CA2246892A1 - Cryptic regulatory elements in plants - Google Patents

Abstract

T-DNA tagging with a promoterless .beta.-glucuronidase (GUS) gene generated transgenic Nicotiana tabacum plants 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.

Description

CRYPTIC REGULATORY ELEMI!:NTS IN PLANTS
Field of Invention This invention relates to cryptic regulator~~ elements within plants.
Background and Prior Art 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, andl 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 unselect~i plant promoters residing at their natural location within the chromosome, and the coding sequence of a mark~:r gene located on the inserted T-DNA (Fobert et al., 1991, Plant Mol. Biol. 17, 837-851).
It has generally been assumed that activation of promoterless or enhancerless marker genes result from T-DNA irvsertions within or immediately adjacent to genes. The recent isolation of severa:~ 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; Yanof<,~ky 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.
Inactive regulatory sequences that are bur ied 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 rave been called "cryptic promoters" (Al-Shawi et al., 1991, Mol. Cedl. 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 int:rons of genes, such as those encoding for yeast actin (Irniger et al., 1992, Nu~~leic Acids Res. 20, 4733-4739), and a mammalian melanoma-associated amtigen (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 regi~~n was assimilated into the exon-intron structure of the present-day gene (Irndger et al., 1992, Nucleic Acids Res. 20, 4733-4739). A cryptic promoter leas 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, 5336-5340. This cryptic promoter is responsible for activation of a N-myc:2, a functional processed gene which arose from retropositon of N-myc transcript (Fourel et al. , 1992, Mol.
Cell. Biol. 12, 5336-534.4). These types of regulatory sequences have not yet been isolated from plants.
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 rnRNA, 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 1~'icotiana 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 (Bone et al., 199, Plant J. 6, 513-523), or by repression of trans~;,ription, as for the 5' UTR of the pollen-specific LAT59 gene from tomato (Curie and McCor~rnick, 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.
The present invention discloses transgenir plants generated by tagging with a promoterless GUS ((3-glucuronidase) T-DPJA 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.
S~xnmary of Invention This invention relates to cryptic regulator: elements within plants.
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.
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 1.0 kb of 'the 5' flanking region did not alter GUS expression, as compared to the entire 1.65 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 ().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.
A second transgenic tobacco plant, T1275, contained a 4.38 kb EcoRIIXbaI 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 analysi;~, 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 by 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 DraI fragment, and a at least one post transcriptional regulatory element located within a NdeI-SmaI fragment.
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 comp rise 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.
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 This invention is also directed to a cryptic. regulatory element comprising a nucleic acid fragment bounded by F,'coRI-SmaI restriction sites defined by the restriction map of Figure 2 (B). Furthermore, this invention relates to a cryptic regulatory element comprising; an XbaI - SmaI fragment, of the restriction map of Figure 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 restrintion map of Figure 2 (B), wherein the fragment is of about 500 bp. This imrention also is directed to a cryptic regulatory element comprising an XbaI and SnaBI fragment, as defined by the restriction map of Figure 2 (B), wherein the fragment is of about 1.5 kb, or a cryptic regulatory element comprising a Hiru~III and SnaBI fragment, defined by the restriction map of Figure 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 Figure 2, wherein the fragment is of about 2 kb.
This invention also embraces a regulatory element characterized in that it is substantially homologous with the sequence ~iefmed by SEQ ID N0:2.
This invention is also directed to a cryptic regulatory element that comprises at least an 18 by contiguous sequence of SEQ ID N~0:2. Furthermore, this regulatory element functions in diverse plant species when introduced on a cloning vector. This invention also relates to a c:himeric gene construct comprising a DNA of interest for which constitutive expression is desired, and a constitutive regulatory element, comprising at least an 18 by contiguous sequence of SEQ ID NO: 2.
This invention also embraces cryptic regulatory elements comprising an XbaI - SmaI fragment, an Xbal - Ndel fragment, an SphI - SmaI fragment, a PstI - SmaI fragment, an SspI - SmaI fragment, a BstYI - SmaI fragment, a DraI - SmaI fragment, a NdeI-SmaI fragment, a XbaI-BstYI fragment, or a BstYI-DraI fragment as defined by Figure 13(C).
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 -'j_ transgenic plants containing chimeric gene constructs, or cloning vectors comprising cryptic plant regulatory elements.
This invention further relates to any trans;;enic plant containing a cryptic regulatory element, having a DNA sequence substantially homologous to SEQ ID NO: 1, or SEQ ID N0:2, operatively linked to a DNA region that is transcribed into RNA.
Also included in the present invention a method of conferring expression of a gene in a plant, 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 a plant 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 in to a plant.
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 N0:2. This invention also relates to the above method wherein the nucleic acid comprises at least a 19 by contiguous sequence of SEQ ID NO:1, or the nucleic acid comprises apt least an 18 by contiguous sequence of SEQ ID N0:2.
.* ,. ., _g_ 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, or analogue thereof, as shown in SEQ ID NO: 2.
This invention also relates to a cloning 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 I)NA sequence, or analogue thereof, as shown in SEQ ID NO: 2 and a gene encoding a protein.
This invention also includes a plant cell which has been transformed with a cloning vector as described above, and to a transgeluc plant containing a cloning vector as described above, operatively linked to a gene encoding a protein.
Brief Description of the Drawings Figure 1 depicts the fluorogenic analyses of GUS expression in the plant T218. Each bar represents the average t 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 (S1) 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.

Figure 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. Figure 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 pBINl9 polylinker to create pT218-1. Fragments truncated at the XbaI, SnaE~I and XbaI sites were also subcloned to create pT218-2, pT218-3 and pT2lf;-4. Figure 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), SmaI (M), Sstl (S).
Figure 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 (~-~) and (Fig. 3b) plants transformed with vectors pT218-1 (O-O), pT218-2 (0-0), pT218-3 (0-~ and pT218-4 (0-O) which are illustrated in Figure 2. The 2 day delay in the pesak of GUS activity during seed development, seen with the pT218-2 transformant, likely reflects greenhouse variation conditions.
Figure 4 shows GUS activity in 12 dpa seeds of independent transformants produced with vectors pT218-1 (O), pT218-2 (~), pT218-3 (~
and pT218-4 (0). The solid markers indicate the plants shown in Figure 3 (b) and the arrows indicate the average values for plants transformed with pT218-1 or pT218-2.
Figure 5 shows the mapping of the T218 GUS fusion termini and expression of the region surrounding the insertion site in untransformed plants.
Figure 5 (A) shows the mapping of the GUS mR:(~A termini in plant T218.
The antisense RNA probe from subclone #4 (Figure 2) was used for hybridization with total RNA of tissues from untransformed plants (10 ~cg) and from plant T218 (30 ~,g). Arrowheads indicate the anticipated position of protected fragments if transcripts were initiated at the same sites as the GUS fusion. Figure 5 (B) shows the results of au RNase protection assay using the antisense (relative to the orientation of the GtlS coding region) RNA
probe from subclone a (see Figure 7) against 30 wg total RNA of tissues from untransformed plants. The abbreviations used ar~~ as follows: P, untreated RNA probe; -, control assay using the probe and tRNA only; L, leaves from untransformed plants; 8, 10, 12, seeds from untr;~nsformed plants at 8, 10, and 12 dpa, respectively; T10, seeds of plant T218 at 10 dpa; +, control hybridization against unlabelled in vitro-synthesi~:ed sense RNA from subclone c (panel a) or subclone a (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.
Figure 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 by 993. This site on pT218 is immediately followed by a 12 by 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 n-.iinor transeriptional 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 CART
boxes are marked with boxes. The location of direct (1-5) and indirect (6-8) repeats are indicated by arrows.
Figure 7 shows the base composition of rf:gion 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 ~~robes (a-e) used in RNase protection assays are indicated beneath the graph , Figure 8 shows the Southern blot analyse; of the insertion site in Nicotiana species. DNA from N. tomentosiformis (N torn), N. sylvestris (N
syl), and N. tabacum (N tab) were digested with HindIII (H), XbaI (X) and EcoRI (E) and hybridized using probe #2 (Figure 2). Lambda HindIII markers (kb) are indicated.
Figure 9 shows the AT content of 5' non-coding regions of plant genes.
A program was written in PASCAL to scan Genl3ank release 75.0 and to calculate the AT contents of the S' 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 + l to +200 were compared.
Shorter sequences were also accepted if they wens at least 190 by 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.
Figure 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;I, immature embryos (fj, mature embryos (g), and seed cross-sections (h).
Figure 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, days post anthesis (S2).
Figure 12 shows the restriction map of the cryptic regulatory element of pT1275. Figure 12 (A) shows the plant DNA fused with GUS. Figure 12 (B) shows the restriction map of the plant DNA. The arrow indicates the GUS
mRNA start site within the cryptic regulatory region.
Figure 13 shows deletion constructs of thE; T1275 regulatory element.
Figure 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 pT 1275 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.
Figure 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 Figure 13 (A)). Figure 13 (C) shows the restriction map of the plant DNA of pT1275 upstream from the GUS insertion site.
Figure 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-Gl:lS-nos (S plants). Figure 14 (A) shows the levels of GUS expression in leave; from randomly selected plants containing either T1275-GUS-nos (left-hand side) or 35S-GUS-nos (right-hand side). Figure 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). Figure 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; untrar~sformed control (SR1) and GUS produced in E. coli (Ec).
Figure 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.
Figure 16 shows the region surrounding tlae insertion site in untransformed plants, positions of various probe, used for RNase protection assays, and results of the RNase protection assay. Figure 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 BstYl site, not the IP; **: probe 7 included 600bp of the T1275 plant sequence and 400 by of the GUS gene). Figure 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-CiUS-nos (10 pg RNA) and untransformed tobacco (30 pg RNA). Undigested prohe (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 T12'75-GUS-nos or between about 446 to +169 of untransformed tobacco. The protected fragment in transformed plants is about 596 by (upper arrowhead) and, if present, accummlated transcripts initiated at this site in untransformed plants are predicted to protect a iFragment of about 169 by (lower arrowhead). Upper band in RNA-containing lanes was added to samples to indicate loss of sample during assay.

Figure 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 71275-GUS-nos, or 35S-GUS-nos constructs, with or without the Ndel Smal fragment (see Figure 15).
Figure 17 (A) shows the level of accumulated GI1S mRNA measured by RlVase protection assay and densitometry of autoradiogr;~ms in leaves from the same randomly selected plants containing either 71275-GUS-nos, 71275-N-GUS-nos. Figure 17 (B) shows the level of accumulat~:d GUS mRIVA measured by RlVase protection for 35S-GUS-nos or 35S+N-GUS-nos. Figure 17 (C) shows the ratio between GUS specific activity and mRNA levels in leaves of individual, regenerated, greenhouse-grown traps~;enic plants containing T 1275-GUS-nos, 71275-N-GUS-nos, 35S-GUS-nos, or 35S+N-GUS-nos constructs.
Detailed Description of the Preferred Embodiments 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.
T-DNA tagging with a promoterless (3-glcGCUronidase (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 devel~~ping seed coats, 7218, and another plant that expressed GUS in all organs, 71275 (see co-pending patent applications US serial No. 08/593121 and PCT/C".A97/00064, both of which are incorporated by reference).

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:
~ 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 ~~f the 5' flanking tobacco DNA (see Figure 2). This region spans from nucleotide 1 to nucleotide 467 of SEQ ID NO: 1.
~ In T1275, a regulatory region was identif.ed 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 Figure 13), negative regulatory elements between XbaI and BstYI, a transcriptional enhancer between BstYI and DraI, and a translational enhancer regulatory element between the .NdeI-SmaI sites.
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.
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.
The term cryptic regulatory element refer, 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 XXX but are capable of being functional when positioned adjacent to a gene.
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 ~Nhich are capable of mediating organ specificity, or controlling developmental o~~ temporal gene activation.
Furthermore, "regulatory element" includes pronnoter 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. "Regulatory elements" ass 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 RIVA 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 yromoter 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 trans~~ribed 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.
An example of a cryptic regulatory element of the present invention, which is not to be considered limiting in any mariner, is an organ-specific, and temporally-specific element obtained from plant '.0218. 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 Figure 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:l. 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 1-46"7, or 1-993, of SEQ ID NO:1.
Furthermore, the seed-coat specific nucleotide sequence may be defined as comprising at least a 19 by fragment of nucleotides 1-467, or 1-993 as defined within SEQ ID NO:1.

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 loc,~lized between XbaI - SmaI, as identified by the restriction map of Figure 12 (B) or a fragment thereof.
Furthermore, the constitutive regulatory element may be defined as a nucleic acid fragment localized between XbaI - SmaI as i~dentifred by the restriction map of Figure 13 (C) or a fragment thereof. The: constitutive cryptic regulatory element may also be defined by a nucleotide sequence comprising at least an 18 by fragment of the regulatory region defined in SEQ ID N0:2, or by a nucleic acid comprising from about 80% similarity to the nucleotide sequence of SEQ ID N0:2.
Another cryptic regulatory element of the present invention includes, but is not limited to, a post-transcriptional or trar~slational enhancer regulatory element localized between NdeI - SmaI (see Figure 15), or the post-transcriptional or translational enhancer regulatory element may comprise the nucleotide sequence as defined by nucleotides 2086-2224 of SEQ ID N0:2 or an analog thereof, or the element may comprise 80 % similarity to the nucleotide sequence of nucleotides 2086-2224 of SEQ ID N0:2.
Furthermore, other regulatory elements of the present invention include negative regulatory elements (for example located within an XbaI-BstYI
fragment as defined by Figure 13 (C)), a transcriptional enhancer localized within the BstYI-DraI fragment of Figure 13 (C) , a core promoter element located within the DraI-NdeI fragment of Figure 13 (C), or a regulatory element or post-transcriptional element downstres~m of the transcriptional start site.
An "analogue" of the above identified cryptic regulatory elements includes any substitution, deletion, or additions t~~ 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.
There are several types of regulatory elements, including those that are developmentally regulated, inducible and constiW tive. 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.
An inducible regulatory element is one th,~t 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.

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:

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 (lVlandel et al, 1995 Plant Mol. Biol. 29: 995-1004).
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.
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 tc~ result in the expression of said exogenous gene.
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 S' AATAAA-3' although variations are not uncommon.

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 .
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 strucaural 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.
To aid in identification of txansfonmed 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 ((3-glucuronidase), or luminescence, such as luciferase are useful.
Also considered part of this invention are transgenic plants containing the chimeric gene construct comprising a regulatory element of the present invention. Methods of regenerating whole plants from plant cells are known in the art. In general, transformed plant cells are cultured in an appropriate medium, which may contain selectivf; agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation c:an be encouraged by employing the appropriate plant hormones in acc~crdance 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.
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. :fn Plant Metabolism, 2d Ed.
DT. Dermis, DH Turpin, DD Lefebrve, DB Lay:aell (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). The present invention further includes a suitable vector comprising the chimeric gene construct.
The DNA sequences of the present invention thus include the DNA
sequences of SEQ ID NO: 1 and 2, the regulatory regions and fragments thereof, as well as analogues of, or nucleic acid ~;equences comprising about 80% similarity with the nucleic acids as defined in SEQ ID NO's: 1 and 2.
Analogues (as defined above), include those DN~~ sequences which hybridize under stringent hybridization conditions (see Maliiatis et al. , in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, p.
387-389) to the DNA sequence of SEQ ID NO: 1 or 2, provided that said sequences maintain at least one regulatory property of the activity of the regulatory element as defined herein.

An example of one such stringent hybridi;~ation conditions may be hybridization in 4XSSC at 65 °C, followed by washing in O.1XSSC at 65 °C for an hour. Alternatively an exemplary stringent hybridization condition could be in 50% formamide, 4XSSC at 42°C. Analogues also include those DNA
sequences which hybridize to the sequences of SEQ ID NO: 1 or 2 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 4XSSC at 50°C or with 30-40 % formamide at 42 ° C .
There are several lines of evidence that suggest that the seed coat-specific expression of GUS activity in the plant T'218 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.
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.
Similarly, results indicate that the constih~tive 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 Figure 16, Table 2). RNase protection assays were performed using RNA from organs of untransformed tobacco and probes that spanned the T 1275 sequence from about -2055 by to +1200 by 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 (Figure 16; see also PCT C.'A97/00064, which is incorporated by reference). Furthermore, GenB~ink 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 -141 E. and -1308, nucleotides 636-746 of SEQ ID N0:2; and between about -541 and -395, nucleotides 1513-1659 of SEQ ID N0:2 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 activatf;d a cryptic promoter.
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, soybean, alfalfa, pea, arabidopsis, corn, wheat and barley (Table 3), indicating that this regulatory element is useful for directing gene expression in both dicot and monocot plants.
The transcriptional start site was delimited by RNase protection assay to a single position about 220 by 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 TIC A+, A C/A CIA A/C/T A A
A/T). Sequence similarity is not detected about 30 by 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).
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) 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 (-1305-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 (-1!~7-GUS-nos; Table 6).
5' deletions of the promoter (see Figures l~s(A) and (B) and analysis by transient expression using biolistics showed that the promoter was active within a fragment 62 by from the transcriptional start site indicating that the core promoter has a basal level of expression (see Table 5). Deleaion of a fragment containing the transcriptional start site (see -62(-tsr)/GUS/no~; in Figure 13 (B); Table 5) did not eliminate expression, however deletions to -12 by and further (i.e. +30) did eliminate expression indicating that the region defined by -(62-12) by (nucleotides 1992-2042 of SEQ ID N0:2) contained the core promoter. DNA
sequence searches did not reveal conventional core promoter motifs found in plant genes such as the TATA box.

A number of the 5' promoter deletion clonea (Figure 13 (A)) 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 (nucleotides 1857- of SEQ ID N0:2). Histochemical analysis of organs sampled from the transgenic plants indicated GUS expression in leaf, seeds and flowers as a minimum as not all the organs were analyzed.
A comparison of GUS specific activities in the leaves of transgenic tobacco SR1 transformed with the T1275-GUS-nos gene and the 355-GUS-nos genes revealed a similar range of values (Figure. 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 (Figure. 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 (Figure 14(B) suggesting the existence of a post-transcriptional regulatory element in the mRNA leader sequence.
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 leaves. Deletion of the NdeI-SmaI fragment from the T1275-GUS-nos gene (Figure 15) resulted in a 46-fold reduction in the amount of GUS specific activity that could be detected in leaves of transgenic tobacco cv Delgold (see Tablf; 7). Addition of the same fragment to a 355-GUS-nos gene (Figure 15) construct increased the amount of GUS specific activity by 4-fold (see Table 7). The; data is consistent with the presence of a post-transcriptional regulatory element in this fragment.
The Ndel Smal regulatory elements situated downstream of the transcriptional start site functions both at a transcriptional, and post-transcriptional level. The levels of rnRNA examined from transgenic tobacco plants transformed with either T1275-GUS-nos, T1275-N-GUS-nos, 35S-GUS-nos, or 35S+N-GUS-nos, are higher in transgenic ~alants comprising the Ndel Smal fragment under the control of the T1275 promoter but lower in those under control of the 35S promoter, than in plants comprising constructs that lack this region (Figure 17 (A)). This indicates that this region functions by either modulating transcriptional rates, or the stability of the transcript, or both.
The Ndel Smal region also functions post-~.ranscriptionally. The ratio of GUS specific activity to relative RNA level in individual transgenic tobacco plants that lack the Ndel Smal fragment is lower, and when averaged indicates an eight fold reduction in GUS activity per RNA, than in plants comprising this region (Figure 17 (B)). Similarly, an increase, by an average of six fold, in GUS
specific activity is observed when the Ndel Smal region is added within the untranslated region (Figure 17 (B)). The GUS specific activity:relative RNA
levels are similar in constructs containing the Nde~! Smal fragment (T1275-GUS-nos and 35S+N-GUS-nosy. These results indicate that the Ndel Smal 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 efflc iency of RNA containing the Ndel to Smal fragment (see Table 8). Furthermorf;, the levels of protein produced using mRNAs comprising the Ndel-Smal fragment are greater than those produced using the known translational enhancer of Alfalfa Mosaic Virus RNA4 (refJ. These results indicate that this region functions post-transcriptionally, as a translational enhancer.
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 promoterles;~ marker gene activation in plants. It is interesting to note that transcription~~l GUS fusions in Arabidopsis occur at much greater frequencies (54%) than translational fusions (1.6% , Kertbundit et al., 1991, Proc. Natl. Acad. Sci. L'SA 88, 5212-5216). The possibility that cryptic promoters may account for some fusions was recognized by Lindsey et al. (1993, Transgenic Res. 2, 33-f7).
The cryptic regulatory elements of the present invention may be used to control the expression of any given gene within a plant. Furthermore, the cryptic 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. Some examples of such uses, which are not to be considered limiting, include:
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.
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.
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.
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.

5. Expression of genes in seed coats ~~oding for insecticidal proteins such as a-amylase inhibitor or protease inhibitor.
6. Partitioning of seed metabolites such as glucosinolates into seed coats for nematode resistance.
7. Nucleotide fragments of the regulatory region of at least 19 by as a probe in order to identify analogous regions within other plants etc.
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 Ca:MV promoter have been duplicated or combil~ed with other promoter fragments to produce chimeric promoters with desired properties (e.g. U.S. 5,4~~1,288, 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.
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.
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
Transfer of binary constructs to Agrobact~~rium and leaf disc transformation of Nicotiana tabacum SRl were performed as described by Fobert et al. (1991, Plant Mol. Biol. 17, 837-851'.). Plant tissue was maintained on 100 ~,g/ml kanamycin sulfate (Sigma) throughout in vitro culture.
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 transfonnants in a range of plant organs, T1275 was selected which exhibited high level, constitutive expression of GUS.
Characterization of a Seed Coat-Specific GUS Fusion - T218 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 i nitial 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-lei 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 F 1 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°C.
Tissues analyzed by histological assay were at the same developmental stages as those listed above. Different hand-cut ~;ections 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).
Tissue-specific patterns of GUS expression were only found in seeds.
For instance, GUS activity in plant T218 (Figure 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 tissuf~ analyzed which included leaf, stem, root, anther, ovary, petal and sepal (Figure: 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).
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 fu~~ions transmitted to seeds maternally and not by pollen. As shown in Tablf: 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 Nlol. Biol. 14, 633-635) which is active in both embryo and endosperm (data not: shown).
Table 1. GUS activity in seeds at 14 days post anthesis.
Cross (JUS Acs ivit,~!
nmol~e MUlminlmg Protein T218 T218 1.09 t 0.39 T218 WTa 3.02 t 0.19 WT T218 0.04 t 0.005 WT WT 0.04 t 0.005 NAP-5b NAP-5 14.6 t 7.9 NAP-5 WT 3.42 t 1.60 WT NAP-5 2.91 t 1.97 a WT, untransformed plants b Transgenic tobacco plants with the GUS gene fused to the napin, BngNAPl, promoter (Baszczynski and Fallis, 1990, Plant Mol. Biol. 14, 633-635).
Cloning and Analysis of the Seed Coat-Specific' GUS Fusion 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 ~?coRI 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, CA), 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 #l, Figure 2). This sequence, contained in a 260 by SstIlEcoRI
restriction fragment from pPRF-101 (Fobert et a~'., 1991, Plant Mol. Biol. 17, 837-851), was labelled with [a-32P]-dCTP (NEN) using random priming (Stratagene). After plaque purification, phage D;VA was isolated (Sambrook et al., 1989, A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press), mapped and subcloned into pGEM-4Z (Promega).
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, Figure 2), using the nos 3' fragment as probe (probe #1, Figure 2). ~Co 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 pBIN 19 (Bevan, 1984, Nucl.
Acid Res. 12, 8711-8721), as shown in Figure 2. Several transgenic plants were produced by Agrobacterium-mediated transi:ormation 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 pB1101 (Jefferson, 198',x, 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 dLeveloping seeds showed that expression was restricted to seeds between 10 and 17 dpa, reaching a maximum at 12 dpa (Figure 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.
To locate regions within the flanking plant DNA responsible for seed coat-specificity, truncated derivatives of the GUS fusion were generated (Figure 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, Figure 2) did not alter expression compared with the entire 4.7 kb GUS fusion (Figures 3b and 4). Further deletion of the DNA, to the SnaBI rE;striction site approximately 0.5 kb, 5' of the insertion site (pT218-3, Figure :Z), resulted in the complete loss of GUS activity in developing seeds (Figure~~ 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 DN~~ (pT218-4, Figures 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).
The transcriptional start site for the GUS gene in plant T218 was determined by RNase protection assays with RNr~ probe #4 (Figure 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 Figures 7 and 2, respectively. A
440bp HindIII fragment of the tobacco acetohydroxyacid synthase SURA gene was used to detect SURA and SURF mRNA. DrfA templates were linearized and transcribed in vitro with either T7 or SP6 po lymerases to generate strand-specific RNA probes using the Promega transcription kit and [a 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, i°lant J. 2, 321-330), using 10-30 ~.g of total RNA per assay. Probe digestion vas done at 30°C for 15 min using 30 ~,g mln RNase A (Boehringer Mannheim) and 100 units ml-' RNase T1 (Boehringer Mannheim). Figure 5 shows that two termini were mapped in the plant DNA. The major 5' terminus is situated at an adenine residue, 122 by upstream of the T-DNA insertion site (Figure 6). The sequence at this transcriptional start site is similar to the consensus sequence for plant genes (C/TTC IATCA; Joshi, 1987 Nucleic Acids Res. 15, 6643-6653). A TATA
box consensus sequence is present 37 by upstrearn of this start site (Figure 6).
The second, minor terminus mapped 254 by from the insertion site in an area where no obvious consensus motifs could be identified (Figure 6).
The tobacco DNA upstream of the insertion site is very AT-rich ( > 75 % , see Figure 7). A search for promoter-like motifs and scaffold attachment regions (SAR), which are often associated with promoters (Breyne et al., 1992, Plant Cell 4, 463-471; Gasser and I,aemmli, 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.
Cloning and Analysis of the Insertion Site from Untransformed Plants A lambda DASH genomic library was prepared from DNA of untransformed N. tabacum SRl plants by Stratag~~ne for cloning of the insertion site corresponding to the gene fusion in plant T2i18. The screening of 500,000 plaques with probe #2 (Figure 2) yielded a single lambda clone. The EcoRI
and XbaI fragments were subcloned in pGEM-4Z. to generate pIS-1 and pIS-2.
Figure 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 p:fS-1, were identical over a length of more than 2.5 kb, from the insertion site to their 5' ends, except for a 12 by filler DNA insert of unknown origin at the T-DNA border (Figure 6 and data not shown). The presence of filler DNA is ;~ 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 (Figure 6) and therefore could not account for thf: promoter activity associated with this region. The region of pIS-1 and pIS-2, 3' of the insertion site is also very AT-rich (Figure 7).
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). T~~ determine whether the region surrounding the insertion site was transcribed in ~antransformed 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 F~IVA 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 HCI, 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. 'Che RNA was precipitated overnight in 2M LiCI at 0°C, collected by centri~Fugation, washed in 70%
ethanol and resuspended in water. Northern blot hybridization was performed as described in Gottlob-McHugh et al. (1992, Plcznt Physiol. 100, 820-825).
Probe #3 (Figure 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 rc;gion 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 (Figure 7). Even after lengthy exposures, protected fragments could not be detected with RNA
from 8, 10, 12 dpa seeds or leaves of untransforrned plants (see Figure 5 for examples with two of the probes tested). The spE;ciflc 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 SL'RB mRNA which are expressed at relatively low abundance (data not s)hown). Conditions used in the present work were estimated to be sensitive enough to detect low-abundance messages representing 0.001-0.01 % of total mRTTA levels (Ouellet et al., 1992, Plant J. 2, 321-330). Therefore, the region flanl;ing the site of T-DNA
insertion does not appear to be transcribed in untransformed plants.
Genomic Origins of the Insertion Site Southern blots were performed to determine if the insertion site is conserved among Nicotiana species. Genomic DNA (5 ~,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 labeled, essentially as described in Rutledge: et al. (1991, Mol. Gen.
Genet. 229, 31-40). High-stringency washes were in 0.2 x SSC at 65 °C while low-stringency washes were in 2 x SSC at room ~:emperature. In Figure 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 (Figure 2). Single hybridizing fragments of identical size; were detected in N.
tabacum and N. tomentosiformis DNA digested with Hind:(II, XbaI and EcoRI, but not in N. sylvestris. Hybridizations with pIS-2 (Figure 8) which spans the same region but includes DNA 3' of the insertion site :Melded the same results.
They did not reveal hybridizing bands, even under conditions of reduced stringency, in additional Nicotiana species including N. rushca, N. glutinosa, N.

megalosiphon and N. debneyi (data not shown). Probe #3 (Figure 2) revealed the presence of moderately repetitive DNA specific to the 1V. 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 rf:gions that encode essential genes.
Characterization of a Constitutive GUS fusion -T1275 From the transgenic plants produced (see above), one of these, T1275, was chosen for detail~l study because of its high level and constitutive expression of GUS (see also US patent application 08/593,121 and PCT/CA97100064, both of which are incorporated by reference).
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), ~~tem (S), root (R), anther (A), petal (P), ovary (O), sepal (Se), seeds 10 days past anthesis (S1) and seeds days post-anthesis (S2), were collected from plants grown in the greenhouse and analyzed.
GUS activity in plant T1275 was found in all tissues. Figure 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).
Constitutive GUS expression was confirmed with the more sensitive fluorogenic assay of plant tissue from transformed plant T1275. These results are shown in Figure 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 Molecu lar Biology, 17 : 837-851 ) GUS activity in transformed plants containing pBa121 (Clontech), which contains a CaMV 35S - GUS - nos chimeric gene, was as high as 18,770 t 2450 (pmole MU per minute per mg protein).
Cloning and Analysis of the Constitutive Promoter - GUS Fusion Genomic DNA was isolated from leaves according to Hattori et al.
(1987, Anal. Biochem. 165, 70-74). Ten ~.g of T'1275 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 lambdaGF:M-2 arms previously digested with EcoRI and XbaI and phosphatase-treated. About 40,000 plaques were transferred to a nylon membrax>e (Hybond, Amersham) and screened with the sap-labelled 2kb GUS insert isolated form 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 Figure 12) was isolated from the lambda phage and cloned into pTZl9R previously digested with XbaI and EcoRI and treated with intestinal <;alf phosphatase.
The plant DNA sequence within the clone:, SEQ ID N0:2, 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).

The T1275 Regulatory Element is Cryptic The 4.2kb fragment containitng about 2.2k:b of the T 1275 promoter fused to the GUS gene and the nos 3' was isolated) by digesting pTZ-T1275 with S HindIII and EcoRI. The isolated fragment was li,gated 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 SRl were performed as described above. GUS activity was examined in several organs of many independent transgenic lines. GUS rnRNA 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 Figure lfi (A). Assays were performed using the RPAII kit (Ambion CA) as described b;y 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.
RNase protection assays performed with 1Z1VA 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 (Figure 16 (B)). The insertion site, including by 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 Figure 16 (A). RNA probes were prepared that spanned the entire region as shown in Figure 16 (A). Rr'ase 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 pl;~nt cryptic regulatory element.
Table 2.
Summary of the RNase Protection Assays of the insertion site in untransformed tobacco. Sce Figure 16 (.~) 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 tIRNA
7-EcoRI faint bands, all in tRNA
Constitutive Activity of the T1275 Regulatory Element For analysis of transient expression of Gt7S activity mediated by biolistics (Sandford et al, 1983, Methods Enzymol, 217: 483-509), the Xbal -EcoRl fragment was subcloned in pUCl9 and G~JS activity was detected by staining with X-Gluc as described above. Leaf tissue of greenhouse-grown plants or cell suspension cultures were 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, and cell suspensions of oat, corn, wheat and barley exhibited GUS-positive blue spots after transient lbombardment-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.

Transient Expression of GUS Activity in Tissues of Diverse Plant Species Ti~ue Source Species GUS Activity ~

Leaf Soybean + + +

Alfalfa + +

Arabidopsis +

Leaf disc Tobacco + +

B. napes +

Pea +

Cell Cultures Oat +

Corn +

Wheat +

Barley + +

* Numbers of blue spots: 1 - 10 (+), 10 - 100 (-i- +), 100 - 400 (+ + +) For analysis of GUS expression in different organs, lines derived from progeny of the above lines were examined in detail. Table 4 shows the GUS
specific activities in one of these plants. It is exyressed in leaf, stem, root, developing seeds and the floral organs, sepals, pedals, anthers, pistils and ovaries at varying levels, confirming constitutive expression. Introduction of the same vector into B. napes 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;ure 16 (B)) and the level of mRNA was similar except in flower buds where it was lower (Table 4).

GUS Specific Activity and Relative RNA Levels in the Organs of Progeny of Transgenic Line T64 Organ Relative GUS GUS Specific &NA Activity Levels in T64 (picomollMUlmin/mg protein) Progeny (grey scale units) Transformed Untransformed 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 - 715 2.59 ND*
buds 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 Identification of Regulatory Elements within the Full Length T1275 Regulat~ry Element An array of deletions of the full length regulatory region of T1275 were prepared, as identified in Figures 13 (A) and (B), for further analysis of the cryptic regulatory element.
5' deletions of the promoter (see Figures 13(A) and (B) and analysis by transient expression using biolistics showed that the promoter was active within a fragment 62bp from the transcriptional start site indicating that the core promoter has a basal level of expression (see Tablie 5).
Table 5.
Transient GUS activity detected in soybean leaves by staining with X-gluc after particle bombardment. Vectors illustrated in Figures. 13 (A) and (B).
Genes (iUS staining 1. T1275-GUS-nos 2. -1639-GUS-nos v 3. -1304-GUS-nos v 4. -684-GUS-nos v 5. -394-GUS-nos v 6. -197-GUS-nos 7. -62-GUS-nos +

8. -62(-tsr)-GUS-nos 9. -12-GUS-nos -10.
+30-GUS-nos -Deletion of a fragment containing the trar~scriptional start site (see -62(-tsr)/GUS/nos in Figure 14(B), Table 5) did not eliminate expression, however deletions to -12 by and further (ie+30) did eliminate expression indicating that the region defined by -(62-12) (nucleotides 1992-2042 of SEQ ID N0:2) by contained the core promoter. DNA sequence se~~rches did not reveal conventional core promoter motifs within this region as are typically found in plant genes, such as the TATA box.
A number of the 5' promoter deletion clones (Figure 13(A)) 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 (nucleotide 1857 of SEQ ID N0:2).
Table 6.
GUS specific activities in leaves of greenhouse-grown transgenic tobacco, SRl, transformed with the T1275-GUS-nos gene fusion and 5' deletion clones (see Figure 13 A). MeanfSE(n) Genes GLIS specific activities pmoles MU/min/mg protein 1. T1275-GUS-nos a!83~171 (27) 2. -1639-GUS-nos '~87~188 (26) 3. -1403-GUS-nos E~32~217 (10) 4. -683-GUS-nos in progress 5. -394-GUS-nos 16271340 (13) 6. -197-GUS-nos 47574 (27) Histochemical analysis of organs sampled from tlae transgenic plants indicated GUS expression in leaf, seeds and flowers as a minimum as not all the organs were analyzed.
Activity of the T1275 Regulatory Element Analysis of leaves of randomly-selected, l;reenhouse-grown plants regenerated from culture revealed a wide range of GUS specific activities (Figure 14 (A); T plants). Plants transformed with pBI 121 (CLONETECH) which contains the 35S GUS-nos gene yielded comparable specific activity levels (Figure 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;ure. 14(C)).

Generally, the level of GUS mRNA in the: leaves as determined by RNase protection (Figure 14 (B)) correlated with the GUS specific activities, however, the level of GUS mRNA was about 60 i:old (mean of 13 measurements) lower in plants transformed with the T1275-GUS-nos gene (Figure 14(B)) when compared with plants transformed with 35S-GUS-nos.
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.
Post-Transcriptional Regulatory Elements witlxin T1275 An experiment was performed to determine the presence of a post-transcriptional regulatory element within the T 12 7 5 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 activit:~), mRNA stability or transcription.
Deletion of the Ndel-Smalfragment from the T1275-GUS-nos gene (Figure 15; T1275-N-GUS-nos; including nucleotides 2086-2224 of SEQ ID
N0:2) resulted in a 46-fold reduction in the amount of GUS specific activity that could be detected in leaves of transgenic tobacco c:v Delgold (see Table 7).
Addition of the same fragment to a 35S-GUS-nos gene (Figure 15; 35S+N-GUS-nos) construct increased the amount of GUS specific activity by 4-fold (see Table 7).

Table 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 Figure 15). Mean~SE(n).
Gene GUS specific activity pmoles MU/min/mg protein 1. T1275-GUS-nos 557+183 (21) 2. T1275-N-GUS-nos 1f.+3 (22) 3. P35S-GUS-nos 1818+692 (15) 4. P35S+N-GUS-nos 699~~+3148 (23) 'The data presented in Table 7 are consistent with the presence of a post-transcriptional regulatory sequence in the NdeI-SmaI fragment.
The Ndel Smal fragment functions as a transcriptional enhancer or mRNA
stability determinant The levels of mRNA were determined in leaves obtained from plants transformed with either T1275-GUS-nos, T1275-rJ-GUS-nos, 35S-GUS-nos, or 35S+N-GUS-nos (Figure 17 (A)). Relative RNA :levels were determined by ribonuclease protection assay (Ambion RPAII Kit ) in the presence of a 32P-CTP
labeled in vitro transcribed probe and autoradiographic quantification using Kodak Digital Science 1D Image Analysis Software. Hybridization conditions used during RNase protection assay were overnigrit at 42-45 degrees in 80%
formamide, 100 mM sodium citrate pH 6.4, 300 m~M sodium acetate pH 6.4, 1 mM EDTA.
The levels of mRNA examined from transi;enic tobacco plants transformed with either T1275-GUS-nos, T1275-rJ-GUS-nos, 35S-GUS-nos, or 35S+N-GUS-nos, were higher in transgenic plants comprising the Ndel Smal 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 (Figure 17 (A)). This indicates that this region functions by either modulating transcriptional rates, or the stability of the transcript, or both.
The Ndel Smal fragment functions as a translational enhancer Analysis were performed in order to determine whether the Ndel Smal region functions post-transcriptionally. The GUS specific activity:relative RNA
level was determined from the GUS specific activity measurements, and relative RNA levels in greenhouse grown transgenic plants (figure 17 (B)). The ratio of GUS specific activity to relative RNA level in individual transgenic tobacco plants comprising the Ndel Smal fragment is higher than in plants that do not comprise this region (Figure 17 {B)). 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 siz fold, in GUS specific activity is observed when the lVdel Smal region is added within the 35S
untranslated region (Figure 17 (B)). The GUS s~~ecific activity:relative RNA
levels are similar in constructs containing the Nde.! Smal fragment (T1275-GUS-nos and 35S+N-GUS-nosy. These results indicate that the Ndel Smal fragment modulates gene expression post-transcriptionally.
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 Figure 1 S 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 BgIII and EcoRl, from pBI525 (Dada et al., 1993, Plant Science 94: 139-149) and ligation -$
with pGEM4Z (Promega) digested with BamHl arid EcoRl. Transcripts were prepared in vitro in the presence of m'G(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.
Translation of transcripts in vitro demonstrate an increase in translational efficiency of RNA containing the Ndel to Smal fragment (see Table 8).
Table 8 In vitro translation of mRNA obtained from transgenic tobacco plants transformed with vectors with or without a Nd~I Smal fragment obtained from the T1275 GUS gene fusion (see Figure 15) using wheat germ eztract.
in vitro translation in vitro transcript fold enhancement T1275-GUS-nos 3.7 T1275-N-GUS-nos 1.0 AMV-GUS-nos 1.9 The levels of protein produced using mRNAs comprising the Ndel Smal 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.

149). These results indicate that this region functions post-transcriptionally, as a translational enhancer.
All scientific publications and patent documents are incorporated herein by reference.

-$1-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 LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Brian Miki (A) NAME: Theresa. Ouellet (A) NAME: Jiro Hattori (A) NAME: Elizabeth Foster (A) NAME: Helene Labbe (A) NAME: Teresa Martin-Heller (A) NAME: Lihing Tian (A) NAME: Daniel Brown (ii) TITLE OF INVENTION: Cryptic Regulatory Elements in Plants (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA
(A) APPLICATION NUMBER: 2,246,892 (B) FILING DATE: September, 09.1998 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1070 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:
(B) CLONE: pT218 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TGGAAGATAA

AACAGTCATC

AATTTTCATG

TTTATTATAG CAACCAAA.AA ATATCGAAAC GTTATAGAGC GATTTGATTG420 AGATACGATT

ATTACTCCTC

TTCAATACTT

TAATTTGAAG

AGCTCTACTA

ACTAAAGGTG

AGATCAGTTA

AACTTTATTT

TGTCAATCAG

AATTTTTATA

TGCTATCGTG

GTCCTGTAGA

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2224 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:
(B) CLONE: pT1275 (xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 2:

CTTA

Claims (48)

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 seed-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 Figure 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 500bp.

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 2053-2224 of SEQ ID NO:2.

28. The cryptic regulatory element of claim 11, comprising nucleotides 2086-2253 of SEQ ID NO:2.

29. The cryptic regulatory element of claim 11, comprising an XbaI - SmaI
fragment of Figure 13 (C).

30. The cryptic regulatory element of claim 29, comprising an SphI - SmaI
fragment.

31. The cryptic regulatory element of claim 29 comprising a PstI - SmaI
fragment.

32. The cryptic regulatory element of claim 29, comprising an SspI - SmaI
fragment.

33. The cryptic regulatory element of claim 29, comprising a BstYI - SmaI
fragment.

34. The cryptic regulatory element of claim 29, comprising a DraI - SmaI
fragment.

35. The cryptic regulatory element of claim 29, comprising a NdeI - SmaI
fragment.

36. The cryptic regulatory element of claim 29, comprising an XbaI-BstYI
fragment

37. The cryptic regulatory element of claim 29, comprising a BstYI-DraI
fragment

38. The cryptic regulatory element of claim 29, comprising an XbaI-Ndel fragment.

39. A method of conferring expression of a gene in a plant, 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 a plant capable of expressing the chimeric gene construct.

40. The method of claim 39, wherein the plant-derived cryptic regulatory element is selected from the group consisting of a seed-coat specific regulatory element, and constitutive regulatory element.

41. The method of claim 40, wherein the seed-coat specific regulatory element comprises a nucleic acid that is substantially homologous with the sequence of SEQ ID NO:1.

42. The method of claim 40, wherein the constitutive regulatory element comprises a nucleic acid that is substantially homologous with the sequence of SEQ ID NO:2.

43. The method of claim 41, wherein the nucleic acid comprises at least a 19 by contiguous sequence of SEQ ID NO:1.

44. The method of claim 42, wherein the nucleic acid comprises at least an 18 by contiguous sequence of SEQ ID NO:2.

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

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

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

48. 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 of claim 1, or a fragment thereof, to produce a chimeric gene construct, and introducing the chimeric gene construct into a plant capable of expressing the chimeric gene construct.