invention is credited to Brown, Daniel Charles William, Foster, Elizabeth, Hattori, Jiro, Labbe, Helene, Martin-Heller, Teresa, Miki, Brian, Ouellet, Therese, Tian, Lining, Wu, Keqiang, Zhang, Peijun.
Application Number | 20010047091 09/747368 |
Document ID | / |
Family ID | 4162812 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010047091 |
Kind Code |
A1 |
Miki, Brian ; et
al. |
November 29, 2001 |
Cryptic regulatory elements obtained from plants
Abstract
T-DNA tagging with a promoterless .beta.-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.
Inventors: |
Miki, Brian; (Ottawa,
CA) ; Ouellet, Therese; (Nepean, CA) ;
Hattori, Jiro; (Ottawa, CA) ; Foster, Elizabeth;
(Nepean, CA) ; Labbe, Helene; (Ottawa, CA)
; Martin-Heller, Teresa; (Gloucester, CA) ; Tian,
Lining; (London, CA) ; Brown, Daniel Charles
William; (Ilderton, CA) ; Zhang, Peijun;
(Nepean, CA) ; Wu, Keqiang; (Nepean, CA) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Family ID: |
4162812 |
Appl. No.: |
09/747368 |
Filed: |
December 22, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09747368 |
Dec 22, 2000 |
|
|
|
PCT/CA99/00578 |
Jun 22, 1999 |
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Current U.S.
Class: |
536/24.1 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8222 20130101; C12N 15/8234 20130101; C12N 15/8216
20130101 |
Class at
Publication: |
536/24.1 |
International
Class: |
C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 9, 1998 |
CA |
2,246,892 |
Claims
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.
Description
FIELD OF INVENTION
[0001] This invention relates to cryptic regulatory elements within
plants.
BACKGROUND AND PRIOR ART
[0002] Bacteria from the genus Agrobacterium have the ability to
transfer specific segments of DNA (T-DNA) to plant cells, where
they stably integrate into the nuclear chromosomes. Analyses of
plants harbouring the T-DNA have revealed that this genetic element
may be integrated at numerous locations, and can occasionally be
found within genes. One strategy which may be exploited to identify
integration events within genes is to transform plant cells with
specially designed T-DNA vectors which contain a reporter gene,
devoid of cis-acting transcriptional and translational expression
signals (i.e. promoterless), located at the end of the T-DNA. Upon
integration, the initiation codon of the promoterless gene
(reporter gene) will be juxtaposed to plant sequences. The
consequence of T-DNA insertion adjacent to, and downstream of, gene
promoter elements may be the activation of reporter gene
expression. The resulting hybrid genes, referred to as
T-DNA-mediated gene fusions, consist of unselected plant promoters
residing at their natural location within the chromosome, and the
coding sequence of a marker gefie located on the inserted T-DNA
(Fobert et al., 1991, Plant Mol. Biol. 17, 837-851).
[0003] It has generally been assumed that activation of
promoterless or enhancerless marker genes result from T-DNA
insertions within or immediately adjacent to genes. The recent
isolation of several T-DNA insertional mutants (Koncz et al., 1992,
Plant Mol. Biol. 20, 963-976; reviewed in Feldmann, 1991, Plant J.
1, 71-82; Van Lijsebettens et al. 1991, Plant Sci. 80. 27-37;
Walden et al., 1991, Plant J. 1: 281-288; Yanofsky et al., 1990,
Nature 346, 35-39), shows that this is the case for at least some
insertions. However, other possibilities exist. One of these is
that integration of the T-DNA activates silent regulatory sequences
that are not associated with genes. Lindsey et al. (1993,
Transgenic Res. 2, 33-47) referred to such sequences as
"pseudo-promoters" and suggested that they may be responsible for
activating marker genes in some transgenic lines.
[0004] Inactive regulatory sequences that are buried in the genome
but with the capability of being functional when positioned
adjacent to genes have been described in a variety of organisms,
where they have been called "cryptic promoters" (Al-Shawi et al.,
1991, Mol. Cell. Biol. 11, 4207-4216; Fourel et al., 1992, Mol.
Cell. Biol. 12, 5336-5344; Irniger et al., 1992. Nucleic Acids Res.
20, 4733-4739; Takahashi et al., 1991, Jpn J. Cancer Res. 82,
1239-1244). Cryptic promoters can be found in the introns of genes,
such as those encoding for yeast actin (Irniger et al., 1992,
Nucleic Acids Res. 20. 4733-4739), and a mammalian
melanoma-associated antigen (Takahashi et al., 1991, Jpn J. Cancer
Res. 82, 1239-1244). It has been suggested that the cryptic
promoter of the yeast actin gene may be a relict of a promoter that
was at one time active but lost function once the coding region was
assimilated into the exon-intron structure of the present-day gene
(Irniger et al. 1992, Nucleic Acids Res. 20, 4733-4739). A cryptic
promoter has also been found in an untranslated region of the
second exon of the woodchuck N-myc proto-oncogene (Fourel et al.,
1992, Mol. Cell. Biol. 12, 5336-5344). This cryptic promoter is
responsible for activation of a N-myc2, a functional processed gene
which arose from retropositon of N-myc transcript (Fourel et al.,
1992, Mol. Cell. Biol. 12, 5336-5344). These types of regulatory
sequences have not yet been isolated from plants.
[0005] Other regulatory elements are located within the 5' and 3'
untranslated regions (UTR) of genes. These regulatory elements can
modulate gene expression in plants through a number of mechanisms
including translation, transcription and RNA stability. For
example, some regulatory elements are known to enhance the
translational efficiency of "mRNA, resulting in an increased
accumulation of recombinant protein by many folds. Some of those
regulatory elements contain translational enhancer sequences or
structures, such as the Omega sequence of the 5' leader of the
tobacco mosaic virus (Gallie and Walbot, 1992, Nucleic Acid res.
20, 4631-4638), the 5' alpha-beta leader of the potato virus X
(Tomashevskaya et al, 1993, J. Gen. Virol. 74,2717-2724), and the
5' leader of the photosystem I gene psaDb of Nicotiana sylvestris
(Yamamoto et al., 1995. J. Biol. Chem 270, 12466-12470). Other 5'
regulatory elements affect gene expression by quantitative
enhancement of transcription, as with the UTR of the thylakoid
protein genes PsaF, PetH and PetE from pea (Bolle et al., 199,
Plant J. 6. 513-523), or by repression of transcription, as for the
5' UTR of the pollen-specific LAT59 gene from tomato (Curie and
McCormick, 1997, Plant Cell 9, 2025-2036). Some 3' regulatory
regions contain sequences that act as mRNA instability
determinants, such as the DST element in the Small Auxin-Up RNA
(SAUR) genes of soybean and Arabidopisis (Newman et al. 1993, Plant
Cell 5. 701-714). Other translational enhancers are also well
documented in the literature (e.g. Helliwell and Gray 1995, Plant
Mol. Bio. vol 29, pp. 621-626; Dickey L. F. al. 1998, Plant Cell
vol 10, 475-484; Dunker B.P. et al. 1997 Mol. Gen. Genet. vol 254,
pp. 291-296). However, there have been no reports of these types of
cryptic regulatory elements, nor have any cryptic regulatory
elements of this kind been isolated from plants.
[0006] The present invention discloses transgenic plants generated
by tagging with a promoterless GUS (.beta.-glucuronidase) T-DNA
vector and the isolation and characterization of cryptic regulatory
elements identified using this protocol. Cloning and
characterization of these insertion sites uncovered unique cryptic
regulatory elements not conserved among related species. In one of
the plants of interest, GUS expression was spatially and
developmentally regulated with in seed tissue. The isolated
regulatory element specific to this tissue has not been previously
isolated or characterized in any manner. In another plant, a novel
constitutive regulatory element was identified that is expressed in
tissues throughout the plant and across a broad range of plant
species. Furthermore, novel non-translated 5' sequences have been
identified that function as post transcriptional regulatory
elements.
SUMMARY OF INVENTION
[0007] This invention relates to cryptic regulatory elements within
plants.
[0008] Several transgenic tobacco plants, including T218 and T1275.
were identified using the method of this invention that contain
novel regulatory elements. These regulatory elements were found not
to be active in the native plant.
[0009] Plant T218 contains a 4.65 kb EcoRI fragment containing the
2.15 kb promoterless GUS-nos gene and 2.5 kb of 5 flanking DNA.
Deletion of the region approximately between 2.5 and 10 kb ot the
5' flanking region did not alter GUS expression, as compared to the
entire 4.65 kb GUS fusion. A further deletion to 0.5 kb of the 5'
flanking site resulted in complete loss of GUS activity. Thus the
region between 1.0 and 0.5 of the 5' flanking region of the tobacco
DNA contains the elements essential to gene activation. This region
is contained within a XbaI--SnaBI restriction site fragment of the
flanking tobacco DNA. Expression of a gene operatively associated
with the regulatory region was only observed in seed tissues, more
specifically seed-coat tissue.
[0010] A second transgenic tobacco plant, T1275, contained a 4.38
kb EcoRI/XbaI fragment containing the 2.15 kb promoterless GUS-nos
gene and 2.23 kb of 5' flanking tobacco DNA (2225 bp). Expression
of the cloned fragment in transgenic tobacco, N. tabacum c.v. Petit
Havana, SRI and transgenic B. napus c.v. Westar was observed in
leaf, stem, root, developing seed and flower. By transient
expression analysis, GUS activity was also observed in leaf tissue
of soybean, alfalfa, Arabidopsis, tobacco, B. napus, pea and
suspension cultured cells of oat, corn, wheat and barley. The
transcription start site for the GUS gene in transgenic tobacco was
located in the plant DNA upstream of the insertion site. A set of
deletions within the plant DNA revealed the presence of a core
promoter element located within a 62 bp region from the
transcriptional start site, the occurrence of at least one negative
regulatory element located within an XbaI-SspI fragment, a
transcriptional enhancer located within the BstYI-Dral fragment,
and at least one post transcriptional regulatory element located
within a NdeI-SmaI fragment.
[0011] This invention therefore provides for isolated nucleic acids
that comprise cryptic regulatory elements within plants. This
invention also is directed to cryptic regulatory elements that
comprise at least one of: a promoter, a core promoter element, a
negative regulatory element, a transcriptional enhancer, a
translational enhancer and a post transcriptional regulatory
element.
[0012] Furthermore, this invention relates to a cryptic regulatory
element comprising a nucleic acid that is substantially homologous
to the nucleotide sequence of SEQ ID NO:1. This invention also
relates to a nucleic acid comprising at least 19 contiguous
nucleotides of nucleotides 1 to 993 of SEQ ID NO:1, or, comprising
a nucleotide sequence consisting of at least 19 contiguous
nucleotides of nucleotides 1 to 467 of SEQ ID NO:1. This invention
also relates to a vector comprising the nucleic acids as defined
above.
[0013] This invention is also directed to a cryptic regulatory
element comprising a nucleic acid fragment bounded by EcoRI-SmaI
restriction sites defined by the restriction map of FIG. 2(B).
Furthermore, this invention relates to a cryptic regulatory element
comprising an XbaI--SmaI fragment, of the restriction map of FIG.
2(B) of about 2 kb. Also considered within the scope of the present
invention is a cryptic regulatory element comprising an XbaI and
SnaBI fragment as defined by the restriction map of FIG. 2(B),
wherein the fragment is of about 500 bp. This invention also is
directed to a cryptic regulatory element comprising an XbaI and
SnaBI fragment, as defined by the restriction map of FIG. 2(B),
wherein the fragment is of about 1.5 kb, or a cryptic regulatory
element comprising a HindIII and SnaBI fragment, defined by the
restriction map of FIG. 2(B), wherein the fragment is of about 1.9
kb. Furthermore, this invention also embraces a cryptic regulatory
element comprising an EcoRI and SnaBI fragment defined by the
restriction map of FIG. 2, wherein the fragment is of about 2
kb.
[0014] This invention also embraces a regulatory element
characterized in that it is substantially homologous with the
sequence defined by SEQ ID NO:2. This invention is also directed to
a cryptic regulatory element that comprises at least an 18 bp
contiguous sequence of SEQ ID NO:2. Furthermore, this regulatory
element functions in diverse plant species when introduced on a
cloning vector. This invention also relates to a chimeric gene
construct comprising a DNA of interest for which constitutive
expression is desired, and a constitutive regulatory element,
comprising at least an 18 bp contiguous sequence of SEQ ID NO:
2.
[0015] This invention also embraces a cryptic regulatory element
comprising an
[0016] XbaI--SmaI fragment (comprising nucleotides 1-2224 of SEQ ID
NO:2), an
[0017] XbaI--NdeI fragment (comprising nucleotides 1-1086 of SEQ ID
NO :2), an
[0018] SphI--SmaI fragment (comprising nucleotides 415-2224 of SEQ
ID NO:2), a
[0019] PstI--SmaI fragment (comprising nucleotides 750-2224 of SEQ
ID NO:2), an
[0020] SspI--SmaI fragment (comprising nucleotides 1370-2224 of SEQ
ID NO:2), a
[0021] BstYI--SmaI fragment (comprising nucleotides 1660-2224 of
SEQ ID NO:2), a
[0022] DraI--SmaI fragment (comprising nucleotides 1875-2224 of SEQ
ID NO:2), a
[0023] NdeI-SmaI fragment (comprising nucleotides 2086-2224 of SEQ
ID NO:2), a
[0024] XbaI-BstYI fragment (comprising nucleotides 1-1660 of SEQ ID
NO:2), a
[0025] BstYI-DraI fragment (comprising nucleotides 1660-1875 of SEQ
ID NO:2), a +1 to Sma1 fragment (comprising nucleotides 2055-2224
of SEQ ID NO:2), Dra1-Nde1 fragment (comprising nucleotides
1875-2086 of SEQ ID NO:2) or a Dra1 to -62 fragment (comprising
nucleotides 1875-1992 of SEQ ID NO:2) as defined in FIG. 13(C).
[0026] This invention is also also directed to a cryptic regulatory
element comprising nucleotides 1-141 of SEQ ID NO:3, nucleotides
1-188 of SEQ ID NO:3, nucleotides 1-97 of SEQ ID NO:4 nucleotides
1-129 of SEQ ID NO:4, nucleotides 1-119 of SEQ ID NO:5, or
nucleotides 1-86 of SEQ ID NO:5.
[0027] This invention also pertains to a transgenic host organism
containing a cryptic regulatory element as defined above
operatively linked to a gene encoding a protein. The host organism
may be selected from the group consisting of a plant, a tree, an
insect, a fungi, a bacteria, a yeast and a non-human animal.
[0028] This invention also includes a plant cell which has been
transformed with a chimeric gene construct, or a cloning vector
comprising a cryptic plant regulatory element. Furthermore, this
invention embraces transgenic plants containing chimeric gene
constructs, or cloning vectors comprising cryptic plant regulatory
elements.
[0029] This invention further relates to any transgenic plant
containing a cryptic regulatory element, having a DNA sequence
substantially homologous to SEQ ID NO: 1, or SEQ ID NO:2 and
operatively linked to a DNA region that is transcribed into
RNA.
[0030] Also included in the present invention is 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 a cryptic regulatory element as defined
above, to produce a chimeric gene construct, and introducing the
chimeric gene construct into the host organism capable of
expressing the chimeric gene construct. This invention also
embraces a method of modulating expression of a gene in a plant,
comprising operatively linking an exogenous DNA of interest, for
which expression is desired with a promoter of interest and the
cryptic regulatory element as defined above and introducing the
chimeric construct into the host organism. Furthermore, the method
of conferring or modulating gene expression may include 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 as defined above to produce a
chimeric gene construct, and introducing the chimeric gene
construct into the host organism capable of expressing the chimeric
gene construct. The host organism may be selected from the group
consisting of a plant, a tree, an insect, a fungi, a bacteria, a
yeast and a non-human animal.
[0031] This invention also relates to the above method wherein the
plant-derived cryptic regulatory element is a seed-coat specific or
constitutive regulatory element. Furthermore, this invention
embraces the above method wherein the seed-coat specific regulatory
element comprises a nucleic acid that is substantially homologous
with the sequence of SEQ ID NO:1, or constitutive regulatory
element comprises a nucleic acid that is substantially homologous
with the sequence of SEQ ID NO:2. This invention also relates to
the above method wherein the nucleic acid comprises at least a 19
bp contiguous sequence of SEQ ID NO:1, or the nucleic acid
comprises at least an 18 bp contiguous sequence of SEQ ID NO:2.
[0032] According to the present invention there is also provided a
seed coat-specific cryptic regulatory element contained within a
DNA sequence, or analogue thereof, as shown in SEQ ID NO: 1.
Furthermore, there is provided a constitutive regulatory element
contained within a DNA sequence, fragment or an analogue thereof,
as shown in SEQ ID NO: 2.
[0033] This invention also relates to a vector containing a seed
coat-specific cryptic regulatory element, which is contained within
a DNA sequence, or analogue thereof, as shown in SEQ ID NO: 1 and a
gene encoding a protein. This invention also relates to a cloning
vector containing a constitutive cryptic regulatory element, which
is contained within a DNA sequence, fragment, or an analogue
thereof, as shown in SEQ ID NO: 2 and a gene encoding a
protein.
[0034] This invention also includes a plant cell which has been
transformed with a vector as described above, and to a transgenic
plant containing a cloning vector as described above, operatively
linked to a gene encoding a protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 depicts the fluorogenic analyses of GUS expression in
the plant T218. Each bar represents the average.+-.one standard
deviation of three samples. Nine different tissues were analyzed:
leaf (L), stem (S), root (R), anther (A), petal (P), ovary (O),
sepal (Se), seeds 10 days post anthesis (SI) and seeds 20 days
post-anthesis (S2). For all measurements of GUS activity, the
fraction attributed to intrinsic fluorescence, as determined by
analysis of untransformed tissues, is shaded black on the graph.
Absence of a black area at the bottom of a histogram indicates that
the relative contribution of the background fluorescence is too
small to be apparent.
[0036] FIG. 2 shows the cloning of the GUS fusion in plant T218
(pT218) and construction of transformation vectors. Plant DNA is
indicated by the solid line and the promoterless GUS-nos gene is
indicated by the open box. The transcriptional start site and
presumptive TATA box are located by the closed and open arrow heads
respectively.
[0037] FIG. 2(A) shows DNA probes #1, 2, 3, and RNA probe #4 (all
listed under the pT218 restriction map). The EcoRI fragment in
pT218 was subcloned in the pBIN19 polylinker to create pT218-1.
Fragments truncated at the XbaI, SnaBI and XbaI sites were also
subcloned to create pT218-2, pT218-3 and pT218-4.
[0038] FIG. 2(B) shows the restriction map of the plant DNA
upstream from the GUS insertion site. Abbreviations for the
endonuclease restriction sites are as follows: EcoRI (E), HindIII
(H), XbaI (X), SnaBI (N), Smal (M), SstI (S).
[0039] FIG. 3 shows the expression pattern of promoter fusions
during seed development. GUS activity in developing seeds (4-20
days postanthesis (dpa)) of (FIG. 3a) plant T218
(.circle-solid.-.circle-solid.) and (FIG. 3b) plants transformed
with vectors pT218-1 (.smallcircle.-.smallcircle.)- , pT218-2
(.quadrature.-.quadrature.), pT218-3 (.gradient.-.gradient.) and
pT218-4 (.DELTA.-.DELTA.) which are illustrated in FIG. 2. The 2
day delay in the peak of GUS activity during seed development, seen
with the pT218-2 transformant, likely reflects greenhouse variation
conditions.
[0040] FIG. 4 shows GUS activity in 12 dpa seeds of independent
transformants produced with vectors pT218-1 (.smallcircle.),
pT218-2 (.quadrature.), pT218-3 (.gradient.) and pT218-4 (.DELTA.).
The solid markers indicate the plants shown in FIG. 3(b) and the
arrows indicate the average values for plants transformed with
pT218-1 or pT218-2.
[0041] FIG. 5 shows the mapping of the T218 GUS fusion termini and
expression of the region surrounding the insertion site in
untransformed plants.
[0042] FIG. 5(A) shows the mapping of the GUS mRNA termini in plant
T218. The antisense RNA probe from subclone #4 (FIG. 2) was used
for hybridization with total RNA of tissues from untransformed
plants (10 .mu.g) and from plant T218 (30 .mu.g). Arrowheads
indicate the anticipated position of protected fragments if
transcripts were initiated at the same sites as the T218 GUS
fusion.
[0043] FIG. 5(B) shows the results of an RNase protection assay
using the antisense (relative to the orientation of the GUS coding
region) RNA probe from subclone e (see FIG. 7) against 30 .mu.g
total RNA of tissues from untransformed plants. The abbreviations
used are as follows: P, untreated RNA probe; -, control assay using
the probe and tRNA only; L, leaves from untransformed plants; 8,
10, 12, seeds from untransformed plants at 8, 10, and 12 dpa,
respectively; T10, seeds of plant T218 at 10 dpa; +, control
hybridization against unlabelled in vitro-synthesized sense RNA
from subclone c (panel a) or subclone e (panel b). The two
hybridizing bands near the top of the gel are end-labelled DNA
fragment of 3313 and 1049 bp. included in all assays to monitor
losses during processing. Molecular weight markers are in number of
bases.
[0044] FIG. 6 provides the nucleotide sequence of pT218 (top line)
(SEQ ID NO: 1) and pIS-1 (bottom line). Sequence identity is
indicated by dashed lines. The T-DNA insertion site is indicated by
a vertical line after bp 993. This site on pT218 is immediately
followed by a 12 bp filler DNA, which is followed by the T-DNA. The
first nine amino acids of the GUS gene and the GUS initiation codon
(*) are shown. The major and minor transcriptional start site is
indicated by a large and small arrow, respectively. The presumptive
TATA box is identified and is in boldface. Additional putative TATA
and CAAT boxes are marked with boxes. The location of direct (1-5)
and indirect (6-8) repeats are indicated by arrows.
[0045] FIG. 7 shows the base composition of region surrounding the
T218 insertion site cloned from untransformed plants. The site of
T-DNA insertion in plant T218 is indicated by the vertical arrow.
The position of the 2 genomic clones pIS-1 and pIS-2, and of the
various RNA probes (a-e) used in RNase protection assays are
indicated beneath the graph.
[0046] FIG. 8 shows the Southern blot analyses of the insertion
site in Nicotiana species. DNA from N. tomentosiformis (N tom). N.
sylvesrris (N syl), and N. tabacum (N tab) were digested with
HindIII (H), XbaI (X) and EcoRI (E) and hybridized using probe #2
(FIG. 2). Lambda HindIII markers (kb) are indicated.
[0047] FIG. 9 shows the AT content of 5' non-coding regions of
plant genes. A program was written in PASCAL to scan GenBank
release 75.0 and to calculate the AT contents of the 5' non-coding
(solid bars) and the coding regions (hatched bars) of all plant
genes identified as "Magnoliophyta" (flowering plants). The region
-200 to -1 and +1 to +200 were compared. Shorter sequences were
also accepted if they were at least 190 bp long. The horizontal
axis shows the ratio of the AT content (%). The vertical axis shows
the number of the sequences having the specified AT content
ratios.
[0048] FIG. 10 shows the constitutive expression of GUS in all
tissues of plant T1275, including leaf segments (a), stem
cross-sections (b), roots (c), flower cross-sections (d), ovary
cross-sections (e), immature embryos (f), mature embryos (g), and
seed cross-sections (h).
[0049] FIG. 11 shows GUS specific activity within a variety of
tissues throughout the plant T1275, including leaf (L), stem (S),
root (R), anther (A), petal (P), ovary (O), sepal (Se), seeds 10
days post anthesis (S1), and seeds, 20 days post anthesis (S2).
[0050] FIG. 12 shows the restriction map of the cryptic regulatory
element of pT1275.
[0051] FIG. 12(A) shows the plant DNA fused with GUS.
[0052] FIG. 12(B) shows the restriction map of the plant DNA. The
arrow indicates the GUS riRNA start site within the cryptic
regulatory region.
[0053] FIG. 13 shows deletion constructs of the T1275 regulatory
element.
[0054] FIG. 13(A) shows the 5' endpoints of each construct as
indicated by the restriction endonuclease site, relative to the
full length T1275 regulatory element, the arrow indicates the
transcriptional start site. Plant DNA is indicated by the solid
line, the promoterless GUS-nos gene is indicated by the open box
and the shaded box indicates the region coding for the amino
terminal peptide fused to GUS. The XbaI fragment in pT1275 was
subcloned to create pT1275-GUS-nos. Deletion constructs truncated
at the SphI, PstI, SspI, BstYI, and DraI sites were also subcloned
to create -1639-GUS-nos, -1304-GUS-nos, -684-GUS-nos, -394-GUS-nos,
and -197-GUS-nos, respectively.
[0055] FIG. 13(B) shows further deletion constructs of -62-GUS-nos,
-12-GUS-nos, -62(-tsr)-GUS-nos and +30-GUS-nos, relative to
-197-GUS-nos (see FIG. 13(A)).
[0056] FIG. 13(C) shows the restriction map of the plant DNA of
pT1275 upstream from the GUS insertion site.
[0057] FIG. 13(D) shows modified constructs of the T1275 regulatory
elements. T1275 is indicated by the open box, the CaMV35S promoter
element is indicated by the black box. The activity of these
constructs is also indicated. GUS activity was determined in
tobacco leaves following transient expression using microparticle
bombardment. TA30-GUS: a TATATAA element was inserted into the -30
position of -62-GUS; TA35S-GUS: the -62 to -20 fragment of -62-GUS
was substituted with the -46 to -20 fragment of the 35S promoter;
GCC-62-GUS: a GCC box was fused with -62-GUS; DRA2-GUS: the -197 to
-62 fragment was repeated; BST2-GUS: the -394 to -62 fragment was
repeated; -46-35S: 35S minimal promoter; DRAI-35S: the -197 to -62
fragment of T1275 was fused with -46-35S; BSTI-35S: the -394 to -62
fragment of T1275 was fused with -46-35S; BST2-35S: two copies of
the -394 to -62 fragment of T1275 were fused with -46-35S.
[0058] FIG. 13(E) shows constructs of the -197 to -62 fragment
fused with the 35S minimal promoter. 46-35S: 35S minimal promoter;
DRAI-35S: the -197 to -62 fragment of T1275 was fused with -46-35S;
DRA1R-35S: the -197 to -62 fragment of T1275 was fused with -46-35S
in a reversed orientation; DRA2-35S: two copies of the -197 to -62
fragment of T1275 were fused with -46-35S.
[0059] FIG. 13(F) shows GUS specific activity of transgenic
Arabidopsis plants. Leaf tissues from Arabidopsis plants
transformed with -47-35S, DRA1-35S, DRA1R-35S and DRA2-35S
constructs were used for GUS assay.
[0060] FIG. 13(G) shows the constitutive expression of GUS in
Arabidopsis plants transformed with DRA1-35S. From left to right:
flower, silque and seedling.
[0061] FIG. 14 shows the GUS specific activity, mRNA, and protein
levels in leaves of individual, regenerated, greenhouse-grown
transgenic plants containing T1275-GUS-nos (T plants), or
35S-GUS-nos (S plants).
[0062] FIG. 14(A) shows the levels of GUS expression in leaves from
randomly selected plants containing either T1275-GUS-nos (left-hand
side) or 35S-GUS-nos (right-hand side).
[0063] FIG. 14(B) shows the level of accumulated GUS mRNA measured
by RNase protection assay and densitometry of autoradiograms in
leaves from the same randomly selected plants containing either
T1275-GUS-nos (left-hand side) or 35S-GUS-nos (right-hand
side).
[0064] FIG. 14(C) shows a Western blot of GUS fusion protein
obtained from T1275-GUS-nos and 35S-GUS-nos plants. Leaf extracts
were equally loaded onto gels and GUS was detected using anti-GUS
antibodies. The molecular weight markers are indicated on the
right-hand side of the gel: untransformed control (SR1) and GUS
produced in E. coli (Ec).
[0065] FIG. 15 shows deletion and insertion constructs of the 5'
untranslated leader region of T1275 regulatory element and
construction of transformation vectors. The constructs are
presented relative to T1275-GUS-nos or 35S-GUS-nos. The arrow
indicates the transcriptional start site. Plant DNA is indicated by
the solid line labeled T1275, the 35S regulatory region by the
solid line labelled CaMV35S, the NdeI--SmaI region by a filled in
box, the shaded box coding for the amino terminal peptide, and the
promoterless GUS-nos gene is indicated by an open box. The deletion
construct removing the NdeI--SmaI fragment of T1275-GUS-nos is
identified as T1275-N-GUS-nos. The NdeI--SmaI fragment from
T1275-GUS-nos was also introduced into 35S-GUS-nos to produce
35S+N-Gus-nos.
[0066] FIG. 16 shows the region surrounding the insertion site in
untransformed plants, positions of various probes used for RNase
protection assays, and results of the RNase protection assay.
[0067] FIG. 16(A) shows a restriction map of the insertion site and
various probes used for the assay (IP: insertion point of GUS in
transformed plants; *: that T1275 probe ended at the BstYI site,
not the IP; **: probe 7 included 600bp of the T1275 plant sequence
and 400 bp of the GUS gene).
[0068] FIG. 16(B) shows results of an RNase protection assay of RNA
isolated from leaf (L), stem (St), root (R), flower bud (F) and
developing seed (Se) tissues of tobacco transformed with
T1275-GUS-nos (10 .mu.g RNA) and untransformed tobacco (30 .mu.g
RNA). Undigested probe (P), tRNA negative control lanes and markers
are indicated. RNase protection assays shown used a probe to detect
sense transcripts between about -446 and +596 of T1275-GUS-nos or
between about -446 to +169 of untransformed tobacco. The protected
fragment in transformed plants is about 596 bp (upper arrowhead)
and, if presents accumulated transcripts initiated at this site in
untransformed plants are predicted to protect a fragment of about
169 bp (lower arrowhead). Upper band in RNA-containing lanes was
added to samples to indicate loss of sample during assay.
[0069] FIG. 17 shows the levels of mRNA, as well as the ratio
between GUS specific activity and mRNA levels in leaves of
individual, regenerated, greenhouse-grown transgenic plants
containing T1275-GUS-nos, or 35S-GUS-nos constructs, with or
without the NdeI-SmaI fragment (see FIG. 15).
[0070] FIG. 17(A) shows the level of accumulated GUS mRNA measured
by RNase protection assay and densitometry of autoradiograms in
leaves from the same randomly selected plants containing either
T1275-GUS-nos, T1275-N-GUS-nos.
[0071] FIG. 17(B) shows the level of accumulated GUS mRNA measured
by RNase protection for 35S-GUS-nos or 35S+N-GUS-nos.
[0072] FIG. 17(C) shows the ratio between GUS specific activity and
mRNA levels in leaves of individual, regenerated, greenhouse-grown
transgenic plants containing T1275-GUS-nos, T1275-N-GUS-nos,
35S-GUS-nos, or 35S+N-GUS-nos constructs.
[0073] FIG. 18 shows the maps of T1275-GUS-nos and
T1275(AN)-GUS-nos.
[0074] FIG. 18(A) shows T1275-GUS-nos (also referred to as
tCUP-GUS-nos).
[0075] FIG. 18(B) shows T1275(AN)-GUS-nos (also referred to as
tCUPdelta-GUS-nos). ".DELTA.N", (also referred to as "dN" or
"deltaN") was created by changing the NdeI site "a" in the leader
sequence of T1275-GUS-nos (FIG. 18(A)) to a BglII site "b" (see
FIG. 18(B)) to eliminate the upstream ATG at nucleotides 2087-2089
or SEQ ID NO:2. A Kozak consensus sequence "c" was constructed at
the initiator MET codon and a NcoI site was added. The
transcriptional start site, determined for T1275, is indicated by
the arrow.
[0076] FIG. 19 shows constructs used for the transient expression
via particle bombardment of corn callus. Maps for 35S-GUS-nos. 35S
(+N)-GUS-nos, 35S (.DELTA.N)-GUS-nos and 35S(+i)-GUS-nos are
presented indicating the "N" region, ADH1 intron, and the arrow
indicates the transcriptional start site. Note that
35S(.DELTA.N)-GUS-nos is referred to as 35S+deltaN-dK-GUS-nos. Also
shown are the associated activities of the constructs in the callus
expressed as a ratio of GUS to luciferase (control) activity.
[0077] FIG. 20 shows maps of the constructs used for transient
expression in yeast. Shown are pYES-GUS-nos (also referred to as
pYEGUS); pYES(+N)-GUS-nos (also referred to as pYENGUS);
pYES(.DELTA.N)-GUS-nos (also referred to as pYEdNGUS) and
pYES(.DELTA.N.sup.M)-GUS-nos (also referred to as pYEdN.sup.MGUS),
which lacks the Kozak consensus sequence.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0078] This invention relates to cryptic regulatory elements
identified in plants. More specifically, this invention relates to
cryptic promoters, negative regulatory elements, transcriptional
enhancer elements and other post transcriptional regulatory
elements identified in plants.
[0079] T-DNA tagging with a promoterless .beta.-glucuronidase (GUS)
gene generated several transgenic Nicotiana tabacum plants that
expressed GUS activity. Examples, which are not to be considered
limiting in any manner, of transgenic plants displaying expression
of the promoterless reporter gene, include a plant that expressed
GUS only in developing seed coats, T218, and another plant that
expressed GUS in all organs, T1275 (see co-pending patent
applications U.S. Ser. No. 08/593121 and PCT/CA97/00064. both of
which are incorporated by reference).
[0080] Cloning and deletion analysis of the GUS fusions in both of
these plants revealed that the regulatory regions were located in
the plant DNA proximal to the GUS gene:
[0081] In T218, a cryptic regulatory region was identified between
an EcoRI-SmaI fragment, and further deletion analyses localized a
cryptic regulatory element to an approximately 0.5 kb region
between a XbaI and a SnaBI restriction endonuclease site of the 5'
flanking tobacco DNA (see FIG. 2). This region spans from
nucleotide 1 to nucleotide 467 of SEQ ID NO: 1.
[0082] In T1275, a regulatory region was identified within an
XbaI--SmaI fragment, which comprises several cryptic regulatory
elements which were localized to several regions throughout the
upstream region and include a minimal promoter region between DraI
and NdeI sites (see FIG. 13), negative regulatory elements between
XbaI and BstYI, a transcriptional enhancer between BstYI and DraI,
and between Dra1-(-62) (nucleotides 1875 to 1992 of SEQ ID NO:2),
and a translational enhancer regulatory element between the
NdeI-SmaI sites (also referred to as "N", see below; SEQ ID NO:3).
Also included are regulatory elements ".DELTA.N" (also referred to
as dN, or deltaN), an element derived from N, that comprises a
Kozack sequence (FIG. 18, SEQ ID NO:4), and .DELTA.N.sup.M, that
lacks a Kozack sequence (SEQ ID NO:5).
[0083] However, it is to be understood that other portions of the
isolated disclosed regulatory elements within T218 and T1275 may
also exhibit activities in directing organ specificity, tissue
specificity, or a combination thereof, or temporal activity, or
developmental activity, or a combination thereof, or other
regulatory attributes including, negative regulatory elements,
enhancer sequences, or post transcriptional regulatory elements,
including sequences that affect stability of the transcription or
initiation complexes or stability of the transcript.
[0084] Thus, the present invention includes cryptic regulatory
elements obtained from plants that are capable of conferring, or
enhancing expression upon gene of interest linked in operative
association therewith. Furthermore, the present invention includes
cryptic regulatory elements obtained from plants capable of
mediating the translational efficiency of a transcript produced
from a gene of interest linked in operative association therewith.
It is to be understood that the cryptic regulatory elements of the
present invention may also be used in combination with other
regulatory elements, either cryptic or otherwise, such as
promoters, enhancers, or fragments thereof, and the like.
[0085] The term cryptic regulatory element refers to regulatory
elements that are inactive in the control of expression at their
native location. These inactive regulatory sequences are buried in
the genome including intergenic regions or regions of genes that
are not involved in the regulation of adjacent sequences but are
capable of being functional when positioned adjacent to a gene.
[0086] By "regulatory element" or regulatory region", it is meant a
portion of nucleic acid typically, but not always, upstream of a
gene, and may be comprised of either DNA or RNA, or both DNA and
RNA. The regulatory elements of the present invention includes
those which are capable of mediating organ specificity, or
controlling developmental or temporal gene activation. Furthermore,
"regulatory element" includes promoter elements, core promoter
elements, elements that are inducible in response to an external
stimulus, elements that are activated constitutively, or elements
that decrease or increase promoter activity such as negative
regulatory elements or transcriptional enhancers, respectively. It
is also to be understood that enhancer elements may be repeated
thereby further increasing the enhancing effect of an enhancer
element on a regulatory region. "Regulatory elements" as used
herein, also includes elements that are active following
transcription initiation or transcription, for example, regulatory
elements that modulate gene expression such as translational and
transcriptional enhancers, translational and transcriptional
repressors, and mRNA stability or instability determinants. In the
context of this disclosure, the term "regulatory element" also
refers to a sequence of DNA, usually, but not always, upstream (5')
to the coding sequence of a structural gene, which includes
sequences which control the expression of the coding region by
providing the recognition for RNA polymerase and/or other factors
required for transcription to start at a particular site. An
example of a regulatory element that provides for the recognition
for RNA polymerase or other transcriptional factors to ensure
initiation at a particular site is a promoter element. A promoter
element comprises a core promoter element, responsible for the
initiation of transcription, as well as other regulatory elements
(as listed above) that modify gene expression. It is to be
understood that nucleotide sequences, located within introns, or 3'
of the coding region sequence may also contribute to the regulation
of expression of a coding region of interest. A regulatory element
may also include those elements located downstream (3') to the site
of transcription initiation, or within transcribed regions, or
both. In the context of the present invention a
post-transcriptional regulatory element may include elements that
are active following transcription initiation, for example
translational and transcriptional enhancers, translational and
transcriptional repressors, and mRNA stability determinants.
[0087] The regulatory elements, or fragments thereof, of the
present invention may be operatively associated with heterologous
regulatory elements or promoters in order to modulate the activity
of the heterologous regulatory element. Such modulation includes
enhancing or repressing transcriptional activity of the
heterologous regulatory element, modulating post-transcriptional
events, or both enhancing or repressing transcriptional activity of
the heterologous regulatory element and modulating
post-transcriptional events. For example, one or more regulatory
elements, or fragments thereof, of the present invention may be
operatively associated with constitutive, inducible, or tissue
specific promoters or fragment thereof to modulate the activity of
such promoters within plant, insect, fungi, bacterial, yeast, or
animal cells.
[0088] An example of a cryptic regulatory element of the present
invention, which is not to be considered limiting in any manner is
an organ-specific, and temporally-specific element obtained from
plant T218. Such an element is a seed-specific regulatory element.
More preferably the element is a seed-coat specific regulatory
element as described herein, or an analogue thereof, or a nucleic
acid fragment localized between EcoRI--SmaI sites, as defined in
restriction map of FIG. 2(B) or a fragment thereof. The seed
coat-specific regulatory element may also be defined by a nucleic
acid comprising substantial homology (similarity) with the
nucleotide sequence comprising nucleotides 1-467, or 1-993, of SEQ
ID NO:1. For example, which is not to be considered limiting in any
manner, the nucleic acid may exhibit 80% similarity to the
nucleotide sequence comprising nucleotides 1-467, or 1-993, of SEQ
ID NO:1. Furthermore, the seed-coat specific nucleotide sequence
may be defined as comprising at least a 19 bp fragment of
nucleotides 1-467. or 1-993 as defined within SEQ ID NO:1.
[0089] Another example of a cryptic regulatory element of an aspect
of the present invention includes, but is not limited to, a
constitutive regulatory element obtained from the plant T1275, as
described herein and analogues or fragments thereof, or a nucleic
acid fragment localized between XbaI--SmaI, as identified by the
restriction map of FIG. 12(B) or a fragment thereof. Furthermore,
the constitutive regulatory element may be defined as a nucleic
acid fragment localized between XbaI--SmaI as identified by the
restriction map of FIG. 13(A) or (C) or a fragment thereof. The
constitutive cryptic regulatory element may also be defined by a
nucleotide sequence comprising at least an 18 bp fragment of the
regulatory region defined in SEQ ID NO:2, or by a nucleic acid
comprising from about 80% similarity to the nucleotide sequence of
SEQ ID NO:2.
[0090] A further regulatory element of the present invention
includes an enhancer element within the -394 to -62 fragment of
T1275 (nucleotides 1660 to 1992 of SEQ ID NO:2). This fragment may
also be duplicated and fused to a regulatory region, for example a
core promoter, producing an increase in the activity of the
regulatory region (see FIG. 13(D)).
[0091] Another cryptic regulatory element of the present invention
includes, but is not limited to, a post-transcriptional or
translational enhancer regulatory element localized between
NdeI--SmaI (see FIG. 15, nucleotides 1-188 of SEQ ID NO:3). The
post-transcriptional or translational enhancer regulatory element
may also comprise the nucleotide sequence as defined by nucleotides
1-141 of SEQ ID NO:3 (nucleotides 2086-2224 of SEQ ID NO:2) or an
analog thereof, or the element may comprise 80% similarity to the
nucleotide sequence of nucleotides 1-141 of SEQ ID NO:3
(nucleotides 2086-2224 of SEQ ID NO:2).
[0092] A shortened fragment of the NdeI--SmaI fragment, referred to
as .DELTA.N, dN or deltaN is also characterized within the present
invention. .DELTA.N was prepared by mutagenesis replacing the out
of frame ATG (located at nucleotides 2087-2089, SEQ ID NO:1) within
the NdeI-SmaI fiagment (see FIG. 18). AN constructs with (SEQ ID
NO:4) or without (SEQ ID NO:5) a Kozak consensus sequence was also
characterized (Tables 10, and 12) and found to exhibit enhancer
activity. Therefore, other cryptic regulatory elements of the
present invention include, but are not limited to,
post-transcriptional or translational enhancers regulatory elements
localized at nucleotides 1-97 of SEQ ID NO:4 and nucleotides 1-86
of SEQ DI NO: 4 or 5. These post-transcriptional or translational
enhancer regulatory elements may comprise the nucleotide sequence
as defined by nucleotides 1-86 of SEQ ID NO:4 or 5 (nucleotides
2170-2224 of SEQ ID NO:2) or an analog thereof, or the element may
comprise 80% similarity to the nucleotide sequence of nucleotides
1-86 of SEQ ID NO:4 or 5 (nucleotides 2170-2224 of SEQ ID NO:2).
Furthermore, these regulatory elements may comprise the nucleotide
sequence as defined by nucleotides 1-97 of SEQ ID NO:4 and
comprising a Kozack sequence or an analog thereof, or the element
may comprise 80% similarity to the nucleotide sequence of
nucleotides 1-97 of SEQ ID NO:4.
[0093] Furthermore, other regulatory elements of the present
invention include negative regulatory elements (for example located
within an XbaI-BstYI fragment as defined by FIG. 13(C); nucleotides
1:1660 of SEQ ID NO:2), a transcriptional enhancer localized within
the BstYI-DraI fragment of FIG. 13(C) (nucleotides 1660-1875 of SEQ
ID NO:2), a core promoter element located within the DraI-NdeI
fragment of FIG. 13(C) (nucleotides 1875-2086 of SEQ ID NO:2), a
transcriptional enhancer within the Dra1 to -62 fragment
(nucleotides 1875-1992 of SEQ ID NO:2; FIGS. 13(D) to (G)), or a
regulatory element or post-transcriptional element downstream of
the transcriptional start site, for example but not limited to the
NdeI-SmaI fragment (nucleotides 1-188 of SEQ ID NO3) and
derivatives and fragments thereof (for example nucleotides 1-141 of
SEQ ID NO:3), including .DELTA.N (nucleotides 1-129 or 1-97 of SEQ
ID NO:4, AN.sup.M (nucleotides 1-119 or 1-86 SEQ ID NO:5), and
nucleotides 1-86 of SEQ ID NO:4 or 5 (nucleotides 2086 to 2170 of
SEQ ID NO:2).
[0094] An "analogue" of the above identified cryptic regulatory
elements includes any substitution, deletion, or additions to the
sequence of a regulatory element provided that said analogue
maintains at least one regulatory property associated with the
activity of the regulatory element. Such properties include
directing organ specificity, tissue specificity, or a combination
thereof, or temporal activity, or developmental activity, or a
combination thereof, or other regulatory attributes including,
negative regulatory elements, enhancer sequences, or sequences that
affect stability of the transcription or translation complexes or
stability of the transcript.
[0095] There are several types of regulatory elements, including
those that are developmentally regulated, inducible and
constitutive. A regulatory element that is developmentally
regulated, or controls the differential expression of a gene under
its control, is activated within certain organs or tissues of an
organ at specific times during the development of that organ or
tissue. However, some regulatory elements that are developmentally
regulated may preferentially be active within certain organs or
tissues at specific developmental stages, they may also be active
in a developmentally regulated manner, or at a basal level in other
organs or tissues within the plant as well.
[0096] An inducible regulatory element is one that is capable of
directly or indirectly activating transcription of one or more DNA
sequences or genes in response to an inducer. In the absence of an
inducer the DNA sequences or genes will not be transcribed.
Typically the protein factor, that binds specifically to an
inducible regulatory element to activate transcription, is present
in an inactive form which is then directly or indirectly converted
to the active form by the inducer. The inducer can be a chemical
agent such as a protein, metabolite, growth regulator, herbicide or
phenolic compound or a physiological stress imposed directly by
heat, cold, salt, or toxic elements or indirectly through the
action of a pathogen or disease agent such as a virus. A plant cell
containing an inducible regulatory element may be exposed to an
inducer by externally applying the inducer to the cell or plant
such as by spraying, watering, heating or similar methods.
[0097] A constitutive regulatory element directs the expression of
a gene throughout the various parts of a plant and continuously
throughout plant development. Examples of known constitutive
regulatory elements include promoters associated with the CaMV 35S
transcript. (Odell et al., 1985, Nature, 313: 810-812), the rice
actin 1 (Zhang et al. 1991, Plant Cell, 3: 1155-1165) and
triosephosphate isomerase 1 (Xu et al, 1994, Plant Physiol. 106:
459-467) genes, the maize ubiquitin 1 gene (Cornejo et al, 1993,
Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6
genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637-646), and the
tobacco translational initiation factor 4A gene (Mandel et al, 1995
Plant Mol. Biol. 29: 995-1004).
[0098] The term "constitutive" as used herein does not necessarily
indicate that a gene under control of the constitutive regulatory
element is expressed at the same level in all cell types, but that
the gene is expressed in a wide range of cell types even though
variation in abundance is often observed.
[0099] The present invention is further directed to a chimeric gene
construct containing a DNA of interest operatively linked to a
regulatory element of the present invention. Any exogenous gene can
be used and manipulated according to the present invention to
result in the expression of said exogenous gene.
[0100] The chimeric gene construct of the present invention can
further comprise a 3' untranslated region. A 3' untranslated region
refers to that portion of a gene comprising a DNA segment that
contains a polyadenylation signal and any other regulatory signals
capable of effecting mRNA processing or gene expression. The
polyadenylation signal is usually characterized by effecting the
addition of polyadenylic acid tracks to the 3' end of the mRNA
precursor. Polyadenylation signals are commonly recognized by the
presence of homology to the canonical form 5' AATAAA-3' although
variations are not uncommon.
[0101] Examples of suitable 3' regions are the 3' transcribed
non-translated regions containing a polyadenylation signal of
Agrobacterium tumor inducing (Ti) plasmid genes, such as the
nopaline synthase (Nos gene) and plant genes such as the soybean
storage protein genes and the small subunit of the
ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. The 3'
untranslated region from the structural gene of the present
construct can therefore be used to construct chimeric genes for
expression in plants.
[0102] The chimeric gene construct of the present invention can
also include further enhancers, either translation or transcription
enhancers, as may be required. These enhancer regions are well
known to persons skilled in the art, and can include the ATG
initiation codon and adjacent sequences. The initiation codon must
be in phase with the reading frame of the coding sequence to ensure
translation of the entire sequence. The translation control signals
and initiation codons can be from a variety of origins, both
natural and synthetic. Translational initiation regions may be
provided from the source of the transcriptional initiation region,
or from the structural gene. The sequence can also be derived from
the regulatory element selected to express the gene, and can be
specifically modified so as to increase translation of the
mRNA.
[0103] To aid in identification of transformed plant cells, the
constructs of this invention may be further manipulated to include
plant selectable markers. Useful selectable markers include enzymes
which provide for resistance to an antibiotic such as gentamycin,
hygromycin, kanamycin, and the like. Similarly, enzymes providing
for production of a compound identifiable by colour change such as
GUS (.beta.-glucuronidase), or luminescence, such as luciferase are
useful.
[0104] Also considered part of this invention are transgenic plants
containing the chimeric gene construct comprising a regulatory
element of the present invention. However, it is to be understood
that the regulatory elements of the present invention may also be
combined with gene of interest for expression within a range of
host organisms. Such organisms include, but are not limited to:
[0105] plants, both monocots and dicots, for example, corn, wheat,
barley, oat, tobacco, Brassica, soybean, pea, alfalfa, potato,
ginseng, Arabidopsis;
[0106] trees, for example peach, spruce;
[0107] yeast, fungi, insects, animal and bacteria cells.
[0108] Methods for the transformation and regeneration of these
organisms are established in the art and known to one of skill in
the art.
[0109] By "gene of interest" it is meant any gene that is to be
expressed within a host organism. Such a gene of interest may
include, but is not limited to, a gene that encodes a
pharmaceutically active protein, for example growth factors, growth
regulators, antibodies, antigens, their derivatives useful for
immunization or vaccination and the like. Such proteins include,
but are not limited to, interleukins, insulin, G-CSF, GM-CSF.
hPG-CSF. M-CSF or combinations thereof, interferons, for example,
interferon-.alpha., interferon-.beta., interferon-.tau., blood
clotting factors, for example, Factor VIII. Factor IX, or tPA or
combinations thereof. A gene of interest may also encode an
industrial enzyme, protein supplement, nutraceutical, or a
value-added product for feed, food, or both feed and food use.
Examples of such proteins include, but are not limited to
proteases, oxidases, phytases, chitinases, invertases, lipases,
cellulases, xylanases, enzymes involved in oil biosynthesis
etc.
[0110] Methods of regenerating whole plants from plant cells are
also known in the art. In general, transformed plant cells are
cultured in an appropriate medium, which may contain selective
agents such as antibiotics, where selectable markers are used to
facilitate identification of transformed plant cells. Once callus
forms, shoot formation can be encouraged by employing the
appropriate plant hormones in accordance with known methods and the
shoots transferred to rooting medium for regeneration of plants.
The plants may then be used to establish repetitive generations,
either from seeds or using vegetative propagation techniques.
[0111] The constructs of the present invention can be introduced
into plant cells using Ti plasmids, Ri plasmids, plant virus
vectors, direct DNA transformation, micro-injection,
electroporation, etc. For reviews of such techniques see for
example Weissbach and Weissbach, Methods for Plant Molecular
Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson
and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and
Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism,
2d Ed. D T. Dennis, D H Turpin, D D Lefebrve, D B Layzell (eds),
Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). The
present invention further includes a suitable vector comprising the
chimeric gene construct.
[0112] The DNA sequences of the present invention thus include the
DNA sequences of SEQ ID NO: 1, 2, 3, 4 and 5, the regulatory
regions and fragments thereof, as well as analogues of, or nucleic
acid sequences comprising about 80% similarity with the nucleic
acids as defined in SEQ ID NO's: 1 to 5. Analogues (as defined
above), include those DNA sequences which hybridize under stringent
hybridization conditions (see Maniatis et al., in Molecular Cloning
(A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, p.
387-389) to any one of the DNA sequence of SEQ ID NO: 1, 2, 3, 4,
or 5, provided that said sequences maintain at least one regulatory
property of the activity of the regulatory element as defined
herein.
[0113] An example of one such stringent hybridization conditions
may be hybridization in 4.times.SSC at 65.degree. C., followed by
washing in 0.1.times.SSC at 65.degree. C. for an hour.
Alternatively an exemplary stringent hybridization condition could
be in 50% formamide, 4.times.SSC at 42.degree. C. Analogues also
include those DNA sequences which hybridize to any one of the
sequences of SEQ ID NO: 1 to 5 under relaxed hybridization
conditions, provided that said sequences maintain at least one
regulatory property of the activity of the regulatory element.
Examples of such non-hybridization conditions includes
hybridization in 4.times.SSC at 50.degree. C. or with 30-40%
formamide at 42.degree. C.
[0114] There are several lines of evidence that suggest that the
seed coat-specific expression of GUS activity in the plant T218 is
regulated by a cryptic regulatory element. The region surrounding
the regulatory element and transcriptional start site for the GUS
gene are not transcribed in untransformed plants. Transcription was
only observed in plant T218 when T-DNA was inserted in cis. DNA
sequence analysis did not uncover a long open reading frame within
the 3.3 kb region cloned. Moreover, the region is very AT rich and
predicted to be noncoding (data not shown) by the Fickett algorithm
(Fickett, 1982, Nucleic Acids Res. 10, 5303-5318) as implemented in
DNASIS 7.0 (Hitachi). Southern blots revealed that the insertion
site is within the N. tomentosiformis genome and is not conserved
among related species as would be expected for a region with an
important gene.
[0115] Furthermore, Northern analysis demonstrate that the
transcript, associated with the regulatory region and corresponding
to the native plant sequence, does not accumulate in developing
seeds or leaves of untransformed plants. This indicates that in
native plants, the regulatory region as defined as pT218, is
silent.
[0116] Similarly, results indicate that the constitutive expression
of GUS activity in the plant T1275 is regulated by a cryptic
regulatory element. RNase protection assays performed on the region
spanning the regulatory element and downstream region did not
reveal a transcript for the sense strand (see FIG. 16, Table 2).
RNase protection assays were performed using RNA from organs of
untransformed tobacco and probes that spanned the T1275 sequence
from about -2055 bp to +1200 bp relative to the transcriptional
start site. In all tissues tested (leaf, stem, root, flower bud,
petal, ovary and developing seed) protected fragments were not
detected, in the sense orientation relative to the GUS coding
region, with all probes (FIG. 16; see also PCT CA97/00064, which is
incorporated by reference). Furthermore, GenBank searches revealed
no significant sequence similarity with the T1275 sequence. An
amino acid identity of about 66% with two open reading frames on
the antisense strand of the genomic sequence of T1275 (between
about -1418 and -1308. nucleotides 636-746 of SEQ ID NO:2; and
between about -541 and -395. nucleotides 1513-1659 of SEQ ID NO:2
relative to the transcriptional start) and an open reading frame of
a partial Arabidopsis expressed sequence (GenBank Accession No.
W43439) was identified. The sequence which lies downstream of
sequences at the T-DNA insertion point in untransformed tobacco
shows no significant similarity in GenBank searches. These data
suggest that this region is silent in untransformed plants and that
the insertion of the T-DNA activated a cryptic promoter.
[0117] Southern analysis indicates that the 2.2 kb regulatory
region of T1275 does not hybridize with DNA isolated from soybean,
potato, sunflower, Arabidopsis, B. napus, B. oleracea, corn, wheat
or black spruce. However, transient assays indicate that this
regulatory region can direct expression of the GUS coding region in
all plant species tested including canola, tobacco, Brassica,
Arabidopsis, soybean, alfalfa, pea, ginseng, potato, corn, wheat,
barley, white spruce and peach (Table 3). indicating that this
regulatory element is useful for directing gene expression in both
dicot and monocot plants as well as trees. Furthermore, regulatory
elements were also found to modulate gene expression in a diverse
range of species including yeast, bacteria and insect cells.
[0118] The transcriptional start site was delimited by RNase
protection assay to a single position about 220 bp upstream of the
translational initiation codon of the GUS coding region in the
T-DNA. The sequence around the transcriptional start site exhibits
similarity with sequences favored at the transcriptional start site
compiled from available dicot plant genes (T/A T/C A.sub.+1 A C/A
C/A A/C/I A A A/T). Sequence similarity is not detected about 30 bp
upstream of the transcriptional start site with the TATA-box
consensus compiled from available dicot plant genes (C T A T A A/T
A T/A A).
[0119] Deletions in the upstream region indicate that negative
regulatory elements and enhancer sequences exist within the full
length regulatory region. For example, deletion of the 5' region to
BstYI (-394 relative to the transcriptional start site; see FIG.
13(C)) resulted in a 3 to 8 fold increase in expression of the gene
associated therewith (see Table 6), indicating the occurrence of at
least one negative regulatory element within the XbaI-BstYI portion
of the full length regulatory element. Other negative regulatory
elements also exist within the XbaI-BstYI fragment as removal of an
XbaI-PstI fragment also resulted in increased activity
(-1403-GUS-nos; Table 6). An enhancer is also localized within the
BstYI-DraI fragment as removal of this region results in a 4 fold
loss in activity of the remaining regulatory region (-197-GUS-nos;
Table 6).
[0120] 5' deletions of the promoter (see FIGS. 13(B) and (C) and
analysis by transient expression using biolistics showed that the
promoter was active within a fragment 62 bp from the
transcriptional start site indicating that the core promoter has a
basal level of expression (see Table 5). Deletion of a fragment
containing the transcriptional start site (see -62(-tsr)/GUS/nos in
FIG. 13(C); Table 5) did not eliminate expression, however
deletions to -12 bp and further (i.e. +30) did eliminate expression
indicating that the region defined by -(62-12) bp (nucleotides
1992-2042 of SEQ ID NO:2) contained the core promoter. DNA sequence
searches did not reveal conventional core promoter motifs found in
plant genes such as the TATA box.
[0121] A number of the 5' promoter deletion clones (FIGS. 13(B) and
(C)) were transferred into tobacco and Arabidopsis by
Agrobacterium-mediated transformation using the vector pRD400.
Analysis of GUS specific activity in leaves of transgenic plants
(see Table 6) confirmed the transient expression data down to the
-197 fragment (nucleotides 1875-2224 of SEQ ID NO:2). Histochemical
analysis of tobacco organs sampled from the transgenic plants
indicated GUS expression in leaf, seeds and flowers. Histochemical
analysis of Arabidopsis organs revealed GUS activity in leaf, stem
flowers and silques when the promoter was deleted to the -394 and
-1 97 fragments (see FIGS. 13(E) to (G)).
[0122] A comparison of GUS specific activities in the leaves of
transgenic tobacco SRl transformed with the T1275-GUS-nos gene and
the 35S-GUS-nos genes revealed a similar range of values (FIG.
14(A)). Furthermore, the GUS protein levels detected by Western
blotting were similar between plants transformed with either gene
when the GUS specific activities were similar (FIG. 14(C)).
Analysis of GUS mRNA levels by RNase protection however revealed
that the levels of mRNA were about 60 fold (mean of 13
measurements) lower in plants transformed with the T1275-GUS-nos
gene (FIG. 14(B) suggesting the existence of a post-transcriptional
regulatory element in the mRNA leader sequence.
[0123] Expression of GUS, under the control of T1275 or a fragment
thereof, or the modulation of GUS expression arising from T1275 or
a fragment thereof, has been observed in a range of species
including corn, wheat, barley, oat, tobacco, Brassica, soybean,
alfalfa, pea, potato, Ginseng, Arabidopsis, peach, spruce, yeast,
fungi, insects and bacterial cells.
[0124] Further analysis confirmed the presence of a regulatory
sequence within the NdeI-SmaI fragment of the mRNA leader sequence
that had a significant impact on the level of GUS specific activity
expressed in all organs tested. Deletion of the NdeI-SmaI fragment
from the T1275-GUS-nos gene (FIG. 15) resulted in about a 46-fold
reduction in the amount of GUS specific activity that could be
detected in leaves of transgenic tobacco cv Delgold (see Table 7).
Similar results were also obserevd in the transgenic tobacco
cultivar SRI and transgenic alfalfa (Table 7). Addition of the same
fragment to a 35S-GUS-nos gene construct (FIG. 15) increased the
amount of GUS specific activity by about 5-fold in transgenic
tobacco and a higher amount in transgenic alfalfa (see Table 7).
Increased GUS activity was observed in organs of tobacco and
alfalfa plants tranformed with constructs containing NdeI-SmaI
fragment (Table 8 and 9).
[0125] A modulation of GUS activity was noted in a variety of
species that were transformed with a regulatory element of the
present invention. For example but not necessarily limited to, the
NdeI-SmaI fragment of T1275 (also referred to as "N") and
derivatives or analogues thereof, produced an increase in activity
within a variety of organisms tested including a range of plants
(Tables 3 and 10, and FIG. 19), white spruce (a conifer; Table 11)
and yeast (Table 12).
[0126] A shortened fragment of the NdeI-SmaI fragment, (referred to
as ".DELTA.N". "dN", or "deltaN") was produced that lacks the
out-of frame upstream .DELTA.TG at nucleotides 2087-2089 of SEQ ID
NO:2 (see FIGS. 18(A) and (B)). Constructs comprising
T1275(AN)-GUS-nos yielded 5 fold greater levels of GUS activity in
leaves of transgenic tobacco compared to plants expressing
T1275-GUS-nos. Furthermore, in corn callus and yeast, AN
significantly increased GUS expression driven by the 35 S promoter
(FIG. 19 and Table 10).
[0127] The NdeI-SmaI regulatory elements situated downstream of the
transcriptional start site functions both at a transcriptional, and
post-transcriptional level. The levels of mRNA observed in
transgenic plants transformed with T1275-GUS-nos are higher than
the levels in plaints transformed with T1275(-N)-GUS-nos. However,
the opposite is true with plants tranformed with 35S-GUS-nos or
35S(+N)-GUS-nos, where higher levels of mRNA are detected in the
absence of the Ndel-SmaI fragment (see FIGS. 17(A) and (B)). This
indicates that this region functions by either modulating
transcriptional rates, or the stability of the transcript, or
both.
[0128] The NdeI-SmaI region also functions post-transcriptionally.
The ratio of GUS specific activity to relative RNA level in
individual transgenic tobacco plants that lack the NdeI-SmaI
fragment is lower, and when averaged indicates an eight fold
reduction in GUS activity per RNA, than in plants comprising this
region (FIG. 17(C)). Similarly, an increase, by an average of six
fold, in GUS specific activity is observed when the NdeI-SmaI
region is added within the 35S untranslated region (FIG. 17(C)).
The GUS specific activity:relative RNA levels are similar in
constructs containing the NdeI-SmaI fragment (T1275-GUS-nos and
35S+N-GUS-nos). These results indicate that the NdeI-SmaI fragment
modulates gene expression post-transcriptionally. Further
experiments suggest that this region is a novel translational
enhancer. Translation of transcripts in vitro demonstrate an
increase in translational efficiency of RNA containing the NdeI to
SmaI fragment (see Table 13). Furthermore, the levels of protein
produced using mRNAs comprising the NdeI-SmaI fragment are greater
than those produced using the known translational enhancer of
Alfalfa Mosaic Virus RNA4. These results indicate that this region
functions post-transcriptionally, as a translational enhancer.
[0129] As this is the first report of cryptic regulatory elements
in plants, it is impossible to estimate the degree to which cryptic
regulatory elements may contribute to the high frequencies of
promoterless marker gene activation in plants. It is interesting to
note that transcriptional GUS fusions in Arabidopsis occur at much
greater frequencies (54%) than translational fusions (1.6%.
Kertbundit et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5212-5216).
The possibility that cryptic promoters may account for some fusions
was recognized by Lindsey et al. (1993, Transgenic Res. 2,
33-47).
[0130] The regulatory elements of the present invention may be used
to control the expression of a gene of interest within desired host
expression system, for example, but not limited to:
[0131] plants, both monocots and dicots, for example, corn,
tobacco, Brassica, soybean, pea, alfalfa, potato, ginseng, wheat,
oat, barley, Arabidopsis;
[0132] trees, for example peach, spruce;
[0133] yeast, fungi, insects, and bacteria.
[0134] Furthermore, the regulatory elements as described herein may
be used in conjunction with other regulatory elements, such as
tissue specific, inducible or constitutive promoters, enhancers, or
fragments thereof, and the like. For example, the regulatory region
or a fragment thereof as defined herein may be used to regulate
gene expression of a gene of interest spatially and developmentally
within developing seed coats, or within a heterologous expression
system, for example yeast, insects, or fungi expression systems.
Some examples of such uses, which are not to be considered
limiting, include:
[0135] 1. Modification of storage reserves in seed coats, such as
starch by the expression of yeast invertase to mobilize the starch
or expression of the antisense transcript of ADP-glucose
pyrophosphorylase to inhibit starch biosynthesis.
[0136] 2. Modification of seed color contributed by condensed
tannins in the seed coats by expression of antisense transcripts of
the phenylalanine ammonia lyase or chalcone synthase genes.
[0137] 3. Modification of fibre content in seed-derived meal by
expression of antisense transcripts of the caffeic acid-o-methyl
transferase or cinnamoyl alcohol dehydrogenase genes.
[0138] 4. Inhibition of seed coat maturation by expression of
ribonuclease genes to allow for increased seed size, and to reduce
the relative biomass of seed coats, and to aid in dehulling of
seeds.
[0139] 5. Expression of genes in seed coats coding for insecticidal
proteins such as .alpha.-amylase inhibitor or protease
inhibitor.
[0140] 6. Partitioning of seed metabolites such as glucosinolates
into seed coats for nematode resistance.
[0141] 7. Nucleotide fragments of the regulatory region of at least
19 bp as a probe in order to identify analogous regions within
other plants.
[0142] 8. Enhancing expression of a gene of interest within a host
organisms of interest. Regulatory regions or fragments thereof,
including enhancer fragments of the present invention, may be
operatively associated with a heterologous nucleotide sequence
including heterologous regulatory regions to increase the
expression of a gene of interest within a host organism. A gene of
interest may include, but is not limited to, a gene that encodes a
pharmaceutically active protein, for example growth factors, growth
regulators, antibodies, antigens, their derivatives useful for
immunization or vaccination and the like. Such proteins include,
but are not limited to, interleukins, insulin, G-CSF, GM-CSF,
hPG-CSF, M-CSF or combinations thereof, interferons, for example,
interferon-.alpha., interferon-.beta., interferon-.tau., blood
clotting factors, for example, Factor VIII, Factor IX, or tPA or
combinations thereof. A gene of interest may also encode an
industrial enzyme, protein supplement, nutraceutical, or a
value-added product for feed, food, or both feed and food use.
Examples of such proteins include, but are not limited to
proteases, oxidases, phytases chitinases, invertases, lipases,
cellulases, xylanases, enzymes involved in oil metabolic and
biosynthetic pathways etc.
[0143] Similarly, a constitutive regulatory element may also be
used to drive the expression within all organs or tissues, or both
of a plant of a gene of interest, and such uses are well
established in the literature. For example, fragments of specific
elements within the 35S CaMV promoter have been duplicated or
combined with other promoter fragments to produce chimeric
promoters with desired properties (e.g. U.S. Pat. Nos. 5,491,288,
5,424,200, 5,322,938, 5,196,525, 5,164,316). As indicated above, a
constitutive regulatory element or a fragment thereof, as defined
herein, may also be used along with other promoter, enhancer
elements, or fragments thereof, translational enhancer elements or
fragments thereof in order to control gene expression. Furthermore,
oligonucleotides of 18 bps or longer are useful as probes or PCR
primers in identifying or amplifying related DNA or RNA sequences
in other tissues or organisms.
[0144] Thus this invention is directed to regulatory elements and
gene combinations comprising these cryptic regulatory elements.
Further this invention is directed to such regulatory elements and
gene combinations in a cloning vector, wherein the gene is under
the control of the regulatory element and is capable of being
expressed in a plant cell transformed with the vector. This
invention further relates to transformed plant cells and transgenic
plants regenerated from such plant cells. The regulatory element,
and regulatory element-gene combination of the present invention
can be used to transform any plant cell for the production of any
transgenic plant. The present invention is not limited to any plant
species, or species other than plant.
[0145] While this invention is described in detail with particular
reference to preferred embodiments thereof, said embodiments are
offered to illustrate but not limit the invention.
EXAMPLES
[0146] Transfer of binary constructs to Agrobacterium and leaf disc
transformation of Nicotiana tabacum SR1 were performed as described
by Fobert et al. (1991, Plant Mol. Biol. 17, 837-851). Plant tissue
was maintained on 100 .mu.g/ml kanamycin sulfate (Sigma) throughout
in vitro culture.
[0147] Nine-hundred and forty transgenic plants were produced.
Several hundred independent transformants were screened for GUS
activity in developing seeds using the fluorogenic assay. One of
these, T218, was chosen for detailed study because of its unique
pattern of GUS expression. Furthermore, following the screening of
transformants in a range of plant organs, T1275 was selected which
exhibited high level, constitutive expression of GUS.
[0148] Characterization of a Seed Coat-specific GUS
Fusion--T218
[0149] Fluorogenic and histological GUS assays were performed
according to Jefferson (Plant Mol. Biol. Rep. , 1987, 5, 387-405),
as modified by Fobert et al. (Plant Mol. Biol., 1991, 17, 837-851).
For initial screening, leaves were harvested from in vitro grown
plantlets. Later flowers corresponding to developmental stages 4
and 5 of Koltunow et al. (Plant Cell, 1990, 2, 1201-1224) and beige
seeds, approximately 12-16 dpa (Chen et al., 1988, EMBO J. 7,
297-302), were collected from plants grown in the greenhouse. For
detailed, quantitative analysis of GUS activity, leaf, stem and
root tissues were collected from kanamycin resistant F1 progeny of
the different transgenic lines grown in vitro. Floral tissues were
harvested at developmental stages 8-10 (Koltunow et al., 1990,
Plant Cell 2, 1201-1224) from the original transgenic plants.
Flowers of these plants were also tagged and developing seeds were
collected from capsules at 10 and 20 dpa. In all cases, tissue was
weighed, immediately frozen in liquid nitrogen, and stored at
-80.degree. C.
[0150] Tissues analyzed by histological assay were at the same
developmental stages as those listed above. Different hand-cut
sections were analyzed for each organ. For each plant, histological
assays were performed on at least two different occasions to ensure
reproducibility. Except for floral organs, all tissues were assayed
in phosphate buffer according to Jefferson (1987, Plant Mol. Biol.
Rep. 5, 387-405), with 1 mM X-Gluc (Sigma) as substrate. Flowers
were assayed in the same buffer containing 20% (v/v) methanol
(Kosugi et al., 1990, Plant Sci. 70, 133-140).
[0151] Tissue-specific patterns of GUS expression were only found
in seeds. For instance, GUS activity in plant T218 (FIG. 1) was
localized in seeds from 9 to 17 days postanthesis (dpa). GUS
activity was not detected in seeds at other stages of development
or in any other tissue analyzed which included leaf, stem, root,
anther, ovary, petal and sepal (FIG. 1). Histological staining with
X-Gluc revealed that GUS expression in seeds at 14 dpa was
localized in seed coats but was absent from the embryo, endosperm,
vegetative organs and floral organs (results not shown).
[0152] The seed coat-specificity of GUS expression was confirmed
with the more sensitive fluorogenic assay of seeds derived from
reciprocal crosses with untransformed plants. The seed coat
differentiates from maternal tissues called the integuments which
do not participate in double fertilization (Esau, 1977, Anatomy of
Seed Plants. New York: John Wiley and Sons). If GUS activity is
strictly regulated, it must originate from GUS fusions transmitted
to seeds maternally and not by pollen. As shown in Table 1, this is
indeed the case. As a control, GUS fusions expressed in embryo and
endosperm, which are the products of double fertilization, should
be transmitted through both gametes. This is illustrated in Table 1
for GUS expression driven by the napin promoter (BngNAPI,
Baszczynki and Fallis, 1990, Plant Mol. Biol. 14, 633-635) which is
active in both embryo and endosperm (data not shown).
1TABLE 1 GUS activity in seeds at 14 days post anthesis. Cross GUS
Activity .female. .male. nmole MU/min/mg Protein T218 T218 1.09
.+-. 0.39 T218 WT.sup.a 3.02 .+-. 0.19 WT T218 0.04 .+-. 0.005 WT
WT 0.04 .+-. 0.005 NAP-5.sup.b NAP-5 14.6 .+-. 7.9 NAP-5 WT 3.42
.+-. 1.60 WT NAP-5 2.91 .+-. 1.97 .sup.aWT. untransformed plants
.sup.bTransgenic tobacco plants with the GUS gene fused to the
napin, BngNAP1, promoter (Baszczynski and Fallis, 1990, Plant Mol.
Biol. 14, 633-635).
[0153] Cloning and Analysis of the Seed Coat-specific GUS
Fusion
[0154] Genomic DNA was isolated from freeze-dried leaves using the
protocol of Sanders et al. (1987, Nucleic Acid Res. 15, 1543-1558).
Ten micrograms of T218 DNA was digested for several hours with
EcoRI using the appropriate manufacturer-supplied buffer
supplemented with 2.5 mM spermidine. After electrophoresis through
a 0.8% TAE agarose gel, the DNA size fraction around 4-6 kb was
isolated, purified using the GeneClean kit (BIO 101 Inc., LaJolla,
Calif.), ligated to phosphatase-treated EcoRI-digested Lambda GEM-2
arms (Promega) and packaged in vitro as suggested by the supplier.
Approximately 125,000 plaques were transferred to nylon filters
(Nytran, Schleicher and Schuell) and screened by plaque
hybridization (Rutledge et al., 1991, Mol. Gen. Genet. 229, 31-40),
using the 3' (termination signal) of the nos gene as probe (probe
#1, FIG. 2). This sequence, contained in a 260 bp SstI/EcoRl
restriction fragment from pPRF-101 (Fobert et al., 1991, Plant Mol.
Biol. 17, 837-851), was labelled with [.alpha.-.sup.32P]-dCTP (NEN)
using random priming (Stratagene). After plaque purification, phage
DNA was isolated (Sambrook et al., 1989, A Laboratory Manual. New
York: Cold Spring Harbor Laboratory Press), mapped and subcloned
into pGEM-4Z (Promega).
[0155] The GUS fusion in plant T218 was isolated as a 4.7 kb EcoRI
fragment containing the 2.2kb promoterless GUS-nos gene at the
T-DNA border of pPRF120 and 2.5 kb of 5' flanking tobacco DNA
(pT218, FIG. 2), using the nos 3' fragment as probe (probe #1, FIG.
2). To confirm the ability of the flanking DNA to activate the GUS
coding region, the entire 4.7 kb fragment was inserted into the
binary transformation vector pBIN19 (Bevan, 1984, Nucl. Acid Res.
12, 8711-8721), as shown in FIG. 2. Several transgenic plants were
produced by Agrobacterium-mediated transformation of leaf discs.
Plants were transformed with a derivative which contained the 5'
end of the GUS gene distal to the left border repeat. This
orientation is the same as that of the GUS gene in the binary
vector pBllOl (Jefferson, 1987, Plant Mol. Biol. Rep. 5, 387-405).
Southern blots indicated that each plant contained 1-4 T-DNA
insertions at unique sites. The spatial patterns of GUS activity
were identical to that of plant T218. Histologically, GUS staining
was restricted to the seed coats of 14 dpa seeds and was absent in
embryos and 20 dpa seeds (results not shown). Fluorogenic assays of
GUS activity in developing seeds showed that expression was
restricted to seeds between 10 and 17 dpa, reaching a maximum at 12
dpa (FIGS. 3(a) and 3(b)). The 4.7 kb fragment therefore contained
all of the elements required for the tissue-specific and
developmental regulation of GUS expression.
[0156] To locate regions within the flanking plant DNA responsible
for seed coat-specificity, truncated derivatives of the GUS fusion
were generated (FIG. 2) and introduced into tobacco plants.
Deletion of the region approximately between 2.5 and 1.0 kb, 5' of
the insertion site (pT218-2, FIG. 2) did not alter expression
compared with the entire 4.7 kb GUS fusion (FIGS. 3b and 4).
Further deletion of the DNA, to the SnaBI restriction site
approximately 0.5 kb, 5' of the insertion site (pT218-3, FIG. 2),
resulted in the complete loss of GUS activity in developing seeds
(FIGS. 3b and 4). This suggests that the region approximately
between 1.0 and 0.5 kb, 5' of the insertion site contains elements
essential to gene activation. GUS activity in seeds remained absent
with more extensive deletion of plant DNA (pT218-4, FIGS. 2, 3b and
4) and was not found in other organs including leaf, stem, root,
anther, petal, ovary or sepal from plants transformed with any of
the vectors (data not shown).
[0157] The transcriptional start site for the GUS gene in plant
T218 was determined by RNase protection assays with RNA probe #4
(FIG. 2) which spans the T-DNA/plant DNA junction. For RNase
protection assays, various restriction fragments from pIS-1, pIS-2
and pT218 were subcloned into the transcription vector pGEM-4Z as
shown in FIGS. 7 and 2, respectively. A 440bp HindIII fragment of
the tobacco acetohydroxyacid synthase SURA gene was used to detect
SURA and SURB mRNA. DNA templates were linearized and transcribed
in vitro with either T7 or SP6 polymerases to generate
strand-specific RNA probes using the Promega transcription kit and
[.alpha.-.sup.32P]CTP as labelled nucleotide. RNA probes were
further processed as described in Ouellet et al. (1992, Plant J. 2,
321-330). RNase protection assays were performed as described in
Ouellet et al., (1992, Plant J. 2, 321-330), using 10-30 .mu.g of
total RNA per assay. Probe digestion was done at 30.degree. C. for
15 min using 30 .mu.g ml.sup.-1 RNase A (Boehringer Mannheim) and
100 units ml.sup.-1 RNase T1 (Boehringer Mannheim). FIG. 5 shows
that two termini were mapped in the plant DNA. The major 5'
terminus is situated at an adenine residue, 122 bp upstream of the
T-DNA insertion site (FIG. 6). The sequence at this transcriptional
start site is similar to the consensus sequence for plant genes
(C/TTC.dwnarw.ATCA; Joshi, 1987 Nucleic Acids Res. 15, 6643-6653).
A TATA box consensus sequence is present 37 bp upstream of this
start site (FIG. 6). The second, minor terminus mapped 254 bp from
the insertion site in an area where no obvious consensus motifs
could be identified (FIG. 6).
[0158] The tobacco DNA upstream of the insertion site is very
AT-rich (>75%, see FIG. 7). A search for promoter-like motifs
and scaffold attachment regions (SAR), which are often associated
with promoters (Brain et al., 1992, Plant Cell 4, 463-471; Gasser
and Laemmli, 1986, Cell 46, 521-530), identified several putative
regulatory elements in the first 1.0 kb of tobacco DNA flanking the
promoterless GUS gene (data not shown). However, the functional
significance of these sequences remains to be determined.
[0159] Cloning and Analysis of the Insertion Site from
Untransformed Plants
[0160] A lambda DASH genomic library was prepared from DNA of
untransformed N. tabacum SR1 plants by Stratagene for cloning of
the insertion site corresponding to the gene fusion in plant T218.
The screening of 500,000 plaques with probe #2 (FIG. 2) yielded a
single lambda clone. The EcoRI and XbaI fragments were subcloned in
pGEM-4Z to generate pIS-1 and pIS-2. FIG. 7 shows these two
overlapping subclones, pIS-1 (3.0 kb) and pIS-2 (1.1 kb), which
contain tobacco DNA spanning the insertion site (marked with a
vertical arrow). DNA sequence analysis (using dideoxy nucleotides
in both directions) revealed that the clones, pT218 and pIS-1, were
identical over a length of more than 2.5 kb, from the insertion
site to their 5' ends, except for a 12 bp filler DNA insert of
unknown origin at the T-DNA border (FIG. 6 and data not shown). The
presence of filler DNA is a common feature of T-DNA/plant DNA
junctions (Gheysen et al., 1991, Gene 94, 155-163). Gross
rearrangements that sometimes accompany T-DNA insertions (Gheysen
et al., 1990, Gene 94, 155-163; and 1991, Genes Dev. 5, 287-297)
were not found (FIG. 6) and therefore could not account for the
promoter activity associated with this region. The region of pIS-1
and pIS-2, 3' of the insertion site is also very AT-rich (FIG.
7).
[0161] To determine whether there was a gene associated with the
pT218 promoter, more than 3.3 kb of sequence contained with pIS-1
and pIS-2 was analyzed for the presence of long open reading frames
(ORFs). However, none were detected in this region (data not
shown). To determine whether the region surrounding the insertion
site was transcribed in untransformed plants, Northern blots were
performed with RNA from leaf, stem, root, flower and seeds at 4, 8,
12, 14, 16, 20 and 24 dpa. Total RNA from leaves was isolated as
described in Ouellet et al., (1992, Plant J. 2, 321-330). To
isolate total RNA from developing seeds, 0.5 g of frozen tissue was
pulverized by grinding with dry ice using a mortar and pestle. The
powder was homogenized in a 50 ml conical tube containing 5 ml of
buffer (1 M Tris HCl, pH 9.0, 1% SDS) using a Polytron homogenizer.
After two extractions with equal volumes of
phenol:chloroform:isoamyl alcohol (25:24:1), nucleic acids were
collected by ethanol precipitation and resuspended in water. The
RNA was precipitated overnight in 2M LiCl at 0.degree. C.,
collected by centrifugation, washed in 70% ethanol and resuspended
in water. Northern blot hybridization was performed as described in
Gottlob-McHugh et al. (1992, Plant Physiol. 100, 820-825). Probe #3
(FIG. 2) which spans the entire region of pT218 5' of the insertion
did not detect hybridizing RNA bands (data not shown). To extend
the sensitivity of RNA detection and to include the region 3' of
the insertion site within the analysis, RNase protection assays
were performed with 10 different RNA probes that spanned both
strands of pIS-1 and pIS-2 (FIG. 7). Even after lengthy exposures,
protected fragments could not be detected with RNA from 8, 10, 12
dpa seeds or leaves of untransformed plants (see FIG. 5 for
examples with two of the probes tested). The specific conditions
used allowed the resolution of protected RNA fragments as small as
10 bases (data not shown). Failure to detect protected fragments
was not due to problems of RNA quality, as control experiments
using the same samples detected acetohydroxyacid synthase (AHAS)
SURA and SURB mRNA which are expressed at relatively low abundance
(data not shown). Conditions used in the present work were
estimated to be sensitive enough to detect low-abundance messages
representing 0.001-0.01% of total mRNA levels (Ouellet et al.,
1992, Plant J. 2, 321-330). Therefore, the region flanking the site
of T-DNA insertion does not appear to be transcribed in
untransformed plants.
[0162] Genomic Origins of the Insertion Site
[0163] Southern blots were performed to determine if the insertion
site is conserved among Nicotiana species. Genomic DNA (5 .mu.g)
was isolated, digested and separated by agarose gel electrophoresis
as described above. After capillary transfer on to nylon filters.
DNA was hybridized, and probes were labelled, essentially as
described in Rutledge et al. (1991, Mol. Gen. Genet. 229, 31-40).
High-stringency washes were in 0.2.times.SSC at 65.degree. C. while
low-stringency washes were in 2.times.SSC at room temperature. In
FIG. 8, DNA of the allotetraploid species N. tabacum and the
presumptive progenitor diploid species N. tomentosiformis and N.
sylvestris (Okamuro and Goldberg, 1985, Mol. Gen. Genet., 198,
290-298) were hybridized with probe #2 (FIG. 2). Single hybridizing
fragments of identical size were detected in N. tabacum and N.
tomentosiformis DNA digested with HindIII, XbaI and EcoRI, but not
in N. sylvestris. Hybridizations with pIS-2 (FIG. 8) which spans
the same region but includes DNA 3' of the insertion site yielded
the same results. They did not reveal hybridizing bands, even under
conditions of reduced stringency, in additional Nicotiana species
including N. rustica, N. glutinosa, N. megalosiphon and N. debneyi
(data not shown). Probe #3 (FIG. 2) revealed the presence of
moderately repetitive DNA specific to the N. tomentosiformis genome
(data not shown). These results suggest that the region flanking
the insertion site is unique to the N. tomentosiformis genome and
is not conserved among related species as might be expected for
regions that encode essential genes.
[0164] Characterization of a Constitutive GUS fusion--T1275
[0165] From the transgenic plants produced (see above), one of
these, T1275, was chosen for detailed study because of its high
level and constitutive expression of GUS (see also U.S. patent
application Ser. No. 08/593,121 and PCT/CA97/00064, both of which
are incorporated by reference).
[0166] Fluorogenic and histological GUS assays were performed as
outlined above. For initial screening, leaves were harvested from
in vitro grown plantlets. Later nine different tissues: leaf (L),
stem (S), root (R), anther (A), petal (P), ovary (O), sepal (Se),
seeds 10 days post anthesis (S1) and seeds 20 days post-anthesis
(S2), were collected from plants grown in the greenhouse and
analyzed.
[0167] GUS activity in plant T1275 was found in all tissues. FIG.
10 shows the constitutive expression of GUS by histochemical
staining with X-Gluc of T1275, including leaf (a), stem (b), root
(c), flower (d), ovary (e), embryos (f and g), and seed (h).
[0168] Constitutive GUS expression was confirmed with the more
sensitive fluorogenic assay of plant tissue from transformed plant
T1275. These results are shown in FIG. 11. GUS expression was
evident in all tissue types including leaf (L), stem (S), root (R),
anther (A), pistil (P), ovary (O), sepal (Se), seeds at 10 dpa (S1)
and 20 dpa (S2). Furthermore, the level of GUS expression in leaves
was comparable to the level of expression in transformed plants
containing the constitutive promoter CaMV 35S in a GUS--nos fusion.
As reported by Fobert et al. (1991, Plant Molecular Biology, 17:
837-851) GUS activity in transformed plants containing pBIl21
(Clontech), which contains a CaMV 35S--GUS--nos chimeric gene, was
as high as 18,770.+-.2450 (pmole MU per minute per mg protein).
[0169] Cloning and Analysis of the Constitutive Promoter--GUS
Fusion
[0170] Genomic DNA was isolated from leaves according to Hattori et
al. (1987, Anal. Biochem. 165, 70-74). Ten .mu.g of T1275 total DNA
was digested with EcoRI and XbaI according to the manufacturer's
instructions. The digested DNA was size-fractionated on a 0.7%
agarose gel. The DNA fragments of about 4 to 6 kb were isolated
from the gel using the Elu-Quick kit (Schleicher and Schuell) and
ligated to lambdaGEM-2 arms previously digested with EcoRI and XbaI
and phosphatase-treated. About 40,000 plaques were transferred to a
nylon membrane (Hybond Amersham) and screened with the
.sup.32P-labelled 2kb GUS insert isolated from pBI121. essentially
as described in Rutledge et al. (1991, Mol. Gen Genet. 229, 31-40).
The positive clones were isolated. The XbaI-EcoRI fragment (see
restriction map FIG. 12) was isolated from the lambda phage and
cloned into pTZ19R previously digested with XbaI and EcoRI and
treated with intestinal calf phosphatase.
[0171] The plant DNA sequence within the clone, SEQ ID NO:2, has
not been previously reported in sequence data bases. It is not
observed among diverse species as Southern blots did not reveal
bands hybridizing with the fragment in soybean, potato, sunflower,
Arabidopsis, B. napus, B. oleracea, corn, wheat or black spruce
(data not shown). In tobacco, Southern blots did not reveal
evidence for gross rearrangements at or upstream of the T-DNA
insertion site (data not shown).
[0172] The T1275 Regulatory Element is Cryptic
[0173] The 4.2kb fragment containing about 2.2 kb of the T1275
promoter fused to the GUS gene and the nos 3' was isolated by
digesting pTZ-T1275 with HindIl and EcoRI. The isolated fragment
was ligated into the pRD400 vector (Datla et al., 1992, Gene,
211:383-384) previously digested with HindIII and EcoRI and treated
with calf intestinal phosphatase. Transfer of the binary vector to
Agrobacterium tumefaciens and leaf disc transformation of N.
tabacum SRI were performed as described above. GUS activity was
examined in several organs of many independent transgenic lines.
GUS mRNA was also examined in the same organ by RNase protection
assay (Melton et al, 1984, Nucleic Acids Res. 121: 7035-7056) using
a probe that mapped the mRNA 5' end in both untransformed and
transgenic tissues. RNA was isolated from frozen-ground tissues
using the TRIZOL Reagent (Life Technologies) as described by the
manufacturer. For each assay 10-30 ug of total RNA was hybridized
to RNA probes described in FIG. 16(A). Assays were performed using
the RPAII kit (Ambion Calif.) as described by the manufacturer. The
protected fragments were separated on a 5% Long Ranger acrylamide
(J. J. Baker, N.J.) denaturing gel which was dried and exposed to
Kodak X-RP film.
[0174] RNase protection assays performed with RNA from leaves,
stem, root, developing seeds and flowers of transgenic tobacco
revealed a single protected fragment in all organs indicating a
single transcription start site that was the same in each organ,
whereas RNA from untransformed tobacco tissues did not reveal a
protected fragment (FIG. 16(B)). The insertion site, including 1200
bp downstream, was cloned from untransformed tobacco as a PCR
fragment and sequenced. A composite restriction map of the
insertion site was assembled as shown in FIG. 16(A). RNA probes
were prepared that spanned the entire region as shown in FIG.
16(A). RNase protection assays did not reveal transcripts from the
sense strand as summarized in Table 2. These data suggest that the
insertion site is transcriptionally silent in untransformed tobacco
and is activated by T-DNA insertion. The region upstream of the
insertion site is therefore another example of a plant cryptic
regulatory element.
2TABLE 2 Summary of the RNase Protection Assays of the insertion
site in untransformed tobacco. See FIG. 16 (A) for probe positions.
Probe Rnase Protection Assay result Looking for "sense" RNAs
(relative to the T1275 promoter) C8-EcoRI many bands. all in tRNA
(negative control) A10-HindIII no bands 2-21-HindIII no bands 1-4
SmaI many bands, all in tRNA 7-EcoRI faint bands, all in tRNA
[0175] Constitutive Activity of the T1275 Regulatory Element
[0176] For analysis of transient expression of GUS activity
mediated by biolistics (Sandford et al, 1983, Methods Enzymol, 217:
483-509), the XbaI--EcoRI fragment was subcloned in pUC19 and GUS
activity was detected by staining with X-Gluc as described above.
Leaf tissue of greenhouse-grown plants or cell suspension cultures
we re examined for the number of blue spots that stained. As shown
in Table 3, the T1275-GUS-nos gene was active in each of the
diverse species examined and can direct expression of a gene of
interest in all plant species tested. Leaf tissue of canola,
tobacco, soybean, alfalfa, pea and Arabidopsis, potato, Ginseng,
peach and cell suspensions of oat, corn, wheat and barley exhibited
GUS-positive blue spots after transient bombardment-mediated assays
and histochemical GUS activity staining. This suggests that the
T1275 regulatory element may be useful for directing gene
expression in both dicot and monocot plants.
3TABLE 3 Transient Expression of GUS Activity in Tissues of Diverse
Plant Species Tissue Source Species GUS Activity* Leaf Soybean +++
Alfalfa ++ Arabidopsis + Potato ++ Ginseng ++ Peach + Leaf disc
Tobacco ++ B. napus + Pea + Cell Cultures Oat + Corn + Wheat +
Barley ++ White spruce ++ *Numbers of blue spots: 1-10 (+), 10-100
++, 100-400 (+++)
[0177] For analysis of GUS expression in different organs, lines
derived from progeny of the above transgenic tobacco lines were
examined in detail. Table 4 shows the GUS specific activities in
one of these plants. It is expressed in leaf, stem, root,
developing seeds and the floral organs, sepals, petals, anthers,
pistils and ovaries at varying levels, confirming constitutive
expression. Introduction of the same vector into B. napus,
Arabidopsis, and alfalfa also revealed expression of GUS activity
in these organs (data not shown) indicating that constitutive
expression was not specific to tobacco. Examination of GUS mRNA in
the tobacco organs showed that the transcription start sites was
the same in each (FIG. 16(B)) and the level of mRNA was similar
except in flower buds where it was lower (Table 4).
4TABLE 4 GUS Specific Activity and Relative RNA Levels in the
Organs of Progeny of Transgenic Line T64 Relative GUS RNA GUS
Specific Activity Levels in T64 (picomol/MU/min/mg protein) Progeny
(grey Transformed Untransformed Organ scale units) Tobacco T64
Tobacco Leaf 1774 988.32 3.02 Stem 1820 826.48 7.58 Root 1636
4078.45 22.18 14 day post 1790 253.21 10.03 anthesis Seeds Flower -
buds 715 2.59 ND* Petals ND* 28.24 1.29 Anthers ND* 4.64 0.35
Pistils ND* 9.76 1.72 Sepals ND* 110.02 2.48 Ovary ND* 4.42 2.71
*Not Done
[0178] Identification of Regulatory Elements within the Full Length
T1275 Regulatory Element
[0179] An array of deletions of the full length regulatory region
of T1275 were prepared, as identified in FIGS. 13(B) and (C), for
further analysis of the cryptic regulatory element.
[0180] 5' deletions of the promoter (see FIGS. 13(B) and (C) and
analysis by transient expression using biolistics showed that the
promoter was active within a fragment 62 bp from the
transcriptional start site indicating that the core promoter has a
basal level of expression (see Table 5).
5TABLE 5 Transient GUS activity detected in soybean leaves by
staining with X-gluc after particle bombardment. Vectors
illustrated in FIGS. 13 (B) and (C). Genes GUS staining 1.
T1275-GUS-nos + 2. -1639-GUS-nos + 3. -1304-GUS-nos + 4.
-684-GUS-nos + 5. -394-GUS-nos + 6. -197-GUS-nos + 7. -62-GUS-nos +
8. -62(-tsr)-GUS-nos + 9. -12-GUS-nos - 10. +30-GUS-nos -
[0181] Deletion of a fragment containing the transcriptional start
site (see -62(-tsr)/GUS/nos in FIG. 14(B), Table 5) did not
eliminate expression, however deletions to -12 bp and further
(ie+30) did eliminate expression indicating that the region defined
by bp -62 to -12 (nucleotides 1992-2042 of SEQ ID NO:2) contained
the core promoter. DNA sequence searches did not reveal
conventional core promoter motifs within this region as are
typically found in plant genes, such as the TATA box.
[0182] A number of the 5' promoter deletion clones (FIGS. 13(B) and
(C)) were transferred into tobacco by Agrobacterium-mediated
transformation using the vector pRD400. Analysis of GUS specific
activity in leaves of transgenic plants (see Table 6) confirmed the
transient expression data down to the -197 fragment (i.e.
nucleotide 1857 SEQ ID NO:2).
6TABLE 6 GUS specific activities in leaves of greenhouse-grown
transgenic tobacco, SR1, transformed with the T1275-GUS-nos gene
fusion and 5' deletion clones (see FIG. 13 A). Mean .+-. SE(n) GUS
specific activities Genes pmoles MU/min/mg protein 1. T1275-GUS-nos
283 .+-. 171 (27) 2. -1639-GUS-nos 587 .+-. 188 (26) 3.
-1304-GUS-nos 632 .+-. 217 (10) 4. -684-GUS-nos nd* 5. -394-GUS-nos
1627 .+-. 340 (13) 6. -197-GUS-nos 475 .+-. 74 (27) *nd = not
determined
[0183] Histochemical analysis of organs sampled from the transgenic
plants indicated GUS expression in leaf, seeds and flowers.
[0184] To determine if enhancer elements exist, fragments -394 to
-62 (nucleotides 1660 to 1992 of SEQ Id NO:20) and -197 to -62
(nucleotides 1875 to 1992 of SEQ ID NO:2) were fused to the -46 35S
core promoter. Both fragments raised the expression of the core
promoter about 150 fold (FIG. 13(D), constructs DRA1-35S and
BST1-35S). Doubling of the -394 to -62 region (nucleotides 1660 to
1992 of SEQ ID NO:2) resulted in a 1.8 fold increase in GUS
activity when fused to T1275 core promoter (BSTI-GUS (-394-GUS) v.
BST2-GUS; FIG. 13(D)), a similar effect is observed when the -394
to -62 region is double and fused to the 35S core promoter
(BST1-35S v. BST2-35S). Doubling of the -197 to -62 fragment
(nucleotides 1875 to 1992 of SEQ ID NO:2) also produced increased
GUS activity when fused to the T1275 core promoter (DRA2-GUS).
[0185] The -197 to -62 fragment (nucleotides 1875 to 1992 of SEQ ID
NO:2; DRA1-35S), the -197 to -62 fragment in reverse orientation,
or inverted (DRA1R-35S), and a repeat of the -197 to -62 fragment
(DRA2-35S) were also fused with the 35S minimal promoter (FIG.
13(E) and used to transform Arabidopsis.
[0186] Arabidopsis plants with immature floral buds and few silques
were transformed with the above constructs by dipping the plant
into a solution containing Agrobacterium rumefaciens. 2.3 g/L MS.
5% (w/v) sucrose and 0.03% Silwet L-77 (Lehle Seeds, Round Rock,
Tex.) for 1-2 min. and allowing the plants to grow and set seed.
Seeds from mature plants were collected, dried at 25.degree. C.,
and sown on sterile media containing 40 .mu.g/mL kanamycin to
select transformants. Surviving plantlets were transferred to soil,
grown and seed collected.
[0187] Constructs comprising the -197 to -62 fragment (nucleotides
1875 to 1992 of SEQ ID NO:2) in regular or inverted orientation
exhibited increased transcriptional enhancer activity, over that of
the minimal promoter (FIG. 13(F). A further increase in activity
was observed when plants were transformed with constructs
comprising repeated regions of this regulatory element (FIG. 13(F).
Tissue staining of transformed plants expressing DRA1-35S indicated
that this construct was expressed constitutively as it was detected
in all tested organs, including flower, silque and seedling (FIG.
13(G)).
[0188] Activity of the T1275 Regulatory Element
[0189] Analysis of leaves of randomly-selected, greenhouse-grown
plants regenerated from culture revealed a wide range of GUS
specific activities (FIG. 14(A); T plants). Plants transformed with
pBI 121 (CLONETECH) which contains the 35S-GUS-nos gene yielded
comparable specific activity levels (FIG. 14(A); S plants).
Furthermore, the GUS protein levels detected by Western blotting
were similar between plants transformed with either gene when the
GUS specific activities were similar (FIG. 14(C)).
[0190] Generally, the level of GUS mRNA in the leaves as determined
by RNase protection (FIG. 14(B)) correlated with the GUS specific
activities, however, the level of GUS mRNA was about 60 fold (mean
of 13 measurements) lower in plants transformed with the
T1275-GUS-nos gene (FIG. 14(B)) when compared with plants
transformed with 35S-GUS-nos.
[0191] Since the levels of protein and the activity of extractable
protein were similar in plants transformed with T1275-GUS-nos or
35S-GUS-nos, yet the mRNA levels were dramatically different, these
results suggested the existence of a regulatory element downstream
of the transcriptional start site in the sequence of T1275-derived
transcript.
[0192] Post-transcriptional Regulatory Elements within T1275
[0193] An experiment was performed to determine the presence of a
post-transcriptional regulatory element within the T1275 leader
sequence. A portion of the sequence downstream from the
transcriptional initiation site was deleted in order to examine
whether this region may have an effect on translational efficiency
(determined by GUS extractable activity). mRNA stability or
transcription.
[0194] Deletion of the Nde1-Sma1 fragment ("N"; SEQ ID NO:3) from
the T1275-GUS-nos gene (FIG. 15; T175-N-GUS-nos: includes
nucleotides 2086-2224 of SEQ ID NO:2) resulted in at least about
46-fold reduction in the amount of GUS specific activity that could
be detected in leaves of transgenic tobacco cv Delgold (see Table
7). Similar results, of about at least a 40 fold reduction in GUS
activity due to the deletion of the Nde1-Sma1 fragment, were
observed in transgenic tobacco cv SRI and transgenic alfalfa (Table
7). Addition of the same fragment (Nde1-Sma1) to a 35S-GUS-nos gene
(FIG. 15; 35S+N-GUS-nos) construct increased the amount of GUS
specific activity by about 5-fold in tobacco, and by a much higher
amount in alfalfa (see Table 7).
7TABLE 7 GUS specific activity in leaves of greenhouse-grown
transgenic tobacco cv Delgold transformed with vectors designed to
assess the presence of cryptic regulatory sequences within the
transcribed sequence derived from the T1275 GUS gene fusion (see
FIG. 15). Mean .+-. SE(n). GUS specific activity pmoles MU/min/mg
protein Delgold Delgold Construct (1) (2) SRI Alfalfa 1.
T1275-GUS-nos 557 .+-. 493 .+-. 805 .+-. 187 .+-. 183 (21) 157 (25)
253 (22) 64 (24) 2. T1275-N-GUS-nos 12 .+-. 12 .+-. 6 .+-. 4 .+-. 3
(22) 3 (27) 2 (25) 0.5 (25) 3. 35S-GUS-nos 1848 .+-. 1347 .+-. 1383
.+-. 17 .+-. 692 (15) 415 (26) 263 (25) 11 (24) 4. 35S+N-GUS-nos
6990 .+-. 6624 .+-. 6192 .+-. 1428 .+-. 3148 (23) 2791 (26) 1923
(24) 601 (24)
[0195] A similar effect was noted in organs tested from transformed
tobacco (Table 8) and alfalfa plants (Table 9)
8TABLE 8 Expression of T1275-GUS-nos (+N) compared with T1275-(-N)-
GUS-nos (-N) in organs of transgenic tobacco. Mean .+-. SE (n = 5).
GUS specific Activity (pmol MU/min/mg/protein) Delgold SRI Organ +N
-N +N -N Leaf 1513 .+-. 222 35 .+-. 4 904 .+-. 138 4 .+-. 1 Flower
360 .+-. 47 38 .+-. 8 175 .+-. 44 28 .+-. 3 Seed 402 .+-. 65 69
.+-. 7 370 .+-. 87 33 .+-. 5
[0196]
9TABLE 9 Expression of T1275-GUS-nos, T1275-(-N)-GUS-nos,
35S-GUS-nos, 35S-GUS(+N)-GUS-nos in organs of transgenic alfalfa.
Mean .+-. SE (n = 5). GUS Specific Activity (pmol Mu/min/mg
protein) Construct Leaf Petiole Stem Flower T1275-GUS 756 .+-. 1126
.+-. 1366.7 .+-. 456.1 .+-. 73.6 72.7 260 160.9 T1275(-N)GUS 5.4
.+-. 1.4 7.6 .+-. 1.2 8.1 .+-. 2.0 7.25 .+-. 1.7 35S-GUS 67.5 .+-.
48.9 .+-. 56.8 .+-. 23.2 .+-. 50.3 23.2 28.7 7.3 355(+N)GUS 5545
.+-. 10791 .+-. 9931 .+-. 1039 .+-. 2015 6194 5496 476.7 Control
3.7 13.2 11.8 18.7
[0197] In transient expression assays using particle bombardment of
tobacco leaves, the Nde1-Sma1 fragment fused to the minimal -46 35S
promoter enhanced basal level of 35S promoter activity by about 80
fold (28.67.+-.2.91 v. 0.33.+-.0.33 relative units; No.blue
units/leaf).
[0198] SEQ ID NO:3 comprises nucleotides 2086 to 2224 of SEQ ID
NO:2. Nucleotides 1-141 of SEQ ID NO3: comprise nucleotides
obtained from the plant portion of T1275 (nucleotides 2086 to 2224
of SEQ ID NO:2). Nucleotides 142-183 of SEQ ID NO:3 comprise vector
sequence between the enhancer fragment and the GUS ATG. The GUS ATG
is located at nucleotides 186-188 of SEQ ID NO:3.
[0199] A shortened fragment of the NdeI-SmaI fragment (see SEQ ID
NO:4), referred to as ".DELTA.N", "dN", or "deltaN" and lacking the
out-of frame upstream ATG at nucleotide 2087-2089 of SEQ ID NO:2,
was also constructed and tested in a variety of species. .DELTA.N
was created by replacing the NdeI site (FIG. 18(A)) within the
leader sequence to a BglII site thereby eliminating the upstream
ATG at position 2086 of SEQ ID NO:2. A Kozak consensus sequence was
also constructed at the initiator MET codon and a Ncol site was
added to facilitate construction with other coding regions (see
FIG. 18(B)). Nucleotides 1-86 of SEQ ID NO:4 (i.e. .DELTA.N with
Kozack sequence) are derived from T1275 (nucleotides 2086-2170 of
SEQ ID NO:2). .DELTA.N also includes a Kozack sequence from
nucleotides 87 to 97 of SEQ ID NO:4, and nucleotides 98 to 126 of
SEQ ID NO:4 comprise the vector sequence between the enhancer
fragment and the GUS ATG. The GUS ATG is located at nucleotides
127-129 of SEQ ID NO:4).
[0200] Constructs comprising .DELTA.N, for example
T1275(.DELTA.N)-GUS-nos- , when introduced into tobacco yielded 5
fold greater levels of GUS activity in leaves of transgenic tobacco
(5291.+-.986 pmolMU/min/mg protein; (n=29) compared to plants
expressing T1275-GUS-nos (1115.+-.299 pmol MU/min/mg protein;
n=29).
[0201] Activity of Ndei-Sma1, N, and .DELTA.N in Other Species
[0202] In monocots, transient expression in corn callus indicated
that the NdeI-SmaI fragment (SEQ ID NO:3) or a shortened. NdeI-SmaI
fragment, .DELTA.N (SEQ ID NO:4), significantly increases GUS
expression driven by the 35 S promoter but not to the higher level
of expression generated in the presence of the ADH1 intron ("i";
FIG. 19 and Table 10).
10TABLE 10 Transient expression analysis of GUS activity in
bombarded corn calli. Luciferase activity was used to normalize the
data. Mean .+-. se (n = 5) Construct Ratio GUS:Luciferase activity
35S GUS-nos 7.4 .+-. 4 35S(+N)-GUS-nos 19 .+-. 5
35S(.DELTA.N)-GUS-nos 18 .+-. 10 35S-i-GUS-nos 66 .+-. 27
[0203] The functionality of the NdeI-SmaI fragment (SEQ ID NO:3)
was also determined in non-plant species. In conifers, for example
white spruce, transient bombardment of cell culture exhibited an
increase in expression (Table 11).
11TABLE 11 Expression of T1275-GUS-nos, T1275(-N)-GUS-nos,
35S-GUS-nos, 35S(+N)-GUS-nos in white spruce embryonal masses
following bombardment (n = 3). Average GUS expression per leaf
Construct (Number of blue spots) T1275-GUS-nos 72.67 .+-. 9.33
T1275(-N)-GUS-nos 21.33 .+-. 4.49 35S-GUS-nos 113.67 .+-. 17.32
35S(+N)-GUS-nos 126.33 .+-. 19.41* *average spot much greater in
size and strength.
[0204] In yeast, the presence of the NdeI-SmaI fragment (SEQ ID
NO:3) or .DELTA.N (SEQ DI NO:4) exhibited strong increase in
expression of the marker gene. A series of constructs comprising a
galactose inducible promoter P.sub.galI, various forms of the
Nde1-Sma1 fragment, and GUS (UidA) were made within the yeast
plasmid pYES2. A full length Nde1-Sma1 fragment N (pYENGUS),
.DELTA.N (containing a Kozak consensus sequence; pYEdNGUS), and
.DELTA.N without a Kozak consensus sequence (pYEdN.sup.MGUS; or
.DELTA.N.sup.M) were prepared (see FIG. 20, and SEQ ID NO:5).
[0205] Nucleotides 1-86 of SEQ ID NO:5 (.DELTA.N.sup.M) comprise a
portion of the enhancer regulatory region obtained from T1275
(nucleotide 2086-2170 of SEQ ID NO:2), while nucleotides 87-116
comprise a vector sequence between the enhancer fragment and the
GUS ATG which is located at nucleotides 117-119 of SEQ ID NO:5.
[0206] These constructs were tested in yeast strain INVSC I using
known transformation protocols (Agatep R. et al. 1998,
http://www.biomednet.com- /db/tto). The yeast were grown in
non-inducible medium comprising raffinose as a carbon source for 48
hr at 30.degree. C. and then transferred onto inducible medium
(galactose as a carbon source). Yeast cells were harvested after 4
hr post induction and GUS activity determined quantitatively. Up to
about a 12 fold increase in activity was observed with constructs
comprising AN. Constructs comprising .DELTA.N.sup.M exhibited even
higher levels of reporter activity. The results indicate that the
NdeI-SmaI fragment (SEQ ID NO:3), .DELTA.N (SEQ ID NO:4) and
.DELTA.N.sup.M (SEQ ID NO:5) are functional in yeast (Table
12).
12TABLE 12 Expression of pYEGUS, pYENGUS, pYEdNGUS, and
pYEdN.sup.MGUS (.DELTA.N, without a Kozak consensus sequence) in
transformed yeast (n = 5). Expt. 1 Expt. 2 Construct Activity
Activity pYES-GUS-nos 93 .+-. 15 407 .+-. 8 pYES(+N)-GUS-nos 753
.+-. 86 1771 .+-. 191 pYES(.DELTA.N)-GUS-nos 1119 .+-. 85 2129 .+-.
166 pYES(.DELTA.N.sup.M)-GUS-nos 1731 .+-. 45 6897 .+-. 536
[0207] Constructs containing .DELTA.N.sup.M (i.e. .DELTA.N lacking
the Kozack sequence; SEQ ID NO:5) were also tested in insect cells.
These constructs comprised the insect virus promoter ie2 (Theilmann
D. A and Stewart S., 1992, Virology 187: pp. 84-96) in the present
or absence of ANM and CAT (chloramphenicol acetyl-transferase) as
the reporter gene. The insect line, Ld652Y, derived from gypsy moth
(Lymantria dispar) was transiently transformed with the above
constructs using liposomes (Campbell M. J. 1995, Biotechniques 18:
pp. 1027-1032; Forsythe I. J. et al 1998, Virology 252: pp. 65-81).
Cells were harvested 48 hours after transformation and CAT activity
quanitatively measured using tritiated acetyl-CoA (Leahy P. et al.
1995 Biotechniques 19: pp. 894-898). The presence of the
translational enhancer was found to significantly modulate the
activity of the insect promoter-reporter gene construct in insect
cells.
[0208] Bacteria were transformed with either pBI221, comprising 35S
promoter and GUS, or 35S-N-GUS , comprising the full length
Nde1-Sma1 fragment (SEQ ID NO:3). Since uidA (GUS) is native to
E.coli, two uidA mutants, uidA1 and uidA2, that do not express
uidA, were used for these experiments (mutants obtained from E.coli
Genetic Center 335 Osborn Memorial Laboratories, Department of
Biology, Box 208104. Yale University, New Haven Conn. 06520-8104).
These bacteria were transformed using standard protocols, and
transformants were assessed by assaying GUS activity from a50 .mu.I
aliquot of an overnight culture. The "N" fragment (35s-N-GUS) was
observed to modulate the activity of the reporter gene in bacterial
cells.
[0209] These data are consistent with the presence of a
post-transcriptional regulatory sequence in the NdeI-SmaI
fragment.
[0210] The NdeI-SmaI Fragment Functions as a Transcriptional
Enhancer or mRNA Stability Determinant
[0211] The levels of mRNA were determined in leaves obtained from
tobacco plants transformed with either T1275-GUS-nos,
T1275-N-GUS-nos, 35S-GUS-nos, or 35S+N-GUS-nos (FIGS. 17(A) and
(B)). Relative RNA levels were determined by ribonuclease
protection assay (Ambion RPAII Kit) in the presence of
.alpha.-.sup.32P-CTP labeled in vitro transcribed probe and
autoradiographic quantification using Kodak Digital Science ID
Image Analysis Software. Hybridization conditions used during RNase
protection assay were overnight at 42-45 degrees in 80% formamide,
100 mM sodium citrate pH 6.4, 300 mM sodium acetate pH 6.4, 1 mM
EDTA.
[0212] The levels of mRNA examined from transgenic tobacco plants
transformed with either T1275-GUS-nos, T1275-N-GUS-nos,
35S-GUS-nos, or 35S+N-GUS-nos, were higher in transgenic plants
comprising the NdeI-SmaI fragment under the control of the T1275
promoter but lower in those under the control of the 35S promoter,
than in plants comprising constructs that lack this region (FIGS.
17(A) and (B)). This indicates that this region functions by either
modulating transcriptional rates, or the stability of the
transcript, or both.
[0213] The NdeI-SmaI Fragment Functions as a Translational
Enhancer
[0214] Analysis were performed in order to determine whether the
NdeI-SmaI region (SEQ ID NO:3) functions post-transcriptionially.
The GUS specific activity:relative RNA level was determined from
the GUS specific activity measurements, and relative RNA levels in
greenhouse grown transgenic plants (FIG. 17(C)). The ratio of GUS
specific activity to relative RNA level in individual transgenic
tobacco plants comprising the NdeI-SmaI fragment is higher than in
plants that do not comprise this region (FIG. 17(C)). Similar
results are obtained when the data are averaged, indicating, an
eight fold reduction in GUS activity per RNA. Similarly, an
increase, by an average of six fold, in GUS specific activity is
observed when the region is added within the 35S untranslated
region (FIG. 17(C)). The GUS specific activity:relative RNA levels
are similar in constructs containing the NdeI-SmaI fragment
(T1275-GUS-nos and 35S+N-GUS-nos). These results indicate that the
NdeI-SmaI fragment (seq idno:3) modulates gene expression
post-transcriptionally.
[0215] Further experiments, involving in vitro translation, suggest
that this region is a novel translational enhancer. For these
experiments, fragments, from approximately 3' of the
transcriptional start site to the end of the terminator, were
excised from the constructs depicted in FIG. 15 using appropriate
restriction endonucleases and ligated to pGEM4Z at an approximately
similar distance from the transcriptional start site used by the
prokaryotic T7 RNA polymerase. Another construct containing the AMV
enhancer in the 5' UTR of a GUS-nos fusion was similarly prepared.
This AMV-GUS-nos construct was created by restriction endonuclease
digestion of an AMV-GUS-nos fusion, with BglII and EcoRI, from
pBI525 (Datla et al., 1993, Plant Science 94: 139-149) and ligation
with pGEM4Z (Promega) digested with BamHI and EcoRI. Transcripts
were prepared in vitro in the presence of m.sup.7G(5')ppp(5')G Cap
Analog (Ambion). Transcripts were translated in vitro in Wheat Germ
Extract (Promega) in the presence of 35S-Methionine and fold
enhancement calculated from TCA precipitable cpms.
[0216] Translation of transcripts in vitro demonstrate an increase
in translational efficiency of RNA containing the NdeI to SmaI
fragment (see Table 13).
13TABLE 13 In vitro translation of mRNA obtained from transgenic
tobacco plants transformed with vectors with or without a NdeI-SmaI
fragment obtained from the T1275 GUS gene fusion (see FIG. 15)
using wheat germ extract. in vitro translation in vitro transcript
fold enhancement T1275-GUS-nos 3.7 T1275-N-GUS-nos 1.0 AMV-GUS-nos
1.9
[0217] The levels of protein produced using mRNAs comprising the
NdeI-SmaI fragment are also greater than those produced using the
known translational enhancer of Alfalfa Mosaic Virus RNA4 (Jobling
S. A. and Gehrke L. 1987, Nature, vol 325 pp. 622-625; Datla R. S.
S. et al 1993 Plant Sci. vol 94, pp. 139-149). These results
indicate that this region functions post-transcriptionally, as a
translational enhancer.
[0218] All scientific publications and patent documents are
incorporated herein by reference.
[0219] The present invention has been described with regard to
preferred embodiments. However, it will be obvious to persons
skilled in the art that a number of variations and modifications
can be made without departing from the scope of the invention as
described in the following claims.
Sequence CWU 1
1
5 1 1070 DNA Nicotiana tabacum 1 tctagacttg tcttttcttt acataatcct
cttcttcttt tttttgttag tttcttctgt 60 tttatccaaa aaacgaatta
ttgattaaga aatacaccag acaagttttt tacttctttt 120 tctttttttt
tttgtggtaa aaaattacac ctggacaagt ttatcacgaa aatgaaaatt 180
gctatttaag ggatgtagtt ccggactatt tggaagataa gtgttaacaa aataaataaa
240 taaaaagttt atacagttag atctctctat aacagtcatc cttatttata
acaatacttt 300 actataaccg tcaaatttat tttgaaacaa aattttcatg
ttatgttact ataacagtat 360 tttattatag caaccaaaaa atatcgaaac
agatacgatt gttatagagc gatttgattg 420 tatcattatc cacatatttt
cgtaagccca attactcctc ctacgtacga tgaaagtaaa 480 ccaatttaaa
gttgcaaaaa tccaatagat ttcaatactt cttcaactgg cgttatgtta 540
ggtaatgact cctttttaac ttttcatctt taatttgaag tttctttcat taaaagaaag
600 tttctagaag agaagtgttt taacacttct agctctacta ttatctgtgt
ttctagaaga 660 aaaatagaaa atgtgtccac ctcaaaaaca actaaaggtg
ggcaaatctc cacctattta 720 ttttattttg gattaattaa gatatagtaa
agatcagtta taaacggagt tttgagttga 780 tacagtgaat tttaagatgt
gtaccgattt aactttattt acatttatgt ttcgcacata 840 taagaagtcc
gatttggaaa tactagattt tgtcaatcag gcaattcatg tggttgaaga 900
atttaagtta tatacaatga tgatataaag aatttttata ctattagtgc aaattaatcg
960 attactaaaa attattattc tattaattta tgctatcgtg cctccccaac
ccgtcgaccg 1020 cggtacccgg tggtcagtcc cttatgttac gtcctgtaga
aaccccaacc 1070 2 2224 DNA Nicotiana tabacum 2 tctagactta
cagaaagtct ctaacacgtg agggaatgat ccctttcctt acctccctgt 60
agagatattg gcttttcaac aactagtaca taaatatgcg actttgaccg tgtatcccca
120 gtcaaaaggg aacttcaccc tcctagttct ttatttccaa catacatggg
gagtaatgct 180 aaatttacat agaagaataa taaaatgaac tgtaactaat
gatgtactgt tccaaagaga 240 tgaggacgtc aacatattta ttccttcagc
ccttttcaga ataataccat aagtagaaga 300 aatggcacat aaaatgaagt
cctcggcaag tcaaatgtaa atctgaaccc acccagctaa 360 cccagtgaac
tcaactttcc tggatagatc agcactcctt catgacattg catgccttct 420
ctttaaagag ccgcttgatc tctgaaaacc aaatgaatct ccacagagag atttcgagct
480 ccatgagacg ccttttggtt cttgatttac taaacctata aaaatgaaag
gaagtaggac 540 aactgcattt tgccgcttaa gatgcttcgg cgctttgtga
attttaagtc atgagaaagt 600 acaatgttgg aatctcacat tagaacaatg
tatttgtaat aacctaggaa agcaaagcta 660 gaagggaggt gcagctaaat
cttcttctac cttgttatcc ttgcatttct tgaggaggag 720 gaactgtcct
cgcaggtgca aaatctgcag tcgcccaaaa ggatattcag aagtatatta 780
caacatgttt aatggttaac caagtgaaag atcaaaatag tcattagaac aaaatgcgtg
840 ctcagagcgt atctactagt tcatcaaccc agtacacatc tctgaatttc
atctcttgcc 900 gttgaactaa gtcaattggt caaagacgca taacatgaga
gacactcata aaacggctga 960 ataacatgca gaagacgtca tgcgccttag
gtctcattat gcatgagatt attagttata 1020 tgctccttca gtttgactag
aaatgaaaaa tcagttaagc ctgtaacgaa atgataacct 1080 gcttcaagaa
gattagacta tttttcataa aatatgcagt gccgtgaaat agatacttaa 1140
tcttaggcag gaaaaatctt ctattgggcc ataataagaa ctaccaatta gaaaggaggt
1200 agaaagctcc gatactgtta tgaaggccat tctaagtgct gatgtgaatt
tcccaataca 1260 aaatgacaac aaaaacaaaa gcctcaatcc taagctagtt
ggggtcgcta tataaatcct 1320 cgacatccat ttaactccac ttggactcct
ttctttccaa tattttaata ttgttagatt 1380 aatcataaaa ttgcttagct
ttctactggc acttaaccta ctgcaaccct cctcttctgg 1440 gattccaaca
caaacaacta agaggaattt gaaaaaaaga aagcaaatgt gagaagagac 1500
aaaatgtaca atgatacctc ttcttgcagc aaaggaggca ggttctctgc tgagacaagg
1560 ttctctattt cctgcaagac cttcgtatct tttattcgag accatgtatg
tggaggtaac 1620 gccagcaata gtgctgtcag cacatcgttg cttgcagggg
atcttctgca agcatctcta 1680 tttcctgaag gtctaacctc gaagatttaa
gatttaatta cgtttataat tacaaaattg 1740 attctagtat ctttaattta
atgcttatac attattaatt aatttagtac tttcaatttg 1800 ttttcagaaa
ttattttact attttttata aaataaaagg gagaaaatgg ctatttaaat 1860
actagcctat tttatttcaa ttttagctta aaatcagccc caattagccc caatttcaaa
1920 ttcaaatggt ccagcccaat tcctaaataa cccaccccta acccgcccgg
tttccccttt 1980 tgatccaggc cgttgatcat tttgatcaac gcccagaatt
tccccttttc cttttttaat 2040 tcccaaacac ccctaactct atcccatttc
tcaccaaccg ccacatatga atcctcttat 2100 ctctcaaact ctctcgaacc
ttcccctaac cctagcagcc tctcatcatc ctcacctcaa 2160 aacccaccgg
aatacatggc ttctcaagcc gtggaaacct tatactcacc tccctttgct 2220 ctta
2224 3 188 DNA Artificial Sequence Description of Artificial
SequenceNdeI-SmaI fragment of T1275 3 catatgaatc ctcttatctc
tcaaactctc tcgaaccttc ccctaaccct agcagcctct 60 catcatcctc
acctcaaaac ccaccggaat acatggcttc tcaagccgtg gaaaccttat 120
actcacctcc ctttgctctt acagtactcg gccgtcgacc gcggtacccg ggtggtcagt
180 cccttatg 188 4 129 DNA delta N, with Kozack sequence 4
agatctatcc tcttatctct caaactctct cgaaccttcc cctaacccta gcagcctctc
60 atcatcctca cctcaaaacc caccggccac catggcctct agaggacccc
gggtggtcag 120 tcccttatg 129 5 119 DNA delta N, without Kozak
sequence 5 agatctatcc tcttatctct caaactctct cgaaccttcc cctaacccta
gcagcctctc 60 atcatcctca cctcaaaacc caccggtcta gaggatcccc
gggtggtcag tcccttatg 119
* * * * *
References