Note: Descriptions are shown in the official language in which they were submitted.
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CRYPTIC REGULATORY ELEMENTS OBTAINED FROM PLANTS
Field of Invention
This invention relates to cryptic regulatory 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, and downstream of, gene
promoter elements may be the activation of reporter gene expression. The
resulting hybrid genes, referred to as T-DNA-mediated gene fusions, consist of
unselected plant promoters residing at their natural location within the
chromosome, and the coding sequence of a marker 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 promoteriess or
enhancerless marker genes result from T-DNA insertions within or immediately
adjacent to genes. The recent isolation of several T-DNA insertional mutants
(Koncz et al., 1992, Plant Mol. Biol. 20, 963-976; reviewed in Feldmann,
1991, Plant J. 1, 71-82; Van Lijsebettens et al., 1991, Plant Sci. 80, 27-37;
Walden et al., 1991, Plant J. 1: 281-288; Yanofsky et al., 1990, Nature 346,
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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 buried in the genome but with the
capability of being functional when positioned adjacent to genes have been
described in a variety of organisms, where they have been called "cryptic
promoters" (Al-Shawi et al., 1991, Mol. Cell. Biol. 11, 4207-4216; Fourel et
al., 1992, Mol. Cell. Biol. 12, 5336-5344; Irniger et al., 1992. Nucleic Acids
Res. 20, 4733-4739; Takahashi et al., 1991, Jpn J. Cancer Res. 82, 1239-
1244). Cryptic promoters can be found in the introns of genes, such as those
encoding for yeast actin (Irniger et al., 1992, Nucleic Acids Res. 20, 4733-
4739), and a mammalian melanoma-associated antigen (Takahashi et al., 1991,
Jpn J. Cancer Res. 82, 1239-1244). It has been suggested that the cryptic
promoter of the yeast actin gene may be a relict of a promoter that was at one
time active but lost function once the coding region was assimilated into the
exon-intron structure of the present-day gene (Irniger et al., 1992, Nucleic
Acids Res. 20, 4733-4739). A cryptic promoter has also been found in an
untranslated region of the second exon of the woodchuck N-myc proto-
oncogene (Fourel et al., 1992, Mol. Cell. Biol. 12, 5336-5344). This cryptic
promoter is responsible for activation of a N-myc2, a functional processed
gene
which arose from retropositon of N-myc transcript (Fourel et al., 1992, Mol.
Cell. Biol. 12, 5336-5344). These types of regulatory sequences have not yet
been isolated from plants.
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,
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transcription and RNA stability. For example, some regulatory elements are
known to enhance the translational efficiency of mRNA, resulting in an
increased accumulation of recombinant protein by many folds. Some of those
regulatory elements contain translational enhancer sequences or structures,
such
as the Omega sequence of the 5' leader of the tobacco mosaic virus (Gallie and
Walbot, 1992, Nucleic Acid res. 20, 4631-4638), the 5' alpha-beta leader of
the
potato virus X (Tomashevskaya et al, 1993, J. Gen. Virol. 74, 2717-2724), and
the 5' leader of the photosystem I gene psaDb of Nicotiana sylvestris
(Yamamoto
et al., 1995, J. Biol. Chem 270, 12466-12470). Other 5' regulatory elements
affect gene expression by quantitative enhancement of transcription, as with
the
UTR of the thylakoid protein genes PsaF, PetH and PetE from pea (Bolle et al.,
199, Plant J. 6, 513-523), or by repression of transcription, as for the 5'
UTR of
the pollen-specific LAT59 gene from tomato (Curie and McCormick, 1997, Plant
Ce119, 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 transgenic plants generated by tagging
with a promoterless GUS (P-glucuronidase) T-DNA vector and the isolation
and characterization of cryptic regulatory elements identified using this
protocol. Cloning and characterization of these insertion sites uncovered
unique
cryptic regulatory elements not conserved among related species. In one of the
plants of interest, GUS expression was spatially and developmentally regulated
with in seed tissue. The isolated regulatory element specific to this tissue
has
not been previously isolated or characterized in any manner. In another plant,
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a novel constitutive regulatory element was identified that is expressed in
tissues throughout the plant and across a broad range of plant species.
Furthermore, novel non-translated 5' sequences have been identified that
function as post transcriptional regulatory elements.
Summary of Invention
This invention relates to cryptic regulatory 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 tbund not to be active in the native
plant.
Plant T218 contains a 4.65 kb EcoRl frm,,ment containing the 2.15 kb
promoterless GUS-nos gene and 2.5 kb of 5' tlanking DNA. Deletion of the
region approximately between 2.5 and 1.0 kh of the 5' flanking region did not
alter GUS expression, as compared to the entire 4.65 kb GUS fusion. A
further deletion to 0.5 kb of the 5' tlanking site resulted in complete loss
of
GUS activity. Thus the region between 1.0 and 0.5 of the 5' flanking region of
the tobacco DNA contains the elements essential to gene activation. This
region is contained within a Xbal - 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
EcoRI/Xbal 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. iabacum c.v. Petit Havana, SRI and
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transgenic B. napus c.v. Westar was observed in leaf, stem, root, developing
seed and flower. By transient expression analysis, GUS activity was also
observed in leaf tissue of soybean, alfalfa, Arabidopsis, tobacco, B. napus,
pea
and suspension cultured cells of oat, corn, wheat and barley. The
transcription
start site for the GUS gene in transgenic tobacco was located in the plant DNA
upstream of the insertion site. A set of deletions within the plant DNA
revealed the presence of a core promoter element located within a 62 bp region
from the transcriptional start site, the occurrence of at least one negative
regulatory element located within an Xbal-Sspl fragment, a transcriptional
enhancer located within the BstYI-Dral fragment, and 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 comprise at least one of: a
promoter, a core promoter element, a negative regulatory element, a
transcriptional enhancer, a translational enhancer and a post transcriptional
regulatory element.
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 EcoRI-Smal restriction sites
defined by the restriction map of Figure 2 (B). Furthermore, this invention
relates to a cryptic regulatory element comprising an Xbal - SmaI fragment, of
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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
Xbal and SnaBI fragment as defined by the restriction map of Figure 2 (B),
wherein the fragment is of about 500 bp. This invention also is directed to a
cryptic regulatory element comprising an Xbal 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 HindIII 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 EcoRl and SnaBI fragment defined by the restriction map of
Figure 2, wherein the fragment is of about 2 kb _
This invention also embraces a reRulatory element characterized in that
it is substantially homologous with the sequence defined by SEQ ID NO:2.
This invention is also directed to a cryptic regulatory element that comprises
at
least an 18 bp contiguous sequence of SEQ ID NO:2. Furthermore, this
regulatory element functions in diverse plant species when introduced on a
cloning vector. This invention also relates to a chimeric gene construct
comprising a DNA of interest for which constitutive expression is desired, and
a constitutive regulatory element, comprising at least an 18 bp contiguous
sequence of SEQ ID NO: 2.
This invention also embraces a cryptic regulatory element conlprising an
XbaI - Smal fragment (comprising nucleotides 1-2224 of SEQ ID NO:2), an
XbaI - Ndel fragment (comprising nucleotides 1-1086 of SEQ ID NO:2), an
Sphl - Smal fragment (comprising nucleotides 415-2224 of SEQ ID NO:2), a
Pstl - Smal fragment (comprising nucleotides 1040-2224 of SEQ ID NO:2), an
Sspl - Smal fragment (comprising nucleotides 1370-2224 of SEQ ID NO:2), a
BstYI - Smal fragment (comprising nucleotides 1660-2224 of SEQ ID NO:2), a
Dral - Smal fragment (comprising nucleotides 1875-2224 of SEQ ID NO:2), a
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Ndel-Smal fragment (comprising nucleotides 2086- 2224 of SEQ ID NO:2), a
Xbal-BstYI fragment (comprising nucleotides 1-1660 of SEQ ID NO:2), a
BstYI-Di-aI fragment (comprising nucleotides 1660-1875 of SEQ ID NO:2), a+1
to Smal fragment (comprising nucleotides 2055-2224 of SEQ ID NO:2), Dral-
Nde I fragment (comprising nucleotides 1875-2086 of SEQ ID NO:2) or a Dra 1
to -62 fragment (comprising nucleotides 1875-1992 of SEQ ID NO:2) as defined
in Figure 13(C).
This invention is also also directed to a cryptic regulatory element
comprising nucleotides 1-141 of SEQ ID NO:3, nucleotides 1-188 of SEQ ID
NO:3, nucleotides 7-97 of SEQ ID NO:4, nucleotides 1-129 of SEQ ID NO:4,
nucleotides 1-119 of SEQ ID NO:5, or nucleotides 7-86 of SEQ ID NO:5
This invention also pertains to a transgenic host organism containing a
cryptic regulatory element as defined above operatively linked to a gene
encoding
a protein. The host organism may be selected from the group consisting of a
plant, a tree, an insect, a fungi, a bacteria, a yeast and a non-human a-
iimal.
This invention also includes a plant cell which has been transformed
with a chimeric gene construct, or a cloning vector comprising a cryptic plant
regulatory element. Furthermore, this invention embraces transgenic plants
containing chimeric gene constructs, or cloning vectors comprising cryptic
plant regulatory elements.
This invention further relates to any transgenic plant containing a
cryptic regulatory element, having a DNA sequence substantially homologous
to SEQ ID NO: 1, or SEQ ID NO:2 and operatively linked to a DNA region
that is transcribed into RNA.
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Also included in the present invention is a method of conferring
expression of a gene in a host organism, comprising operatively linking an
exogenous DNA of interest, for which expression is desired with a cryptic
regulatory element as defined above, to produce a chimeric gene construct, and
introducing the chimeric gene construct into the host organism capable of
expressing the chimeric gene construct. This invention also embraces a method
of modulating expression of a gene in a plant, comprising operatively linking
an exogenous DNA of interest, for which expression is desired with a promoter
of interest and the cryptic regulatory element as defined above and
introducing
the chimeric construct into the host organism. Furthermore, the method of
conferring or modulating gene expression may include operatively linking an
exogenous DNA of interest, for which expression is desired with a promoter of
interest and at least one fragment of the cryptic regulatory element as
defined
above to produce a chimeric gene construct, and introducing the chimeric gene
construct into the host organism capable e>f expressing the chimeric gene
construct. The host organism may be selccted fi-om the group consisting of a
plant, a tree, an insect, a fungi, a bacteria. ayeast and a non-human animal.
This invention also relates to the above method wherein the plant-
derived cryptic regulatory element is a seed-coat specific or constitutive
regulatory element. Furthermore, this invention embraces the above method
wherein the seed-coat specific regulatory element comprises a nucleic acid
that
is substantially homologous with the sequence of SEQ ID NO: 1, or constitutive
regulatory element comprises a nucleic acid that is substantially homologous
with the sequence of SEQ ID NO:2. This invention also relates to the above
method wherein the nucleic acid comprises at least a 19 bp contiguous sequence
of SEQ ID NO: 1, or the nucleic acid comprises at least an 18 bp contiguous
sequence of SEQ ID NO:2.
According to the present invention there is also provided a seed coat-
specific cryptic regulatory element contained within a DNA sequence, or
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analogue thereof, as shown in SEQ ID NO: 1. Furthermore, there is provided
a constitutive regulatory element contained within a DNA sequence, fragment
or an analogue thereof, as shown in SEQ ID NO: 2.
This invention also relates to a vector containing a seed coat-specific
cryptic regulatory element, which is contained within a DNA sequence, or
analogue thereof, as shown in SEQ ID NO: 1 and a gene encoding a protein.
This invention also relates to a cloning vector containing a constitutive
cryptic
regulatory element, which is contained within a DNA sequence, fragment, or
an analogue thereof, as shown in SEQ ID NO: 2 and a gene encoding a protein.
This invention also includes a plant cell which has been transformed
with a vector as described above, and to a transgenic plant containing a
cloning
vector as described above, operatively linked to a gene encoding a protein.
Brief Description of the Drawings
Figure 1 depicts the fluorogenic analyses of GUS expression in the plant
T218. Each bar represents the average one standard deviation of three
samples. Nine different tissues were analyzed: leaf (L), stem (S), root (R),
anther (A), petal (P), ovary (0), sepal (Se), seeds 10 days post anthesis (SI)
and seeds 20 days post-anthesis (S2). For all measurements of GUS activity,
the fraction attributed to intrinsic fluorescence, as determined by analysis
of
untransformed tissues, is shaded black on the graph. Absence of a black area
at the bottom of a histogram indicates that the relative contribution of the
background fluorescence is too small to be apparent.
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
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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 pBIN19 polylinker to create
pT218-1. Fragments truncated at the XbaI, SnaB1 and Xbal sites were also
subcloned to create pT218-2, pT218-3 and pT218-4. 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), Xbal (X), SnaBI (N), Smal (M), SstI (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 (0-0), pT218-2 (0-0), pT218-3 (0-0) and pT218-4 (0-A) which are
illustrated in Figure 2. The 2 day delay in the peak of GUS activity during
seed development, seen with the pT218-2 transformant, likely reflects
greenhouse variation conditions.
Figure 4 shows GUS activity in 12 dpa seeds of independent
transformants produced with vectors pT218-1 (0), pT218-2 (EI), pT218-3 (V)
and pT218-4 (A). 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 mRNA 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 g) and
from plant T218 (30 g). Arrowheads indicate the anticipated position of
protected fragments if transcripts were initiated at the same sites as the
T218
GUS fusion. Figure 5 (B) shows the results of an RNase protection assay using
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the antisense (relative to the orientation of the GUS coding region) RNA probe
from subclone e (see Figure 7) against 30 g total RNA of tissues from
untransformed plants. The abbreviations used are as follows: P, untreated
RNA probe; -, control assay using the probe and tRNA only; L, leaves from
untransformed plants; 8, 10, 12, seeds from untransformed plants at 8, 10, and
12 dpa, respectively; T10, seeds of plant T218 at 10 dpa; +, control
hybridization against unlabelled in vitro-synthesized sense RNA from subclone
c (panel a) or subclone e (panel b). The two hybridizing bands near the top of
the gel are end-labelled DNA fragment of 3313 and 1049 bp, included in all
assays to monitor losses during processing. Molecular weight markers are in
number of bases.
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 bp 993. This
site
on pT218 is immediately followed by a 12 bp filler DNA, which is followed by
the T-DNA. The first nine amino acids of the GUS gene and the GUS
initiation codon (*) are shown. The major and minor transcriptional start site
is
indicated by a large and small arrow, respectively. The presumptive TATA
box is identified and is in boldface. Additional putative TATA and CAAT
boxes are marked with boxes. The location of direct (1-5) and indirect (6-8)
repeats are indicated by arrows.
Figure 7 shows the base composition of region surrounding the T218
insertion site cloned from untransformed plants. The site of T-DNA insertion
in plant T218 is indicated by the vertical arrow. The position of the 2
genomic
clones pIS-1 and pIS-2, and of the various RNA probes (a-e) used in RNase
protection assays are indicated beneath the graph.
Figure 8 shows the Southern blot analyses of the insertion site in
Nicotiana species. DNA from N. tomentosiformis (N tom), N. sylvestris (N
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syl), and N. tabacum (N tab) were digested with HindIIl (H), Xbal (X) and
EcoRI (E) and hybridized using probe #2 (Figure 2). Lambda HindIIl 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 GenBank release 75.0 and to
calculate the AT contents of the 5' non-coding (solid bars) and the coding
regions (hatched bars) of all plant genes identified as "Magnoliophyta"
(flowering plants). The region -200 to -1 and +1 to +200 were compared.
Shorter sequences were also accepted if they were at least 190 bp long. The
horizontal axis shows the ratio of the AT content (%o ). 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), immature embryos (f),
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 (0), sepal (Se), seeds 10 days post anthesis (S1), and seeds,
20
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
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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 Xbal fragment in pT1275 was subcloned to create
pT1275-GUS-nos. Deletion constructs truncated at the Sphl, PstI, SspI,
BstYI, and Dral 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 13 (D) shows modified
constructs of the T1275 regulatory elements. T1275 is indicated by the open
box, the CaMV35S promoter element is indicated by the black box. The
activity of these constructs is also indicated. GUS activity was determined in
tobacco leaves following transient expression using microparticle
bombardment. TA30-GUS: a TATATAA element was inserted into the -30
position of -62-GUS; TA35S-GUS: the -62 to -20 fragment of -62-GUS was
substituted with the -46 to -20 fragment of the 35S promoter; GCC-62-GUS: a
GCC box was fused with -62-GUS; DRA2-GUS: the -197 to -62 fragment was
repeated; BST2-GUS: the -394 to -62 fragment was repeated; -46-35S: 35S
minimal promoter; DRAI-35S: the -197 to -62 fragment of T1275 was fused
with -46-35S; BSTI-35S: the -394 to -62 fragment of T1275 was fused with -
46-35S; BST2-35S: two copies of the -394 to -62 fragment of T1275 were
fused with -46-35S. Figure 13 (E) shows constructs of the -197 to -62
fragment fused with the 35S minimal promoter. -46-35S: 35S minimal
promoter; DRAI-35S: the -197 to -62 fragment of T1275 was fused with -46-
35S; DRA1R-35S: the -197 to -62 fragment of T1275 was fused with -46-35S
in a reversed orientation; DRA2-35S: two copies of the -197 to -62 fragment of
T1275 were fused with -46-35S. Figure 13 (F) shows GUS specific activity of
transgenic Arabidopsis plants. Leaf tissues from Arabidopsis plants
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transformed with -47-35S, DRA1-35S, DRA1R-35S and DRA2-35S constructs
were used for GUS assay. Figure 13 (G) shows the constitutive expression of
GUS in Arabidopsis plants transformed with DRAl-35S. From left to right:
flower, silque and seedling.
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-GUS-nos (S plants). Figure 14
(A) shows the levels of GUS expression in leaves from randomly selected
plants containing either T1275-GUS-nos (left-hand side) or 35S-GUS-nos
(right-hand side). Figure 14 (B) shows the level of accumulated GUS niRNA
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; untransforined 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 Ndel - Smal 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 - Smal
fragment of T1275-GUS-nos is identified as T1275-N-GUS-nos. The Ndel -
Smal fragment from T1275-GUS-nos was also introduced into 35S-GUS-nos to
produce 35S+N-Gus-nos.
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Figure 16 shows the region surrounding the insertion site in
untransformed plants, positions of various probes used for RNase protection
assays, and results of the RNase protection assay. 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 bp 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-GiJS-nos (10 g RNA) and
untransformed tobacco (30 g RNA). Undigested probe (P), tRNA negative control
(-)
lanes and markers are indicated. RNase protection assays shown used a probe to
detect
sense transcripts between about -446 and +596 of T1275-GUS-nos or between
about -
446 to +169 of untransformed tobacco. The protected fragment in transformed
plants is
about 596 bp (upper arrowhead) and, if present, accumulated transcripts
initiated at this
site in untransformed plants are predicted to protect a fragment of about 169
bp (lower
arrowhead). Upper band in RNA-containing lanes was added to samples to
indicate loss
of sample during assay.
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 T1275-GUS-nos, or 35S-GUS-
nos constructs, with or without the NdeI- Smal fragment (see Figure 15).
Figure 17 (A) shows the level of accumulated GUS mRNA measured by RNase
protection assay and densitometry of autoradiograms in leaves from the same
randomly selected plants containing either T1275-GUS-nos, T1275-N-GUS-
nos. Figure 17 (B) shows the level of accumulated GUS mRNA measured by
RNase protection for 35S-GUS-nos or 35S+N-GiJS-nos. Figure 17 (C) shows
the ratio between GUS specific activity and mRNA levels in leaves of
individual, regenerated, greenhouse-grown transgenic plants containing T1275-
GUS-nos, T1275-N-GUS-nos, 35S-GUS-nos, or 35S+N-GUS-nos constructs.
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Figure 18 shows the maps of T1275-GUS-nos and T1275(ON)-GUS-
nos. Figure 18(A) shows T1275-GUS-nos (also referred to as tCUP-GUS-nos).
Figure 18 (B) shows T1275(AN)-GUS-nos (also referred to as tCUPdelta-GUS-
nos). "ON", (also referred to as "dN" or "deltaN") was created by changing
the Ndel site "a" in the leader sequence of T1275-GUS-nos (Figure 18(A)) to a
BglI1 site "b" (see Figure 18(B)) to eliminate the upstream ATG at nucleotides
2087-2089 or SEQ ID NO:2. A Kozak consensus sequence "c" was
constructed at the initiator MET codon and a Ncol site was added. The
transcriptional start site, determined for T1275, is indicated by the arrow.
Figure 19 shows constructs used for the transient expression via particle
bombardment of corn callus. Maps for 35S-GUS-nos, 35S (+N)-GUS-nos,
35S (ON)-GUS-nos and 35S(+i)-GUS-nos are presented indicating the "N"
region, ADH1 intron, and the arrow indicates the transcriptional start site.
Note
that 35S(ON)-GUS-nos is referred to as 35S+deltaN-dK-GUS-nos. Also
shown are the associated activities of the constructs in the callus expressed
as a
ratio of GUS to luciferase (control) activitV.
Figure 20 shows maps of the constructs used for transient expression in
yeast. Shown are pYES-GUS-nos (also referred to as pYEGUS); pYES(+N)-
GUS-nos (also referred to as pYENGUS); pYES(ON)-GUS-nos (also referred
to as pYEdNGUS) and pYES(ON"t)-GUS-nos (also referred to as
pYEdN'"GUS), which lacks the Kozak consensus sequence.
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.
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T-DNA tagging with a promoterless P-glucuronidase (GUS) gene
generated several transgenic Nicotiana tabacum plants that expressed GUS
activity. Examples, which are not to be considered limiting in any maruler, of
transgenic plants displaying expression of the promoterless reporter gene,
include a plant that expressed GUS only in developing seed coats, T218, and
another plant that expressed GUS in all organs, T1275 (see co-pending patent
applications US serial No. 5,824,872 and PCT/CA97/00064.
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-
Smal fragment, and further deletion analyses localized a cryptic
regulatory element to an approximately 0.5 kb region between a Xbal
and a SnaBI restriction endonuclease site of 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 identified within an XbaI - Smal
fragment, which comprises several cryptic regulatory elements which
were localized to several regions throughout the upstream region and
include a minimal promoter region between Dral and NdeI sites (see
Figure 13), negative regulatory elements between Xbal and BstYI, a
transcriptional enhancer between BstYI and Dra1, and between Di-a1-(-
62) (nucleotides 1875 to 1992 of SEQ ID NO:2), and a translational
enhancer regulatory element between the Ndel-Snzal sites (also referred
to as "N", see below; SEQ ID NO:3). Also included are regulatory
elements "AN" (also referred to as dN, or deltaN), an element derived
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from N, that comprises a Kozack sequence (Figure 18, SEQ ID NO:4),
and ON"', that lacks a Kozack sequence (SEQ ID NO:5).
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 eff'iciency 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 refers to regulatory elements that
are inactive in the control of expression at their native location. These
inactive
regulatory sequences are buried in the genome including intergenic regions or
regions of genes that are not involved in the regulation of adjacent sequences
but are capable of being functional when positioned adjacent to a gene.
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
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elements of the present invention includes those which are capable of
mediating
organ specificity, or controlling developmental or temporal gene activation.
Furthermore, "regulatory element" includes promoter elements, core promoter
elements, elements that are inducible in response to an external stimulus,
elements that are activated constitutively, or elements that decrease or
increase
promoter activity such as negative regulatory elements or transcriptional
enhancers, respectively. It is also to be understood that enhancer elements
may
be repeated thereby further increasing the enhancing effect of an enhancer
element on a regulatory region. "Regulatory elements" as used herein, also
includes elements that are active following transcription initiation or
transcription, for example, regulatory elements that modulate gene expression
such as translational and transcriptional enhancers, translational and
transcriptional repressors, and mRNA stability or instability determinants. In
the context of this disclosure, the term "regulatory element" also refers to a
sequence of DNA, usually, but not always, upstream (5') to the coding
sequence of a structural gene, which includes sequences which control the
expression of the coding region by providing the recognition for RNA
polymerase and/or other factors required for transcription to start at a
particular
site. An example of a regulatory element that provides for the recognition for
RNA polymerase or other transcriptional factors to ensure initiation at a
particular site is a promoter element. A promoter element comprises a core
promoter element, responsible for the initiation of transcription, as well as
other regulatory elements (as listed above) that modify gene expression. It is
to
be understood that nucleotide sequences, located within introns, or 3' of the
coding region sequence may also contribute to the regulation of expression of
a
coding region of interest. A regulatory element may also include those
elements
located downstream (3') to the site of transcription initiation, or within
transcribed regions, or both. In the context of the present invention a post-
transcriptional regulatory element may include elements that are active
following transcription initiation, for example translational and
transcriptional
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enhancers, translational and transcriptional repressors, and mRNA stability
determinants.
The regulatory elements, or fragments thereof, of the present invention
may be operatively associated with heterologous regulatory elements or
promoters in order to modulate the activity of the heterologous regulatory
element. Such modulation includes enhancing or repressing transcriptional
activity of the heterologous regulatory element, modulating post-
transcriptional
events, or both enhancing or repressing transcriptional activity of the
heterologous regulatory element and modulating post-transcriptional events.
For example, one or more regulatory elements, or fragments thereof, of the
present invention may be operatively associated with constitutive, inducible,
or
tissue specific promoters or fragment thereof, to niodulate the activity of
such
promoters within plant, insect, fungi, bacterial, yeast, or animal cells.
An example of a cryptic regulatorv elemeiu of the present invention,
which is not to be considered limiting in any manner, is an organ-specific,
and
temporally-specific element obtained f--oi1i plant T218. Such an element is a
seed-specific regulatory element. More preferably, the element is a seed-coat
specific regulatory element as described herein, or an analogue thereof, or a
nucleic acid fragment localized between EcoRI - Smal 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:1. For example, which is not to be considered
limiting in any manner, the nucleic acid may exhibit 80% similarity to the
nucleotide sequence comprising nucleotides 1-467, or 1-993, of SEQ ID NO: 1.
Furthermore, the seed-coat specific nucleotide sequence may be defined as
comprising at least a 19 bp fragment of nucleotides 1-467, or 1-993 as defined
within SEQ ID NO: 1.
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Another example of a cryptic regulatory element of an aspect of the
present invention includes, but is not limited to, a constitutive regulatory
element obtained from the plant T1275, as described herein and analogues or
fragments thereof, or a nucleic acid fragment localized between Xbal - 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 Xbal - Smal as identified by the restriction
map of Figure 13 (A) or (C) or a fragment thereof. The constitutive cryptic
regulatory element may also be defined by a nucleotide sequence comprising at
least an 18 bp fragment of the regulatory region defined in SEQ ID NO:2, or
by a nucleic acid comprising from about 80% similarity to the nucleotide
sequence of SEQ ID NO:2.
A further regulatory element of the present invention includes an
enhancer element within the -394 to -62 fragment of T1275 (nucleotides 1660
to 1992 of SEQ ID NO:2). This fragment may also be duplicated and fused to
a regulatory region, for example a core promoter, producing an increase in the
activity of the regulatory region (see Figure 13 (D)).
Another cryptic regulatory element of the present invention includes,
but is not limited to, a post-transcriptional or translational enhancer
regulatory
element localized between Ndel - Smal (see Figure 15, nucleotides 7-188 of
SEQ ID NO:3). The post-transcriptional or translational enhancer regulatory
element may also comprise the nucleotide sequehce as defined by nucleotides 1-
141 of SEQ ID NO:3 (nucleotides 2084-2224 of SEQ ID NO:2) or an analog
thereof, or the element may comprise 80% similarity to the nucleotide sequence
of nucleotides 1-141 of SEQ ID NO:3 (nucleotides 2086-2224 of SEQ ID
NO:2).
A shortened fragment of the Ndel - SmaI fragment, referred to as ON,
dN or deltaN is also characterized within the present invention. AN was
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prepared by mutagenesis replacing the out of frame ATG (located at nucleotides
2087-2089, SEQ ID NO: 1) within the Ndel-Smal fragment (see Figure 18).
ON constructs with (SEQ ID NO:4) or without (SEQ ID NO:5) a Kozak
consensus sequence was also characterized (Tables 10, and 12) and found to
exhibit enhancer activity. Therefore, other cryptic regulatory elements of the
present invention include, but are not limited to, post-transcriptional or
translational enhancers regulatory elements localized at nqcleotides 1-97 of
SEQ ID NO:4 and nucleotides 1-86 of SEQ DI NO: 4 or 5. These post-
transcriptional or translational enhancer regulatory elements may comprise the
nucleotide sequence as defined by nucleotides 1-86 of SEQ ID NO:4 or 5
(nucleotides 2170-2224 of SEQ ID NO:2) or an analog thereof, or the element
may comprise 80% similarity to the nucleotide sequence of nucleotides 1-86 of
SEQ ID NO:4 or 5 (nucleotides 2170-2224 of SEQ ID NO:2). Furthermore,
these regulatory elements may comprise the nucleotide sequence as defined by
nucleotides 1-97 of SEQ ID NO:4 and comprising a Kozack sequence or an
analog thereof, or the element may comprise 80% similarity to the nucleotide
sequence of nucleotides 1-97 of SEQ ID NO:4.
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); nucleotides 1-1660 of SEQ ID NO:2), a
transcriptional enhancer localized within the BstYl-Di-al fragment of Figure
13
(C) (nucleotides 1660-1875 of SEQ ID NO:2), a core promoter element located
within the Dral-Ndel fragment of Figure 13 (C) (nucleotides 1875- 2084 of SEQ
ID NO:2), a transcriptional enhancer within the Dral to -62 fragment
(nucleotides 1875-1992 of SEQ ID NO:2; Figures 13 (D) to (G)), or a
regulatory element or post-transcriptional element downstream of the
transcriptional start site, for example but not limited to the Ndel-SmaI
fragment
(nucleotides 1-188 of SEQ ID N03) and derivatives and fragments thereof (for
example nucleotides 1-141 of SEQ ID NO:3), including ON (nucleotides 1-129
or 7-97 of SEQ ID NO:4, ON"' (nucleotides 1-119 or 7-86 SEQ ID NO:5), and
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nucleotides 7-86 of SEQ ID NO:4 or 5(nucleotides 2084 to 2170 of SEQ ID
NO:2).
An "analogue" of the above identified cryptic regulatory elements
includes any substitution, deletion, or additions to the sequence of a
regulatory
element provided that said analogue maintains at least I 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 constitutive. Ai-egulatory 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,
soine 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 that is capable of directly or
indirectly activating transcription of one or more DNA sequences or genes in
response to an inducer. In the absence of an inducer the DNA sequences or
genes will not be transcribed. Typically the protein factor, that binds
-= specifically to an inducible regulatory element to activate transcription,
is
present in an inactive form which is then directly or indirectly converted to
the
active form by the inducer. The inducer can be a chemical agent such as a
protein, metabolite, growth regulator, herbicide or phenolic compound or a
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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:
1155-
1165) and triosephosphate isomerase 1 (Xu et al, 1994, Plant Physiol. 106:
459-467) genes, the maize ubiquitin 1 gene (Cornejo et a], 1993, Plant Mol.
Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al,
1995, Plant Mol. Biol. 29: 637-646), and the tobacco translational initiation
factor 4A gene (Mandel et al, 1995 Plant Mol. Bial. 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 to 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
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or gene expression. The polyadenylation signal is usually characterized by
effecting the addition of polyadenylic acid tracks to the 3' end of the mRNA
precursor. Polyadenylation signals are commonly recognized by the presence
of homology to the canonical form 5' AATAAA-3' although variations are not
uncommon.
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 structural gene. The sequence
can
also be derived from the regulatory element selected to express the gene, and
can be specifically modified so as to increase translation of the mRNA.
To aid in identification of transformed plant cells, the constructs of this
invention may be further manipulated to include plant selectable markers.
Useful selectable markers include enzymes which provide for resistance to an
antibiotic such as gentamycin, hygromycin, kanamycin, and the like.
Similarly, enzymes providing for production of a compound identifiable by
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colour change such as GUS (P-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. However, it is to be understood that the regulatory elements of the
present invention may also be combined with gene of interest for expression
within a range of host organisms. Such organisms include, but are not limited
to:
= plants, both monocots and dicots, for example, corn, wheat,
barley, oat, tobacco, Brassica, soybean, pea, alfalfa, potato,
ginseng, Arabidopsis;
= trees, for example peach, spruce;
= yeast, fungi, insects, animal and bacteria cells.
Methods for the transformation and regeneration of these organisms are
established in the art and known to one of skill in the art.
By "gene of interest" it is meant any gene that is to be expressed within
a host organism. Such a gene of interest may include, but is not limited to, a
gene that encodes a pharmaceutically active protein, for example growth
factors, growth regulators, antibodies, antigens, their derivatives useful for
immunization or vaccination and the like. Such proteins include, but are not
limited to, interleukins, insulin, G-CSF, GM-CSF, hPG-CSF. M-CSF or
combinations thereof, interferons, for example, interferon-a, interferon-B,
interferon-i, blood clotting factors, for example, Factor VIII, Factor IX, or
tPA or combinations thereof. A gene of interest may also encode an industrial
enzyme, protein supplement, nutraceutical, or a value-added product for feed,
food, or both feed and food use. Examples of such proteins include, but are
not limited to proteases, oxidases, phytases, chitinases, invertases, lipases,
cellulases, xylanases, enzymes involved in oil biosynthesis etc.
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Methods of regenerating whole plants from plant cells are also known in
the art. In general, transformed plant cells are cultured in an appropriate
medium, which may contain selective agents such as antibiotics, where
selectable markers are used to facilitate identification of transformed plant
cells.
Once callus forms, shoot formation can be encouraged by employing the
appropriate plant hormones in accordance with known methods and the shoots
transferred to rooting medium for regeneration of plants. The plants may then
be used to establish repetitive generations, either from seeds or using
vegetative
propagation techniques.
The constructs of the present invention can be introduced into plant cells
using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA
transformation, micro-injection, electroporation, etc. For reviews of such
techniques see for example Weissbach and Weissbach, Methods for Plant
Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988);
Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and
Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed.
DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (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, 2, 3, 4 and 5, the regulatory regions and
fragments thereof, as well as analogues of, or nucleic acid sequences
comprising about 80% similarity with the nucleic acids as defined in SEQ ID
NO's: 1 to 5. Analogues (as defined above), include those DNA sequences
which hybridize under stringent hybridization conditions (see Maniatis et al.,
in
Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory,
1982, p. 387-389) to any one of the DNA sequence of SEQ ID NO: 1, 2, 3, 4,
or 5, provided that said sequences maintain at least one regulatory property
of
the activity of the regulatory element as defined herein.
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An example of one such stringent hybridization conditions may be
hybridization in 4XSSC at 65 C, followed by washing in 0.1XSSC 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 any one of the sequences of SEQ ID NO: 1 to 5
under relaxed hybridization conditions, provided that said sequences maintain
at
least one regulatory property of the activity of the regulatory element.
Examples of such non-hybridization conditions includes hybridization in
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 T218 is regulated by a
cryptic
regulatory element. The region surrounding the regulatory element and
transcriptional start site for the GUS gene are not transcribed in
untransformed
plants. Transcription was only observed in plant T218 when T-DNA was
inserted in cis. DNA sequence analysis did not uncover a long open reading
frame within the 3.3 kb region cloned. Nloreover. 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 ainong 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 constitutive expression of GUS
activity in the plant T1275 is regulated by a cryptic regulatory element.
RNase
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protection assays perforrhed 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 T1275 sequence from
about -2055 bp to +1200 bp relative to the transcriptional start site. In all
tissues
tested (leaf, stem, root, flower bud, petal, ovary and developing seed)
protected
fragments were not detected, inthe sense orientation relative to the GUS
coding
region, with all probes (Figure 16; see also PCT CA97/00064.
Furthermore, GenBank searches revealed no
significant sequence similarity with the T1275 sequence. An amino acid
identity
of about 66% with two open reading frames on the antisense strand of the
genomic sequence of T1275 (between about -1418 and -1308, nucleotides 636-
746 of SEQ ID NO:2; and between about -541 and -395, nucleotides 1513-1659
of SEQ ID NO:2 relative to the transcriptional start) and an open reading
frame of
a partial Arabidopsis expressed sequence (GenBank Accession No. W43439) was
identified. The sequence which lies downstream of sequences at the T-DNA
insertion point in untransformed tobacco shows no significant similarity in
GenBank searches. These data suggest that this region is silent in
untransformed
plants and that the insertion of the T-DNA activated a cryptic promoter.
Southern analysis indicates that the 2.2 kb regulatory region of T1275
does not hybridize with DNA isolated froni soybean, potato, sunflower,
Arabidopsis, B. napus, B. oleracea, corn, wheat or black spruce. However,
transient assays indicate that this regulatory region can direct expression of
the
GUS coding region in all plant species tested including canola. tobacco,
Brassica,
ArabidOpsls, soybean. alfalfa, pea, ginseng, potato, corn, wheat, barley,
white
spruce and peacll (Table 3), indicating that this regulatory element is useful
for
directing gene expression in both dicot and monocot plants as well as trees.
Furthermore, regulatory elements were also found to modulate gene expression
in a diverse range of species including yeast, bacteria and insect cells.
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The transcriptional start site was delimited by RNase protection assay to a
single position about 220 bp upstream of the translational initiation codon of
the
GUS coding region in the T-DNA. The sequence around the transcriptional start
site exhibits similarity with sequences favored at the transcriptional start
site
compiled from available dicot plant genes (T/A T/C A+, A C/A C/A A/C/T A A
A/T). Sequence similarity is not detected about 30 bp upstream of the
transcriptional start site with the TATA-box consensus compiled from available
dicot plant genes (C T A T A A/T A T/A A).
Deletions in the upstream region indicate that negative regulatory
elements and enhancer sequences exist witliin the full length reaulatory
region.
For example, deletion of the 5' region to BstYI (-394 relative to the
transcriptional start site; see Figure 13 (C)) resulted in a 3 to 8 fold
increase in
expression of the gene associated therewith (see Table 6), indicating the
occurrence of at least one negative regulatory element within the Xbal-BstYt
portion of the full length regulatory element. Other negative regulatory
elements also exist within the Xbal- BstYl fragment as removal of an Xbal-Pstl
fragment also resulted in increased activity (-1403-GUS-nos; Table 6). An
enhancer is also localized within the BstYI-Dral fragment as removal of this
region results in a 4 fold loss in activity of the remaining regulatory region
(-
197-GUS-nos; Table 6).
5' deletions of the promoter (see Figures 13(B) and (C) and analysis by
transient expression using biolistics showed that the promoter was active
within a
fragment 62 bp from the transcriptional start site indicating that the core
promoter
has a basal level of expression (see Table 5). Deletion of a fragment
containing
the transcriptional start site (see -62(-tsr)/GUS/nos in Figure 13 (C); Table
5) did
not eliminate expression, however deletions to -12 bp and further (i.e. +30)
did
eliminate expression indicating that the region defined by -(62-12) bp
(nucleotides 1992-2042 of SEQ ID NO:2) contained the core promoter. DNA
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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 clones (Figures 13 (B) and (C))
were transferred into tobacco and Arabidopsis by Agrobacterium-mediated
transformation using the vector pRD400. Analysis of GUS specific activity in
leaves of transgenic plants (see Table 6) confirmed the transient expression
data
down to the -197 fragment (nucleotides 1875-2224 of SEQ ID NO:2).
Histochemical analysis of tobacco organs sampled from the transgenic plants
indicated GUS expression in leaf, seeds and flowers. Histochemical analysis of
Arabidopsis organs revealed GUS activity in leaf, stem flowers and silques
when
the promoter was deleted to the -394 and -197 fragments (see Figures 13 (E) to
(G)).
A comparison of GUS specific activities in the leaves of transgenic
tobacco SR1 transformed with the T1275-GUS-nos gene and the 35S-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.
Expression of GUS, under the control of T1275 or a fragment thereof, or
the modulation of GUS expression arising from T1275 or a fragment thereof, has
been observed in a range of species including corn, wheat, barley, oat,
tobacco,
Brassica, soybean, alfalfa, pea, potato, Ginseng, Arabidopsis, peach, spruce,
yeast, fungi, insects and bacterial cells.
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Further analysis confirmed the presence of a regulatory sequence within
the NdeI-Smal fragment of the mRNA leader sequence that had a significant
impact on the level of GUS specific activity expressed in all organs tested.
Deletion of the Ndel-SmaI fragment from the T1275-GUS-nos gene (Figure 15)
resulted in about a 46-fold reduction in the amount of GUS specific activity
that
could be detected in leaves of transgenic tobacco cv Delgold (see Table 7).
Similar results were also obserevd in the transgenic tobacco cultivar SR1 and
transgenic alfalfa (Table 7). Addition of the same fragment to a 35S-GUS-nos
gene construct (Figure 15) increased the amount of GUS specific activity by
about 5-fold in transgenic tobacco and a higher amount in transgenic alfalfa
(see
Table 7). Increased GUS activity was observed in organs of tobacco and alfalfa
plants tranfonned with constructs containing A'cfel-Smal fragment (Table 8 and
9).
A modulation of GUS activity was noted in a variey of species that were
transformed with a regulatory element of the prescnt invention. For example
but
not necessarily limited to, the Ndel-Smul frag ment c7f'"1,1275 (also referred
to as
"N") and derivatives or analogues thereot'. produced an increase in activity
within
a variety of organisms tested including a range of plants (Tables 3 and 10,
and
Figure 19), white spruce (a conifer; Table 11) and veast (Table 12).
A shortened fragment of the Ndel -SmaI fragment, (referred to as "ON",
"dN", or "deltaN") was produced that lacks the out-of frame upstream ATG at
nucleotides 2087-2089 of SEQ ID NO:2 (see Figure 18(A) and (B)). Constructs
comprising T1275(L1N)-GUS-nos yielded 5 fold greater levels of GUS activity in
leaves of transgenic tobacco compared to plants expressing T1275-GUS-nos.
Furthermore, in corn callus and yeast, AN significantly increased GUS
expression driven by the 35 S promoter (Figure 19 and Table 10).
The Ndel-Smal regulatory elements situated downstream of the
transcriptional start site functions both at a transcriptional, and post-
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transcriptional level. The levels of mRNA observed in transgenic plants
transformed with T1275-GUS-nos are higher than the levels in plants
transformed
with T1275(-N)-GUS-nos. However, the opposite is true with plants tranformed
with 35S-GUS-nos or 35S(+N)-GUS-nos, where higher levels of mRNA are
detected in the absence of the Ndel-SmaI fragment (see Figures 17 (A) and
(B)).
This indicates that this region functions by either modulating transcriptional
rates, or the stability of the transcript, or both.
The NdeI-SmaI region also functions post-transcriptionally. The ratio of
GUS specific activity to relative RNA level in individual transgenic tobacco
plants that lack the NdeI-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 (C)). Similarly, an increase, by an average of six fold, in
GUS specific activity is observed when the NdeI-SmaI region is added within
the 35S untranslated region (Figure 17 (C)). The GUS specific
activity:relative
RNA levels are similar in constructs containing the NdeI-SmaI fragment
(T1275-GUS-nos and 35S+N-GUS-nos). These results indicate that the NdeI-
Smai fragment modulates gene expression post-transcriptionally. Further
experiments suggest that this region is a novel translational enhancer.
Translation of transcripts in vitro demonstrate an increase in translational
efficiency of RNA containing the NdeI to Smal fragment (see Table 13).
Furthermore, the levels of protein produced using mRNAs comprising the
NdeI-SmaI fragment are greater than those produced using the known
translational enhancer of Alfalfa Mosaic Virus RNA4. These results indicate
that this region functions post-transcriptionally, as a translational
enhancer.
As this is the first report of cryptic regulatory elements in plants, it is
impossible to estimate the degree to which cryptic regulatory elements may
contribute to the high frequencies of promoterless marker gene activation in
plants. It is interesting to note that transcriptional GUS fusions in
Arabidopsis
occur at much greater frequencies (54%) than translational fusions (1.6%,
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Kertbundit et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5212-5216). The
possibility that cryptic promoters may account for some fusions was recognized
by Lindsey et al. (1993, Transgenic Res. 2, 33-47).
The regulatory elements of the present invention may be used to control
the expression of a gene of interest within desired host expression system,
for
example, but not limited to:
= plants, both monocots and dicots, for example, corn, tobacco,
Brassica, soybean, pea, alfalfa, potato, ginseng, wheat, oat,
barley, Arabidopsis;
= trees, for example peach, spruce;
= yeast, fungi, insects, and bacteria.
Furthermore, the regulatory elements as described herein may be used in
conjunction with other regulatory elements, such as tissue specific, inducible
or
constitutive promoters, enhancers, or fragments thereof, and the like. For
example, the regulatory region or a fragment thereof as defined herein may be
used to regulate gene expression of a gene of interest spatially and
developmentally within developing seed coats, or within a heterologous
expression system, for example yeast, insects, or fungi expression systems.
Some examples of such uses, which are not to be considered limiting, include:
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.
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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 coding 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 bp as
a probe in order to identify analogous regions within other
plants.
8. Enhancing expression of a gene of interest within a host
organisms of interest. Regulatory regions or fragments thereof,
including enhancer fragments of the present invention, may be
operatively associated with a heterologous nucleotide sequence
including heterologous regulatory regions to increase the
expression of a gene of interest within a host organism. A gene
of interest may include, but is not limited to, a gene that encodes
a pharmaceutically active protein, for example growth factors,
growth regulators, antibodies, antigens, their derivatives useful
for immunization or vaccination and the like. Such proteins
include, but are not limited to, interleukins, insulin, G-CSF,
GM-CSF, hPG-CSF, M-CSF or combinations thereof,
interferons, for example, interferon-a, interferon-B, interferon-i,
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blood clotting factors, for example, Factor VIII, Factor IX, or
tPA or combinations thereof. A gene of interest may also
encode an industrial enzyme, protein supplement, nutraceutical,
or a value-added product for feed, food, or both feed and food
use. Examples of such proteins include, but are not limited to
proteases, oxidases, phytases chitinases, invertases, lipases,
cellulases, xylanases, enzymes involved in oil metabolic and
biosynthetic pathways etc.
Similarly, a constitutive regulatory element may also be used to drive
the expression within all organs or tissues, or both of a plant of a gene of
interest, and such uses are well established in the literature. For example,
fragments of specific elements within the 35S CaMV promoter have been
duplicated or combined with other promoter fragments to produce chimeric
promoters with desired properties (e.g. U.S. 5,491,288, 5,424,200,
5,322,938, 5,196,525, 5,164,316). As indicated above, a constitutive
regulatory element or a fragment thereof', as defined herein, may also be used
along with other promoter, enhancer elements, or fragments thereof ,
translational enhancer elements or fragments thereof in order to control gene
expression. Furthermore, oligonucleotides of 18 bps or longer are useful as
probes or PCR primers in identifying or amplifying related DNA or RNA
sequences in other tissues or organisms.
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
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plant cell for the production of any transgenic plant. The present invention
is
not limited to any plant species, or species other than plant.
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 Agrobacterium and leaf disc
transformation of Nicotiana tabacum SR1 were performed as described by
Fobert et al. (1991, Plant Mol. Biol. 17, 837-851). Plant tissue was
maintained on 100 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 transformants in a range of plant
organs, T1275 was selected which exhibited high level, constitutive expression
of GUS.
Characterization of a Seed Coat-Speciflc 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. (PlantMol. Biol., 1991, 17, 837-851). For initial screening, leaves were
harvested from in vitro grown plantlets. Later flowers corresponding to
developmental stages 4 and 5 of Koltunow et al. (Plant Cell, 1990, 2,
1201-1224) and beige seeds, approximately 12-16 dpa (Chen et al., 1988,
EMBO J. 7, 297-302), were collected from plants grown in the greenhouse.
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For detailed, quantitative analysis of GUS activity, leaf, stem and root
tissues
were collected from kanamycin resistant Fl 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 sections were analyzed for
each
organ. For each plant, histological assays were performed on at least two
different occasions to ensure reproducibility. Except for floral organs, all
tissues were assayed in phosphate buffer according to Jefferson (1987, Plant
Mol. Biol. Rep. 5, 387-405), with 1 mM X-Gluc (Sigma) as substrate. Flowers
were assayed in the same buffer containing 20% (v/v) methanol (Kosugi et al.,
1990, Plant Sci. 70, 133-140).
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 tissue 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
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strictly regulated, it must originate from GUS fusions transmitted to seeds
maternally and not by pollen. As shown in Table 1, this is indeed the case. As
a control, GUS fusions expressed in embryo and endosperm, which are the
products of double fertilization, should be transmitted through both gametes.
This is illustrated in Table 1 for GUS expression driven by the napin promoter
(BngNAPI, Baszczynki and Fallis, 1990, Plant Mol. Biol. 14, 633-635) which
is active in both embryo and endosperm=
Table 1. GUS activity in seeds at 14 days post anthesis.
Cross GUS Activity
nmole MU/min/mg Protein
T218 T218 1.09 0.39
T218 WTI' 3.02 0.19
WT T218 0.04 0.005
WT WT 0.04 0.005
NAP-5' NAP-5 14.6 7.9
NAP-5 WT 3.42 1.60
WT NAP-5 2.91 1.97
WT, untransformed plants
Transgenic tobacco plants with the GUS gene fused to the
napin, BngNAP1, 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 EcoRI using the appropriate
manufacturer-supplied buffer supplemented with 2.5 mM spermidine. After
electrophoresis through a 0.8% TAE agarose gel, the DNA size fraction around
4-6 kb was isolated, purified using the GeneClean kit (BIO 101 Inc., LaJolla,
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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 #1, Figure 2). This sequence, contained in a 260 bp SstI/EcoRl
restriction fragment from pPRF-101 (Fobert et al., 1991, Plant Mol. Biol. 17,
837-851), was labelled with [a 32P]-dCTP (NEN) using random priming
(Stratagene). After plaque purification, phage DNA was isolated (Sambrook et
al., 1989, A Laboratory Manual. New York: Cold Spring Harbor Laboratory
Press), mapped and subcloned into pGEM*-4AZ (Promega).
The GUS fusion in plant T218 was isolated as a 4.7 kb EcoRI fragment
containing the 2.2kb promoterless GUS-170s 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). To confirm the ability of the
flanking DNA to activate the GUS coding region. the entire 4.7 kb fragment
was inserted into the binary transformation vector pBIN19 (Bevan, 1984, Nucl.
Acid Res. 12, 8711-8721), as shown in Figure 2. Several transgenic plants
were produced by Agro6acterium-mediated transformation of leaf discs. Plants
were transformed with a derivative which contained the 5' end of the GUS gene
distal to the left border repeat. This orientation is the same as that of the
GUS
gene in the binary vector pB1101 (Jefferson, 1987, Plant Mol. Biol. Rep. 5,
387-405). Southern blots indicated that each plant contained 1-4 T-DNA
insertions at unique sites. The spatial patterns of GUS activity were
identical to
that of plant T218. Histologically, GUS staining was restricted to the seed
coats of 14 dpa seeds and was absent in embryos and 20 dpa seeds (results not
shown). Fluorogenic assays of GUS activity in developing seeds showed that
expression was restricted to seeds between 10 and 17 dpa, reaching a maximum
at 12 dpa (Figure 3 (a) and 3 (b)). The 4.7 kb fragment therefore contained
all
*Trademark
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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 restriction site approximately
0.5 kb, 5' of the insertion site (pT218-3, Figure 2), resulted in the complete
loss of GUS activity in developing seeds (Figures 3b and 4). This suggests
that
the region approximately between 1.0 and 0.5 kb, 5' of the insertion site
contains elements essential to gene activation. GUS activity in seeds remained
absent with more extensive deletion of plant DNA (pT218-4, 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.
The transcriptional start site for the GUS gene in plant T218 was
determined by RNase protection assays with RNA 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 SURB mRNA. DNA templates were linearized
and transcribed in vitro with either T7 or SP6 polymerases to generate strand-
specific RNA probes using the Promega transcription kit and [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, Plant J. 2, 321-330), using
10-
30 g of total RNA per assay. Probe digestion was done at 30 C for 15 min
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using 30 g ml-' 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
bp 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 I ATCA; Joshi, 1987 Nucleic Acids Res. 15, 6643-6653). A TATA
box consensus sequence is present 37 bp upstream of this start site (Figure
6).
The second, minor terminus mapped 254 bp 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 (Brain et
al., 1992, Plant Cell 4, 463-471; Gasser and Laemmli, 1986, Cell 46, 521-
530), identified several putative regulatory elements in the first 1.0 kb of
tobacco DNA flanking the promoterless GUS gene.
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 SR1 plants by Stratagene for cloning of the insertion
site corresponding to the gene fusion in plant T218. The screening of 500,000
plaques with probe #2 (Figure 2) yielded a single lambda clone. The EcoRI
and Xbal 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 pIS-1, were identical over a
length of more than 2.5 kb, from the insertion site to their 5' ends, except
for a
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12 bp filler DNA insert of unknown origin at the T-DNA border (Figure 6 and
data not shown). The presence of filler DNA is a common feature of T-
DNA/plant DNA junctions (Gheysen et al., 1991, Gene 94, 155-163). Gross
rearrangements that sometimes accompany T-DNA insertions (Gheysen et al.,
1990, Gene 94, 155-163; and 1991, Genes Dev. 5, 287-297) were not found
(Figure 6) and therefore could not account for the promoter activity
associated
with this region. The region of pIS-1 and pIS-2, 3' of the insertion site is
also
very AT-rich (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. To determine whether the region
surrounding the insertion site was transcribed in untransformed plants,
Northern blots were performed with RNA from leaf, stem, root, flower and
seeds at 4, 8, 12, 14, 16, 20 and 24 dpa. Total RNA from leaves was isolated
as described in Ouellet et al., (1992, Plant J. 2, 321-330). To isolate total
RNA from developing seeds, 0.5 g of frozen tissue was pulverized by grinding
with dry ice using a mortar and pestle. The powder was homogenized in a 50
ml conical tube containing 5 ml of buffer (1 M Tris HC1, pH 9.0, 1 % SDS)
using a Polytron homogenizer. After two extractions with equal volumes of
phenol:chloroform:isoamyl alcohol (25:24:1), nucleic acids were collected by
ethanol precipitation and resuspended in water. The RNA was precipitated
overnight in 2M LiCI at 0 C, collected by centrifugation, washed in 70%
ethanol and resuspended in water. Northern blot hybridization was performed
as described in Gottlob-McHugh et al. (1992, Plant Physiol. 100, 820-825).
Probe #3 (Figure 2) which spans the entire region of pT218 5' of the insertion
-= did not detect hybridizing RNA bands. To extend the
sensitivity of RNA detection and to include the region 3' of the insertion
site
within the analysis, RNase protection assays were performed with 10 different
RNA probes that spanned both strands of pIS-1 and pIS-2 (Figure 7). Even
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after lengthy exposures, protected fragments could not be detected with RNA
from 8, 10, 12 dpa seeds or leaves of untransformed plants (see Figure 5 for
examples with two of the probes tested). The specific conditions used allowed
the resolution of protected RNA fragments as small as 10 bases.
Failure to detect protected fragments was not due to problems of RNA
quality, as control experiments using the same samples detected
acetohydroxyacid synthase (AHAS) SURA and SURB mRNA which are
expressed at relatively low abundance. Conditions used in the
present work were estimated to be sensitive enough to detect low-abundance
messages representing 0.001-0.01 % of total rnRNA levels (Ouellet et al.,
1992,
Plant J. 2, 321-330). Therefore, the region flanking the site of T-DNA
insertion does not appear to be transcribed in untransformed plants.
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 labelled, 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 temperature. 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 witll HindIII, Xbal 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 yielded the same results.
They
did not reveal hybridizing bands, even under conditions of reduced stringency,
in additional Nicotiana species including N. rustica, N. glutinosa, N.
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megalosiphon and N. debneyi. Probe #3 (Figure 2) revealed
the presence of moderately repetitive DNA specific to the N. tomentosiformis
genome. These results suggest that the region flanking the
insertion site is unique to the N. tomentosiformis genome and is not conserved
among related species as might be expected for regions that encode essential
genes.
Characterization of a Constitutive GUS fusion -T1275
From the transgenic plants produced (see above), one of these, T1275,
was chosen for detailed study because of its high level and constitutive
expression of GUS (see also US Patent 5,824,872 and PCT/CA97/00064).
Fluorogenic and histological GUS assays were performed as outlined
above. For initial screening, leaves were harvested from in vitro grown
plantlets. Later nine different tissues: leaf (L), stem (S), root (R), anther
(A),
petal (P), ovary (0), sepal (Se), seeds 10 days post anthesis (S1) and seeds
20
days post-anthesis (S2), were collected from plants grown in the greenhouse
and analyzed.
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 (0),
sepal
(Se), seeds at 10 dpa (Si) and 20 dpa (S2). Furthernnore, the level of GUS
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expression in leaves was comparable to the level of expression in transformed
plants containing the constitutive promoter CaMV 35S in a GUS - nos fusion.
As reported by Fobert et al. (1991, Plant Molecular Biology, 17: 837-851)
GUS activity in transformed plants containing pBI121 (Clontech), which
contains a CaMV 35S - GUS - nos chimeric gene, was as high as 18,770
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 T1275 total DNA was digested
with EcoR1 and Xbal according to the manufacturer's instructions. The
digested DNA was size-fractionated on a 0.7% agarose gel. The DNA
fragments of about 4 to 6 kb were isolated from the gel using the Elu-Quick
kit
(Schleicher and Schuell) and ligated to lambdaGEM-2 arms previously digested
with EcoRI and Xbal and phosphatase-treated. About 40,000 plaques were
transferred to a nylon membrane (Hybond*,Amersham) and screened with the
32P-labelled 2kb GUS insert isolated from pBI121, essentially as described in
Rutledge et al. (1991, Mol. Gen Genet. 229, 31-40). The positive clones were
isolated. The XbaI-EcoRI fragment (see restriction map Figure 12) was
isolated from the lambda phage and cloned into pTZ19R previously digested
with Xbal and EcoRI and treated with intestinal calf phosphatase.
The plant DNA sequence within the clone, SEQ ID NO:2, has not been
previously reported in sequence data bases. It is not observed among diverse
species as Southern blots did not reveal bands hybridizing with the fragment
in
soybean, potato, sunflower, Arabidopsis, B. napus, B. oleracea, corn, wheat or
black spruce. In tobacco, Southern blots did not reveal
evidence for gross rearrangements at or upstream of the T-DNA insertion site .
*Trademark
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The T1275 Regulatory Element is Cryptic
The 4.2kb fragment containing about 2.2kb of the T1275 promoter
fused to the GUS gene and the nos 3' was isolated by digesting pTZ-T1275 with
HindIll and EcoRI. The isolated fragment was ligated into the pRD400 vector
(Datla et al., 1992, Gene, 211:383-384) previously digested with HindIIl and
EcoRI and treated with calf intestinal phosphatase. Transfer of the binary
vector to Agrobacterium tumefaciens and leaf disc transformation of N.
tabacuin SR1 were performed as described above. GUS activity was examined
in several organs of many independent transgenic lines. GUS mRNA was also
examined in the same organ by RNase protection assay (Melton et al, 1984,
Nucleic Acids Res. 121: 7035 - 7056) using a probe that mapped the mRNA 5'
end in both untransformed and transgenic tissues. RNA was isolated from
frozen-ground tissues using the TRIZOL* Reagent (Life Technologies) as
described by the manufacturer. For each assay 10 - 30 ug of total RNA was
hybridized to RNA probes described in Figure 16 (A). Assays were performed
using the RPAII kit (Ambion CA) as described by the manufacturer. The
protected fragments were separated on a 5 % Long Ranger* acrylamide (J.J.
Baker, N.J.) denaturing gel which was dried and exposed to Kodak X-RP film.
RNase protection assays performed with RNA from leaves, stem, root,
developing seeds and flowers of transgenic tobacco revealed a single protected
fragment in all organs indicating a single transcription start site that was
the
same in each organ, whereas RNA from untransformed tobacco tissues did not
reveal a protected fragment (Figure 16 (B)). The insertion site, including
1200
bp downstream, was cloned from untransformed tobacco as a PCR fragment
and sequenced. A composite restriction map of the insertion site was
assembled as shown in Figure 16 (A). RNA probes were prepared that spanned
the entire region as shown in Figure 16 (A). RNase protection assays did not
reveal transcripts from the sense strand as summarized in Table 2. These data
suggest that the insertion site is transcriptionally silent in untransformed
*Trademark
r N, ~ 'f1
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tobacco and is activated by T-DNA insertion. The region upstream of the
insertion site is therefore another example of a plant cryptic regulatory
element.
Table 2.
Summary of the RNase Protection Assays of the insertion site in
untransformed tobacco. See Figure 16 (A) for probe positions.
Probe Rnase Protection Assay result
Looking for "sense" RNAs (relative to the T1275 promoter)
C8-EcoRI many bands. all in tRNA (negative control)
A 10-HindIIl no bands
2-21-HindIII no bands
1-4 Smal many bands. all in tRNA
7-EcoRl faint bands. all in tRNA
Constitutive Activity of the T1275 Regulatory Element
For analysis of transient expression of GUS activity mediated by
biolistics (Sandford et al, 1983, Methods Enzymol, 217: 483-509), the XbaI -
EcoRI fragment was subcloned in pUC 19 and GUS 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-GiJS-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
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and Arabidopsis, potato, Ginseng, peach and cell suspensions of oat, corn,
wheat and barley exhibited GUS-positive blue spots after transient
bombardment-mediated assays and histochemical GUS activity staining. This
suggests that the T1275 regulatory element may be useful for directing gene
expression in both dicot and monocot plants.
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TABLE 3
Transient Expression of GUS Activity in Tissues of Diverse Plant Species
Tissue Source Species GUS Activity *
Leaf Soybean + + +
Alfalfa + +
Arabidopsis +
Potato + +
Ginseng + +
Peach +
Leaf disc Tobacco + +
B. napus +
Pea +
Cell Cultures Oat +
Corn +
Wheat +
Barley + +
White spruce + +
* Numbers of blue spots: 1 - 10 (+), 10 - 100 (++), 100 - 400 (+ + +)
For analysis of GUS expression in diffet-ent organs, lines derived from
progeny of the above transgenic tobacco lines were examined in detail. Table 4
shows the GUS specific activities in one of these plants. It is expressed in
leaf,
stem, root, developing seeds and the floral organs, sepals, petals, anthers,
pistils and ovaries at varying levels, confirming constitutive expression.
Introduction of the same vector into B. napus, Ai-abidopsis, and alfalfa also
revealed expression of GUS activity in these organs 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 (Figure 16 (B)) and the level of mRNA was similar except in
flower buds where it was lower (Table 4).
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TABLE 4
GUS Specific Activity and Relative RNA Levels in the
Organs of Progeny of Transgenic Line T64
Organ Relative GUS RNA GUS Specific Activity
Levels in T64 (picomol/MU/min/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 - buds 715 2.59 ND*
Petals ND* 28.24 1.29
Anthers ND* 4.64 0.35
Pistils ND* 9.76 1.72
Sepals ND* 110.02 2.48
Ovary ND* 4.42 2.71 11
* Not Done
Identification of Regulatory Elements within the Full Length T1275
Regulatory Element
An array of deletions of the full length regulatory region of T1275 were
prepared, as identified in Figures 13 (B) and (C), for further analysis of the
cryptic regulatory element.
5' deletions of the promoter (see Figures 13(B) and (C) and analysis by
transient expression using biolistics showed that the promoter was active
within
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a fragment 62bp from the transcriptional start site indicating that the core
promoter has a basal level of expression (see Table 5).
Table 5.
Transient GUS activity detected in soybean leaves by staining with X-gluc
after particle bombardment. Vectors illustrated in Figures. 13 (B) and (C).
Genes GUS staining
1. T1275-GUS-nos +
2. -1639-GUS-nos +
3. -1304-GUS-nos +
4. -684-GUS-nos +
5. -394-GUS-nos
6. -197-GUS-nos +
7. -62-GUS-nos +
8. -62(-tsr)-GUS-nos +
9. -12-GUS-nos -
10. +30-GUS-nos -
Deletion of a fragment containing the transcriptional start site (see -62(-
tsr)/GUS/nos in Figure 14(B), Table 5) did not eliminate expression, however
deletions to -12 bp and further (ie+30) did eliminate expression indicating
that
the region defined by bp -62 to -12 (nucleotides 1992-2042 of SEQ ID NO:2)
contained the core promoter. DNA sequence searches did not reveal
conventional core promoter motifs within this region as are typically found in
plant genes, such as the TATA box.
A number of the 5' promoter deletion clones (Figures 13 (B) and (C))
were transferred into tobacco by Agrobacterium-mediated transformation using
the vector pRD400. Analysis of GUS specific activity in leaves. of transgenic
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plants (see Table 6) confirmed the transient expression data down to the -197
fragment (i.e. nucleotide 1857 SEQ ID NO: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). Mean SE(n)
Genes GUS specific activities
pmoles MU/min/mg protein
1. T1275-GUS-nos 283 171 (27)
2. -1639-GUS-nos 587t188 (26)
3. -1304-GUS-nos 632t217 (10)
4. -684-GUS-nos nd*
5. -394-GUS-nos 1627 340 (13)
6. -197-GUS-nos 475 74 (27)
* nd = not determined
Histochemical analysis of organs sampled from the transgenic plants indicated
GUS expression in leaf, seeds and flowers.
To determine if enhancer elements exist, fragments -394 to -62
(nucleotides 1660 to 1992 of SEQ Id NO:20) and -197 to -62 (nucleotides 1875
to 1992 of SEQ ID NO:2) were fused to the -46 35S core promoter. Both
fragments raised the expression of the core promoter about 150 fold (Figure 13
(D), constructs DRA1-35S and BST1-35S). Doubling of the -394 to -62 region
(nucleotides 1660 to 1992 of SEQ ID NO:2) resulted in a 1.8 fold increase in
GUS activity when fused to T1275 core promoter (BSTI-GUS (-394-GUS) v.
BST2-GUS; Figure 13 (D)), a similar effect is observed when the -394 to -62
regioii is double and fused to the 35S core promoter (BST1-35S v. BST2-35S).
Doubling of the -197 to -62 fragment (nucleotides 1875 to 1992 of SEQ ID
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NO:2) also produced increased GUS activity when fused to the T1275 core
promoter (DRA2-GUS).
The -197 to -62 fragment (nucleotides 1875 to 1992 of SEQ ID NO:2;
DRA1-35S), the -197'to -62 fragment in reverse orientation, or inverted
(DRA1R-35S), and a repeat of the -197 to -62 fragment (DRA2-35S) were also
fused with the 35S minimal promoter (Figure 13 (E) and used to transform
Arabidopsis.
Arabidopsis plants with immature floral buds and few silques were
transformed with the above constructs by dippina the plant into a solution
containing Agrobacterium tumefa.ciens, 2.3 g/L MS, 5% (w/v) sucrose and
0.03% Silwet L-77 (Lehle Seeds, Round Rock. TX) for 1-2 min. and allowing
the plants to grow and set seed. Seeds f'roni mature plants were collected,
dried
at 25 C, and sown on sterile media containing 40ua/mL kanamycin to select
transformants. Surviving plantlets were ti-ansferred to soil, grown and seed
collected.
Constructs comprising the -197 to -62 fragment (nucleotides 1875 to
1992 of SEQ ID NO:2) in regular or invei-ted orientation exhibited increased
transcriptional enhancer activity, ovet- that of the minimal promoter (Figure
13
(F). A further increase in activity was observed when p!ants were transformed
with constructs comprising repeated regions of this regulatory element (Figure
13 (F). Tissue staining of transformed plants expressing DRAl-35S indicated
that this construct was expressed constitutively as it was detected in all
tested
organs, including flower, silque and seedling (Figure 13 (G)).
Activity of the T1275 Regulatory Element
Analysis of leaves of randomly-selected, greenhouse-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
*Trademark
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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 (Figure. 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 fold (mean of 13
measurements) lower in platits transformed with the T1275-GUS-nos gene
(Figure 14(B)) when compared witli 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 downstreanl of the transcriptional start site in the
sequence
of T1275-derived transcript.
Post-Transcriptional Regulatory Elements within T1275
An experiment was performed to determine the presence of a post-
transcriptional regulatory element within the T1275 leader sequence. A portion
of the sequence downstream from the transcriptioilal initiation site was
deleted in
order to examine whether this region may have an effect on translational
efficiency (determined by GUS extractable activity), mRNA stability or
transcription.
Deletion of the Nde 1-Sma I fragment ("N"; SEQ ID NO:3) from the
T1275-GUS-nos gene (Figure 15; T1275-N-GUS-nos; includes nucleotides 2084-
2224 of SEQ ID NO:2) resulted in at least about 46-fold reduction in the
amount
of GUS specific activity that could be detected in leaves of transgenic
tobacco ev
Delgold (see Table 7). Similar results, of about at least a 40 fold reduction
in
GUS activity due to the deletion of the Nde 1-Srrta 1 fragment, were observed
in
transgenic tobacco cv SR1 and transgenic alfalfa (Table 7). Addition of the
same
fragment (Ndel-Srna1) to a 35S-GUS-nos gene (Figure 15; 35S+N-GUS-nos)
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construct increased the amount of GUS specific activity by about 5-fold in
tobacco, and by a much higher amount in alfalfa (see Table 7).
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).
Construct GUS specific activity
pmoles Mli!min/mg protein
Delgold (1) DelQold (2) SR1 Alfalfa
1. T1275-GUS-nos 557t183 (21) 493f157 (25) 805f253 (22) 187t64 (24)
2. T1275-N-GUS-nos 12f3 (22) 12--;-3 (27) 6t2 (25) 4 0.5 (25)
3. 35S-GUS-nos 1848 692(15) 1347 415 (26) 1383t263 (25) 17t1 1(24)
4. 35S+N-GUS-nos 6990 3148(23) 6624 2791 (26) 6192 1923(24) 1428t601 (24)
A similar effect was noted in organs tested from transformed tobacco
(Table 8) and alfalfa plants (Table 9)
Table 8
Expression of T1275-GUS-nos (+N) compared with T1275-(-N)-GUS-nos
(-N) in organs of transgenic tobacco. Mean SE(n=5).
Organ GUS specific Activity (pmol MU/minimg/protein)
Delgold SR 1
+N -N +N -N
Leaf 1513f222 35 4 904 138 4 1
Flower 360t47 38 8 175 44 28 3
Seed 402 65 69 7 370 87 33 5
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Table 9
Expression of T1275-GUS-nos, T1275-(-N)-GUS-nos, 35S-GUS-nos, 35S-
GUS(+N)-GUS-nos in organs of transgenic alfalfa. Mean SE(n=5).
Construct GUS Specific Activity (pmol Mu/min/mg protein)
Leaf Petiole Stem Flower
T1275-GUS 756 73.6 1126 72.7 1366.7 260 456.1 160.9
T1275(-N)GUS 5.4 1.4 7.6 1.2 8.1 2.0 7.25 1.7
35S-GUS. 67.5t50.3 48.9 23.2 56.8 28.7 23.2 7.3
35S(+N)GUS 5545f2015 10791 6194 9931 5496 1039 476.7
Control 3.7 13.2 11.8 18.7
,15
In transient expression assays using particle bombardment of tobacco
leaves, the Ndel-Smal fragment fused to the minimal -46 35S promoter
enhanced basal level of 35S promoter activity by about 80 fold (28.67 2.91 v.
0.33 0.33 relative units; No.blue units/leaf).
SEQ ID NO:3 comprises nucleotides 2084 to 2224 of SEQ ID NO:2.
Nucleotides 1-141 of SEQ ID N03: comprise nucleotides obtained from the plant
portion of T1275 (nucleotides 2084 to 2224 of SEQ ID NO:2). Nucleotides 142-
183 of SEQ ID NO:3 comprise vector sequence between the enhancer fragment
and the GUS ATG. The GUS ATG is located at nucleotides 186-188 of SEQ ID
NO:3.
A shortened fragment of the NdeI-Smal fragment (see SEQ ID NO:4),
referred to as "L1N", "dN", or "deltaN" and lacking the out-of frame upstream
ATG at nucleotide 2087-2089 of SEQ ID NO:2, was also constructed and tested
in a variety of species. AN was created by replacing the Ndel site (Figure
18(A))
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within the leader sequence to a Bgl1I site thereby eliminating the upstream
ATG
at position 2086 of SEQ ID NO:2. A Kozak consensus sequence was also
constructed at the initiator MET codon and a Ncol site was added to facilitate
construction with other coding regions (see Figure 18(B)). Nucleotides 7-86 of
SEQ ID NO:4 (i.e. ON with Kozack sequence) are derived from T1275
(nucleotides 2091-2170of SEQ ID NO:2). AN also includes a Kozack sequence
from nucleotides 87 to 97 of SEQ ID NO:4, and nucleotides 98 to 126 of SEQ ID
NO:4 comprise the vector sequence between the enhancer fragment and the GUS
ATG. The GUS ATG is located at nucleotides 127-129 of SEQ ID NO:4).
Constructs comprising AN, for exanlple T 1275(ON)-GUS-nos, when
introduced into tobacco yielded 5 fold gt-eater le\,els of GUS activity in
leaves of
transgenic tobacco (5291f986 prnolMU/minimw pi-otein; (n=29) compared to
plants expressing T1275-GUS-nos (11 15_299 pmol MU/min/mg protein; n=29).
Activity of Ndei-Smal, N, and ON in other species
In monocots, transient expression in co--n callus indicated that the Nclel-
Snzal fragment (SEQ ID NO:3), or a shortcneci .\'clrl-.S'n1al h-agment, AN
(SEQ ID
NO:4), significantly increases GUS expression driven by the 35 S promoter, but
not to the higher level of expression genci-ated in the presence of the ADH1
intron ("i"; Figure 19 and Table 10).
Table 10
Transient expression analysis of GUS activity in bombarded corn calli.
Luciferase activity was used to normalize the data. Mean se (n=5)
Construct Ratio GUS:Lt.iciferase activity
35S GUS-nos 7.4- 4
35S(+N)-GUS-nos 19 5
35S (AN)-GUS-nos 18 10
35S-i-GUS-nos 66 27
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The functionality of the Nde1-Smal fragment (SEQ ID NO:3) was also
determined in non-plant species. In conifers, for example white spruce,
transient
bombardment of cell culture exhibited an increase in expression (Table 11).
Table 11
Expression of T1275-GUS-nos, T1275(-N)-GUS-nos, 35S-GUS-nos, 35S
(+ N)-GUS-nos in white spruce embryonal masses following bombardment
(n = 3).
Construct Average GUS expression per leaf
(Number of blue spots)
T1275-GUS-nos 72.67 +9.33
T1275(-N)-GUS-nos 21.33 4.49
35S-GUS-nos 113.67 + 17.32
35S(+N)-GUS-nos 126.33 19.41
''average spot much greater in size and strength.
In yeast, the presence of the Nclel-Sniul fragment (SEQ ID NO:3) or ON
(SEQ DI NO:4) exhibited strong increase in expression of the marker gene. A
series of constructs comprising a galactose inducible promoter Pg;,,,, various
forms of the Nde 1-Smal fragment, and GUS (UidA) were made within the yeast
plasmid pYES2. A full length Nde 1-Snurl fragment N (pYENGUS), ON
(containing a Kozak consensus sequence; pYEdNGUS), and'ON without a
Kozak consensus sequence (pYEdN'GUS; or AN"') were prepared (see Figure
20, and SEQ ID NO:5).
Nucleotides 7-86 of SEQ ID NO:5 (ON"' ) comprise a portion of the
enhancer regulatory region obtained from T1275 (nucleotide 2091-2170 of SEQ
ID NO:2), while nucleotides 87-116 comprise a vector sequence between the
enhancer fragment and the GUS ATG which is located at nucleotides 117-119
of SEQ ID NO:5.
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These constructs were tested in yeast strain INVSCI using known
transformation protocols. The yeast were grown in non-inducible
medium comprising raffinose as a carbon source for 48hr at 30 C and then
transferred onto inducible medium (galactose as a carbon source). Yeast cells
were harvested after 4 hr post induction and GUS activity determined
quantitatively. Up to about a 12 fold increase in activity was observed with
constructs comprising N. Constructs comprising AN"' exhibited even higher
levels of i'eporter activity. The results indicate that the Nde 1-Sma 1
fragment
(SEQ ID NO:3), ON (SEQ ID NO:4) and AN"' (SEQ ID NO:5) are functional in
yeast (Table 12).
Table 12
Expression of pYEGUS, pYENGUS, pYEdNGUS, and pYEdN"GUS (ON,
without a Kozak consensus sequence) in transformed yeast (n=5).
Construct Expt. I Expt. 2
Activity Activity
pYES-GUS-nos 93 15 407 8
pYES(+N)-GUS-nos 753 86 1771 191
pYES(ON)-GUS-nos 1119 85 2129 166
pYES(ON")-GUS-nos 1731 45 6897 536
Constructs containing ON"' (i.e. AN lacking the Kozack sequence; SEQ
ID NO:5) were also tested in insect cells. These constructs comprised the
insect
virus promoter,ie2 (Theilmann D.A and Stewart S., 1992, Virology 187: pp.
84-96) in the present or absence of AN"1 and CAT (chloramphenicol acetyl-
transferase) as the reporter gene. The insect line, Ld652Y, derived from gypsy
30 moth (Lynzantria dispar) was transiently transformed with the above
constructs
using liposomes (Campbell M.J. 1995, Biotechniquesr18: pp. 1027-1032;
Forsythe I.J. et al 1998, Virology 252: pp. 65-81). Cells were harvested 48
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hours after transformation and CAT activity quanitatively measured using
tritiated acetyl-CoA (Leahy P. et al. 1995 Biotechniques 19: pp. 894-898). The
presence of the translational enhancer was found to significantly modulate the
activity of the insect promoter-reporter gene construct in insect cells.
Bacteria were transformed with either pBI221, comprising 35S promoter
and GUS, or 35S-N-GUS , comprising the full length Ndel-Smal fragment
(SEQ ID NO:3). Since uidA (GUS) is native to E.coli, two uidA mutants,
uidAl and uidA2, that do not express uidA, were used for these experiments
(mutants obtained from E.coli Genetic Center 335 Osborn Memorial
Laboratories, Department of Biology, Box 208104, Yale University, New
Haven CT 06520-8104). These bacteria were transformed using standard
protocols, and transformants were assessed by assaying GUS activity from a50
,ul aliquot of an overnight culture. The "N" fragment (35s-N-GUS) was
observed to modulate the activity of the reporter gene in bacterial cells.
These data are consistent with the presence of a post-transcriptional
regulatory sequence in the Ndel-Smal fragment.
The NdeI-SmaI fragment functions as a transcriptional enhancer or mRNA
stability determinant
The levels of mRNA were determined in leaves obtained from tobacco
plants transformed with either T1275-GUS-nos, T1275-N-GUS-nos, 35S-GUS-
nos, or 35S+N-GUS-nos (Figures 17 (A) and (B)). 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 1 D Image Analysis Software.
Hybridization conditions used during RNase protection assay were overnight at
42-45 degrees in 80% formamide, 100 mM sodium citrate pH 6.4, 300 mM
sodium acetate pH 6.4, 1 mM EDTA.
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The levels of mRNA examined from transgenic tobacco plants
transformed with either T1275-GUS-nos, T1275-N-GUS-nos, 35S-GUS-nos, or
35S+N-GUS-nos, were higher in transgenic plants comprising the NdeI-SmaI
fragment under the control of the T1275 promoter but lower in those under the
control of the 35S promoter, than in plants comprising constructs that lack
this
region (Figures 17 (A) and (B)). This indicates that this region functions by
either modulating transcriptional rates, or the stability of the transcript,
or both.
The NdeI-SmaI fragment functions as a translational enhancer
Analysis were performed in order to deterinine whether the Ndel-Smal
region (SEQ ID NO:3) functions post-transcriptionally. 'I,he GUS specific
activity:relative RNA level was determined from the GUS specific activity
measurements, and relative RNA levels in oreenhouse grown transgenic plants
(figure 17 (C)). The ratio of GUS specitic 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 (C)). Similar
results are
obtained when the data are averaged, indicating an ei-ht fold reduction in GUS
activity per RNA. Similarly, an increase. by an average of six fold, in GUS
specific activity is observed when the :\'del-.S'mal region is added within
the 35S
untranslated region (Figure 17 (C)). The GUS specific activity:relative RNA
levels are similar in constructs containing the Ndel-SnzaI fragment (T1275-GUS-
nos and 35S+N-GUS-nos). These results indicate that the Ndel-Smal fragment
(seq idno:3) 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 15 using appropriate
restriction
endonucleases and ligated to pGEM4Z at an approximately similar distance from
the transcriptional start site used by the prokaryotic T7 RNA polymerase.
Another construct containing the AMV enhancer in the 5' UTR of a GUS-nos
fusion was similarly prepared. This AMV-GUS-nos construct was created by
CA 02331842 2004-09-16
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restriction endonuclease digestion of an AMV-GUS-nos fusion, with Bgl11 and
EcoRI, from pB1525 (Datla et al., 1993, Plant Science 94: 139-149) and
ligation
with pGEM4Z (Promega) digested with BamHl and EcoRl. Transcripts were
re ared in vitro in the resence of m'G 5' 5' G Cap Analo Ambion
, P P presence ) 8( )=
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 NdeI to Snzal fragment (see Table 13 ).
Table 13
In vitro translation of mRNA obtained from transgenic tobacco plants
transformed with vectors with or without a NdeI-Smal fragment obtained
from the T1275 GUS gene fusion (see Figure 15) using wheat germ extract.
in vitro translation
in vitro transcript fold enhancement
T1275-GUS-nos 3.7
T1275-N-GUS-nos 1.0
AMV-GUS-nos 1.9
The levels of protein produced using mRNAs comprising the Ndel-Smal
fragment are also greater than those produced using the lcnown translational
enhancer of Alfalfa Mosaic Virus RNA4 (Jobling S.A. and Gehrke L. 1987,
Nature, vo1325 pp. 622-625; Datla R.S.S. et al 1993 Plant Sci. vol 94, pp. 139-
=-~ 149). These results indicate that this region functions post-
transcriptionally, as a
translational enhancer.
CA 02331842 2000-12-22
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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.
CA 02331842 2001-06-22
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SEQUENCE LISTING
<110> Miki Dr., Brian
Oullet Dr., Therese
Hattori Dr., Jiro
Foster, Elizabeth
Labbe, Helene
Martin-Heller, Teresa
Lining, Tian
Brown Dr., Daniel
Zhang, Peijun
Wu, Keqiang
<120> Cryptic Regulatory Elements Obtained from Plants
<130> 08-869947CA4
<140> 2,331,842
<141> 1999-06-22
<150> 09/102,312
<151> 1998-06-22
<150> 2,246,892
<151> 1998-09-09
<160> 5
<170> PatentIn Ver. 2.0
<210> 1
<211> 1070
<212> DNA
<213> Nicotiana tabacum
<400> 1
tctagacttg tcttttcttt acataatcct cttcttcttt tttttgttag tttcttctgt 60
tttatccaaa aaacgaatta ttgattaaga aatacaccag acaagttttt tacttctttt 120
tctttttttt tttgtggtaa aaaattacac ctggacaagt ttatcacgaa aatgaaaatt 180
gctatttaag ggatgtagtt ccggactatt tggaagataa gtgttaacaa aataaataaa 240
taaaaagttt atacagttag atctctctat aacagtcatc cttatttata acaatacttt 300
actataaccg tcaaatttat tttgaaacaa aattttcatg ttatgttact ataacagtat 360
tttattatag caaccaaaaa atatcgaaac agatacgatt gttatagagc gatttgattg 420
tatcattatc cacatatttt cgtaagccca attactcctc ctacgtacga tgaaagtaaa 480
ccaatttaaa gttgcaaaaa tccaatagat ttcaatactt cttcaactgg cgttatgtta 540
ggtaatgact cctttttaac ttttcatctt taatttgaag tttctttcat taaaagaaag 600
tttctagaag agaagtgttt taacacttct agctctacta ttatctgtgt ttctagaaga 660
aaaatagaaa atgtgtccac ctcaaaaaca actaaaggtg ggcaaatctc cacctattta 720
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WO 99/67389 PCT/CA99/00578
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ttttattttg gattaattaa gatatagtaa agatcagtta taaacggagt tttgagttga 780
tacagtgaat tttaagatgt gtaccgattt aactttattt acatttatgt ttcgcacata 840
taagaagtcc gatttggaaa tactagattt tgtcaatcag gcaattcatg tggttgaaga 900
atttaagtta tatacaatga tgatataaag aatttttata ctattagtgc aaattaatcg 960
attactaaaa attattattc tattaattta tgctatcgtg cctccccaac ccgtcgaccg 1020
cggtacccgg tggtcagtcc cttatgttac gtcctgtaga aaccccaacc 1070
<210> 2
<211> 2224
<212> DNA
<213> Nicotiana tabacum
<400> 2
tctagactta cagaaagtct ctaacacgtg agggaatgat ccctttcctt acctccctgt 60
agagatattg gcttttcaac aactagtaca taaatatgcg actttgaccg tgtatcccca 120
gtcaaaaggg aacttcaccc tcctagttct ttatttccaa catacatggg gagtaatgct 180
aaatttacat agaagaataa taaaatgaac tgtaactaat gatgtactgt tccaaagaga 240
tgaggacgtc aacatattta ttccttcagc ccttttcaga ataataccat aagtagaaga 300
aatggcacat aaaatgaagt cctcggcaag tcaaatgtaa atctgaaccc acccagctaa 360
cccagtgaac tcaactttcc tggatagatc agcactcctt catgacattg catgccttct 420
ctttaaagag ccgcttgatc tctgaaaacc aaatgaatct ccacagagag atttcgagct 480
ccatgagacg ccttttggtt cttgatttac taaacctata aaaatgaaag gaagtaggac 540
aactgcattt tgccgcttaa gatgcttcgg cgctttgtga attttaagtc atgagaaagt 600
acaatgttgg aatctcacat tagaacaatg tatttgtaat aacctaggaa agcaaagcta 660
gaagggaggt gcagctaaat cttcttctac cttgttatcc ttgcatttct tgaggaggag 720
gaactgtcct cgcaggtgca aaatctgcag tcgcccaaaa ggatattcag aagtatatta 780
caacatgttt aatggttaac caagtgaaag atcaaaatag tcattagaac aaaatgcgtg 840
ctcagagcgt atctactagt tcatcaaccc agtacacatc tctgaatttc atctcttgcc 900
gttgaactaa gtcaattggt caaagacgca taacatgaga gacactcata aaacggctga 960
ataacatgca gaagacgtca tgcgccttag gtctcattat gcatgagatt attagttata 1020
tgctccttca gtttgactag aaatgaaaaa tcagttaagc ctgtaacgaa atgataacct 1080
gcttcaagaa gattagacta tttttcataa aatatgcagt gccgtgaaat agatacttaa 1140
tcttaggcag gaaaaatctt ctattgggcc ataataagaa ctaccaatta gaaaggaggt 1200
agaaagctcc gatactgtta tgaaggccat tctaagtgct gatgtgaatt tcccaataca 1260
aaatgacaac aaaaacaaaa gcctcaatcc taagctagtt ggggtcgcta tataaatcct 1320
cgacatccat ttaactccac ttggactcct ttctttccaa tattttaata ttgttagatt 1380
aatcataaaa ttgcttagct ttctactggc acttaaccta ctgcaaccct cctcttctgg 1440
gattccaaca caaacaacta agaggaattt gaaaaaaaga aagcaaatgt gagaagagac 1500
aaaatgtaca atgatacctc ttcttgcagc aaaggaggca ggttctctgc tgagacaagg 1560
ttctctattt cctgcaagac cttcgtatct tttattcgag accatgtatg tggaggtaac 1620
gccagcaata gtgctgtcag cacatcgttg cttgcagggg atcttctgca agcatctcta 1680
tttcctgaag gtctaacctc gaagatttaa gatttaatta cgtttataat tacaaaattg 1740
attctagtat ctttaattta atgcttatac attattaatt aatttagtac tttcaatttg 1800
ttttcagaaa ttattttact attttttata aaataaaagg gagaaaatgg ctatttaaat 1860
actagcctat tttatttcaa ttttagctta aaatcagccc caattagccc caatttcaaa 192'0
ttcaaatggt ccagcccaat tcctaaataa cccaccccta acccgcccgg tttccccttt 1980
tgatccaggc cgttgatcat tttgatcaac gcccagaatt tccccttttc cttttttaat 2040
tcccaaacac ccctaactct atcccatttc tcaccaaccg ccacatatga atcctcttat 2100
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ctctcaaact ctctcgaacc ttcccctaac cctagcagcc tctcatcatc ctcacctcaa 2160
aacccaccgg aatacatggc ttctcaagcc gtggaaacct tatactcacc tccctttgct 2220
ctta 2224
<210> 3
<211> 188
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ndel-Smal
fragment of T1275
<400> 3
catatgaatc ctcttatctc tcaaactctc tcgaaccttc ccctaaccct agcagcctct 60
catcatcctc acctcaaaac ccaccggaat acatggcttc tcaagccgtg gaaaccttat 120
actcacctcc ctttgctctt acagtactcg gccgtcgacc gcggtacccg ggtggtcagt 180
cccttatg 188
<210> 4
<211> 129
<212> DNA
<213> Artificial Sequence
<400> 4
agatctatcc tcttatctct caaactctct cgaaccttcc cctaacccta gcagcctctc 60
atcatcctca cctcaaaacc caccggccac catggcctct agaggacccc gggtggtcag 120
tcccttatg 129
<210> 5
<211> 119
<212> DNA
<213> delta N, without Kozak sequence
<400> 5
agatctatcc tcttatctct caaactctct cgaaccttcc cctaacccta gcagcctctc 60
atcatcctca cctcaaaacc caccggtcta gaggatcccc gggtggtcag tcccttatg 119
