Note: Descriptions are shown in the official language in which they were submitted.
CA 02239259 1998-07-31
1 MATRIX ATTACHMENT REGIONS
2
3 BACKGROUND OF THE INVENTION
4 Field Of the Invention
The invention relates to nucleic acid molecules isolated from the 5' flanking
6 region of endosperm-specific storage protein genes of monocotyledonous
plants,
7 and which possess nuclear matrix binding activity.
8 Description of the Related Art
9 In the past fifteen years it has become possible to transfer genes from any
organism into a wide range of crop plants including the major monocotyledonous
11 cereal crops wheat, rice, barley, oat and maize. However, even though the
12 introduced genes can be expressed in the transformed crops, the level of
expression
13 can be very low. Indeed, Peach and Velten (1991 ) found that the majority
of
14 detectable transformants exhibited very low expression. The variation in
expression
is due to the influence of the surrounding chromatin at the site of insertion
of the
16 transgene (position effects). As a result, large numbers of transgenic
plants must
17 be produced in order to be sure of producing a single high-expressing
plant. This is
18 not trivial in cereal crops where the transformation efficiency is only 1
to 5%. In
19 addition to expression variability, there is also the possibility that
transgene silencing
will occur in subsequent generations.
21 For the purposes of crop improvement, it would be highly beneficial to
reduce
22 the numbers of transgenic plants which need to be produced to find a high-
23 expressing plant, and also to ensure that there will be no transgene
silencing in
24 subsequent generations.
In eukaryotes, the DNA is folded into chromosomes in the form of loops.
26 These loops are anchored to a proteinaceous nucleoskeleton, known as the
nuclear
27 matrix or scaffold, by segments of DNA known as matrix attachment regions
28 ("MAR"s). The DNA loop structure, which allows for the unwinding of DNA to
permit
29 access by transcriptional regulatory proteins, has important implications
for gene
regulation and expression. One function of the loop structure and matrix
attachment
31 regions is to insulate genes from the effects of surrounding chromatin,
thereby
32 allowing copy number dependent, position independent expression of adjacent
33 genes. For this reason, various studies have investigated the hypothesis
that MARs
CA 02239259 1998-07-31
1 may allow position independent expression of any introduced genes in
transgenic
2 plants.
3 In several studies, the hypothesis was validated, and MARs were found to
4 increase transgene expression, decrease silencing, prevent silencing after
crossing
or backcrossing to non-transformed plants, normalize expression, and to
provide
6 copy number independent expression (Allen et al. (1996); Allen et al.
(1993); Ulker
7 et al. (1997); Mlynarova et al. (1995); Breyne et al. (1992)). These studies
utilized
8 MARs derived from either animal or tobacco sources.
9 Recently, MARs from other plant species such as maize and rice have been
isolated (Avramova and Bennetzen (1993); Nomura et al.(1997)). The maize MAR
11 was derived from the promoter of an alcohol dehydrogenase gene. The rice
MARs
12 were isolated on the basis of functional binding to the nuclear matrix, and
their
13 association with any transcribed gene is unknown.
14 All of the MARs isolated to date have similar features in that they are AT
rich
(greater than 60%) and contain motifs such as A-boxes, T-boxes, ATATTT boxes
16 and boxes showing homology to DNA topoisomerase II consensus cleavage sites
17 (Breyne et al. (1994). None of these motifs are universal, however, and
different
18 MARs do not have extensive homology, nor are there strictly conserved DNA
19 elements. Specific MAR DNA sequences are therefore species specific and
differ in
their ability to bind the nuclear matrix. Each putative MAR must therefore be
21 investigated by functional assays.
22 Given the low transformation efficiency in cereal crop plants, MARs
functional
23 in cereal crop plants to increase transgene expression, decrease silencing,
prevent
24 silencing after crossing or backcrossing to non-transformed plants,
normalize
expression, and to provide copy number independent expression, would be very
26 useful. The species-specificity of MARs limits the utility of the known
rice and maize
27 MARs for improving transgene expression in monocotyledonous cereal crops
such
28 as wheat (Triticum aestivum). Thus, there is an ongoing need for MARs
functional in
29 cereal crop plants.
2
CA 02239259 1998-07-31
1 SUMMARY OF THE INVENTION
2 The present invention provides a DNA sequence, designated "MAR Bx7",
3 isolated from a region adjacent to a wheat storage protein promoter. The DNA
4 sequence, which is depicted in SEQ ID NO: 1, has features characteristic of
MARs
in that it is AT rich (63%), and contains motifs homologous to the DNA
6 topoisomerase II consensus sequence (position 708), T-box (position 118) and
7 AATATATTT motif (position 437). Proof that this sequence acts as a matrix
8 attachment region was provided by a functional test of binding to the
nuclear matrix.
9 The sequence binds the nuclear matrix and binds more strongly than a
previously
isolated MAR from maize. The sequence has also been demonstrated to have no
11 deleterious effect when placed adjacent to a heterologous promoter,
indicating its
12 utility in enhancing and stabilizing transgene expression.
13 As stated above, MAR Bx7 was isolated from the 5' flanking region of a
wheat
14 storage protein gene, specifically the Bx7 storage protein gene from T.
aestivum
variety Glenlea. In the developing endosperm of cereals, storage proteins are
16 produced which are not found in any other plant part or at any other stage
of
17 development. The genes encoding these proteins are very highly expressed,
with
18 up to two percent of the seed protein attributable to each gene. The genes
are
19 regulated at the transcriptional level, and are very tightly regulated in
that they are
only expressed in the developing endosperm. The tight regulation and high
21 expression of these endosperm-specific storage protein genes, including the
Bx7
22 storage protein gene, indicates that MAR Bx7 is highly effective at
allowing very high
23 expression rates, and would therefore be useful in enhancing the expression
of
24 transgenes in transformed plants.
The present invention provides methods by which the exemplified MAR
26 sequence can be used to identify and isolate other MAR sequences from the
5'
27 flanking region of endosperm-specific storage protein genes of
monocotyledonous
28 plants. The inventors have identified DNA sequences within the 5' flanking
regions
29 of other endosperm-specific storage protein genes that are closely
homologous to
the nucleotide sequence of MAR Bx7. The invention provides methods whereby
31 these additional putative MAR sequences can be assayed for nuclear matrix
binding
32 activity, and for their ability to enhance or stabilize transgene
expression in
3
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1 transgenic monocotyledonous plants. Therefore, broadly stated, the present
2 invention provides an isolated nucleic acid molecule comprising a portion of
a 5'
3 flanking region of an endosperm-specific storage protein gene of a
4 monocotyledonous plant, the isolated nucleic acid molecule possessing
nuclear
matrix binding activity.
6 For use in transforming monocotyledonous plants with a transgene, the MAR
7 sequences of the invention are preferably operably linked to at least one
DNA
8 construct which includes a plant promoter, a coding sequence for the gene to
be
9 expressed in the plant, and a poly(A) addition signal. Preferably, an MAR
sequence
of the invention is operably linked both upstream and downstream of the DNA
11 construct. However, a single MAR sequence either upstream or downstream of
the
12 DNA construct is sufficient to confer the benefit of the presence of the
MAR
13 sequence on expression of the transgene. The present invention therefore
extends
14 to a recombinant nucleic acid molecule comprising the above-referenced
isolated
nucleic acid molecule operably linked to at least one DNA construct
comprising, in
16 the 5' to 3' direction of transcription, a promoter functional in
monocotyledonous
17 plants, a coding sequence expressible in monocotyledonous plants, and a
poly(A)
18 addition signal, the isolated nucleic acid molecule being heterologous to
at least one
19 of the promoter or the expressible coding sequence.
The invention extends to plant vectors containing such recombinant nucleic
21 acid molecules, the plant vectors being useful for transforming
monocotyledonous
22 crop plants with a foreign gene.
23 MARs of the invention can be used to enhance transgene expression, provide
24 copy number independent expression and increase stability of transgenes
over
subsequent generations. The MARs of the invention can be used to flank any
26 promoter/gene combination and inserted into any plant species, preferably
27 monocotyledonous cereal species such as wheat, barley, oat, rice and maize.
The
28 invention therefore further extends to recombinant monocotyledonous plants
29 containing stably integrated into their genomes a recombinant nucleic acid
molecule
as described above.
31 The invention further extends to methods for improving gene expression in
32 monocotyledonous plants. Broadly stated, such a method includes the steps
of:
4
CA 02239259 1998-07-31
1 (a) transforming monocotyledonous plant cells with a recombinant nucleic
acid
2 molecule comprising an isolated nucleic acid molecule comprising a portion
of
3 a 5' flanking region of an endosperm-specific storage protein gene of a
4 monocotyledonous plant, the isolated nucleic acid molecule possessing
nuclear matrix binding activity, operably linked to at least one DNA construct
6 comprising, in the 5' to 3' direction of transcription, a promoter
functional in
7 monocotyledonous plants, a coding sequence expressible in
8 monocotyledonous plants, and a poly(A) addition signal, the isolated nucleic
9 acid molecule being heterologous to at least one of the promoter or the
expressible coding sequence;
11 (b) selecting those plant cells that have been transformed;
12 (c) regenerating transformed plant cells to provide differentiated
transformed
13 plants; and
14 (d) selecting those transformed plants exhibiting improved expression of
the
coding sequence relative to a control plant.
16
17 DETAILED DESCRIPTION OF THE INVENTION
18 In order to provide a clear and consistent understanding of the
specification
19 and claims, including the scope to be given to such terms, the following
definitions
are provided.
21 "Coding sequence" means the part of a gene which codes for the amino acid
22 sequence of a protein, or for a functional RNA such as a tRNA or rRNA.
23 "Complement" or "complementary sequence" means a sequence of
24 nucleotides which forms a hydrogen-bonded duplex with another sequence of
nucleotides according to Watson-Crick base-pairing rules. For example, the
26 complementary base sequence for 5'-AAGGCT-3' is 3'-TTCCGA-5'.
27 "Conditions of moderate stringency" means nucleotide sequence hybridization
28 conditions involving washing first in 2 x sodium phosphate-
ethylenediaminetetracetic
29 acid ("SSPE"), 0.1 % sodium dodecyl sulfate ("SDS") at room temperature for
10
minutes followed by washing in 1 x SSPE, 0.1 % SDS at 65 °C for 15
minutes using
31 Hybond N+ membranes (Amersham Pharmacia, Baie D'Urfe, Quebec, Canada).
32 A "control" or a "control plant" in an experiment to determine whether the
5
CA 02239259 1998-07-31
1 presence of a MAR improves the expression of a transgene, is a plant that
has been
2 transformed with an expression cassette comprising a promoter, a coding
sequence
3 of interest, and a poly(A) addition site, but which is not operably linked
to a MAR.
4 Expression of the coding sequence by the control plant can be compared to
that of a
plant tranformed with the same expression cassette flanked by MARs, in order
to
6 assess the effect of the presence of the MARs. For instance, in the
transient
7 expression experiments described in Example 1 herein, the plants tranformed
with
8 pAct1 d are control plants.
9 "Downstream" means on the 3' side of any site in DNA or RNA.
"Endosperm-specific storage protein" means a storage protein which is
11 deposited in the developing starchy endosperm of grains. Their biological
role is to
12 provide a store of amino acids for germination. The major endosperm-
specific
13 storage proteins in wheat are the gluten proteins. Wheat gluten proteins,
and the
14 major endosperm-specific storage proteins of most other cereals, are
characterized
by their insolubility in water or aqueous solutions of salts, but solubility
in mixtures of
16 alcohols and water.
17 "5' flanking region" means the sequences upstream of the coding part of a
18 eukaryotic gene. This region is not transcribed, but contains sequence
elements
19 essential for the control of gene expression such as TATA-boxes, CAAT-
boxes,
enhancers, and specific binding sites for transcription factors.
21 Two nucleic acid sequences are "heterologous" to one another if the
22 sequences are derived from separate organisms, whether or not such
organisms are
23 of different species, as long as the sequences do not naturally occur
together in the
24 same arrangement in the same organism.
"Improved expression" of a transgene operably linked to a MAR relative to
26 that of a transgene not linked to a MAR includes, without limitation, such
useful
27 properties as increased transgene expression, decreased silencing resulting
from
28 position effects, decreased silencing after crossing or back-crossing to
non-
29 transformed plants, normalized expression, and increased copy number
independent expression.
31 "Monocotyledonous plant" means the class of flowering plants characterized
32 by the presence of a single seed leaf (cotyledon) in the embryo.
6
CA 02239259 1998-07-31
1 "Nuclear matrix" means the filamentous mesh-work located between the inner
2 nuclear membrane and heterochromatin, which provides potential attachment
sites
3 for chromatin and cytoplasmic intermediate filaments. The nuclei of
eukaryotic cells
4 have a double-membrane. The outer membrane forms a continuum with some parts
of the endoplasmic reticulum whereas the inner membrane functions in the
6 organization of chromatin. The nuclear matrix is made up of a family of
interrelated
7 polypeptides known as the nuclear lamins. The nuclear lamins fall into three
major
8 types: the neutral A- and C-lamins, and the acidic B-lamins (molecular
weight range
9 from 62-69 kDa). Less frequently occurring lamins belong to the D and E
categories. The lamins are structurally related to the intermediary filaments,
11 assemble to 10 nm filaments in vivo, and possess the typical coiled coil-
12 configuration of two intertwined a-helices.
13 "Nuclear matrix binding activity" means the property of a DNA sequence to
14 bind to isolated nuclear matrices in vitro such that it remains attached to
the nuclear
matrix after centrifugation and is found in the pellet fraction rather than in
the
16 supernatant fraction under assay conditions in which a control sequence of
17 prokaryotic plasmid cloning vector DNA does not bind to the nuclear matrix
and is
18 found associated exclusively with the supernatant fraction after
centrifugation.
19 "Nucleic acid molecule" means a single- or double-stranded linear
polynucleotide containing either deoxyribonucleotides or ribonucleotides that
are
21 linked by 3'-5'-phosphodiester bonds.
22 Two DNA sequences are "operably linked" if the nature of the linkage does
23 not interfere with the ability of the sequences to effect their normal
functions relative
24 to each other. For instance, a promoter region would be operably linked to
a coding
sequence if the promoter were capable of effecting transcription of that
coding
26 sequence.
27 "Plant" means whole plants, plant parts, and plant cells.
28 A "plant vector" means a cloning vector that is designed to introduce
foreign
29 DNA into a plant's genome and includes a plasmid cloning vehicle
specifically
constructed so as to achieve efficient transcription of the cloned DNA
fragments(s)
31 and translation of the corresponding transcripts) within a target plant
cell. Such
32 vectors may be based on, for example, the Ti-plasmid of Agrobacterium
7
CA 02239259 1998-07-31
1 tumefaciens, or DNA plant viruses.
2 "Poly(A) addition signal" means a hexanucleotide consensus sequence close
3 to the 3'-end of most eukaryotic genes transcribed by RNA polymerase II,
that
4 precedes the site where a poly(A) tail is added to the processed messenger
RNA by
some 10-30 bases, and is transcribed into mRNA.
6 "Promoter" means a cis-acting DNA sequence, generally 80-120 base pairs
7 long and located 5' upstream of the initiation site of a gene, to which RNA
8 polymerase may bind and initiate correct transcription. Eukaryotic promoters
differ
9 for the different DNA-dependent RNA polymerases. RNA polymerase II, which
transcribes structural genes, transcribes a multitude of genes from very
different
11 promoters, which have specific sequences in common (e.g. the TATA box at
about
12 position -25 and the CAAT box at about position -90).
13 A "recombinant nucleic acid molecule", for instance a recombinant DNA
14 molecule, is a novel nucleic acid sequence formed in vitro through the
ligation of two
or more nonhomologous DNA molecules (for example a recombinant plasmid
16 containing one or more inserts of foreign DNA cloned into its cloning site
or its
17 polylinker).
18 "Upstream" means on the 5' side of any site in DNA or RNA.
19 The first step in obtaining an MAR of the present invention is to clone a
cereal
(wheat, barley, maize, oat, rice) storage gene promoter region. These species
are
21 closely related, and all have similarly regulated endosperm specific
storage protein
22 genes. The cloning can be accomplished by methods generally known in the
art
23 including, for example: generating DNA primers for the polymerase chain
reaction
24 ("PCR") using sequences in the Genbank database where many promoter
sequences for storage protein genes are catalogued (for example accession
26 numbers X01130, X17637, X03103, X03042), or using the sequence of MAR Bx7;
27 isolation of a cDNA clone for a storage protein gene and using promoter
walking (for
28 example using a commercially available kit such as Promoter Finder from
Clontech
29 (Palo Alto, CA, USA)); or, probing a genomic library using a cDNA probe for
a
storage protein gene or a probe for a storage protein gene generated by PCR.
This
31 list is not exhaustive, and any method which allows the cloning of a
promoter for a
32 gene could be used. The DNA sequence isolated is then cloned into a high
copy
8
CA 02239259 1998-07-31
1 number cloning plasmid (such as pUCl9) and sequenced using sequencing
2 technology well known in the art, for example, as described by Sanger et al.
(1977).
3 The DNA sequence is examined, and any DNA sequences which have
4 greater than 60% AT content and contain at least one motif with homology to
a DNA
topoisomerase II consensus site, A box, T box or ATATTT box (Breyne et al.,
6 (1994)) are selected as candidates for MAR activity.
7 The DNA sequences must then be tested for their ability to bind the nuclear
8 matrix. Nuclear matrices (also known as nuclear scaffolds) can be isolated
from
9 plant species using either a high salt buffer and Dnase I, or using a
lithium
diiodosalicylate (LIS) method as reviewed by Breyne et al. (1994). The nuclear
11 scaffolds are incubated with in vitro-labelled DNA fragments (for example,
using
12 end-labelling with Klenow fragment and alpha-32P dCTP (Sambrook et al.
(1989)) of
13 the putative MAR as well as a non-MAR control fragment such as a piece of
the
14 prokaryotic cloning vector. Appropriate incubation conditions include, for
instance,
incubating 2 A26o units of nuclear scaffolds for 1 hour at 37° C with
20 ng of in vitro-
16 labelled DNA fragments and 10 ,ug control DNA in a total of 100 ,ul of
17 digestion/binding buffer (20 mM Hepes, pH7.4/20mM Kcl/70mM NaCI/lOmM
18 MgCh/1 % thiodoglycol/0.2 mM phenylmethylsulfonyl fluoride/aprotinin at 5
~cg/ml)
19 (Hall et al. (1991 )). The mixture is centrifuged (for example at 2000 x g
for 10
minutes (Hall et al. (1991 )) and the DNA is isolated from the pellet and
supernatant
21 fractions (for example by incubation in 50 ~I of lysis buffer (1 %
SDS/proteinase K at
22 500 ,ug/ml/20mM ethylenediamine tetraacetic acid ("EDTA"), pH 8.0/20mM Tris-
CI,
23 pH 8.0) for 16 hours at 37 degrees C)). The two fractions are separated by
gel
24 electrophoresis on a 1 % agarose Tris/acetate/EDTA ("TAE") gel (Sambrook et
al.,
(1989)), and the gel is then dried and subjected to autoradiography using
standard
26 techniques as described by Sambrook et al. (1989).
27 The putative fragment is confirmed as an MAR if the labelled fragment is
28 found in the pellet fraction. Variations in this assay are possible and can
be found
29 for example in Hall et al. (1991), Van der Geest (1994); and Avramova and
Bennetzen (1993). Other variations still involving isolation of nuclear
matrices and
31 evaluation of the ability of the putative MAR fragment to bind to the
matrices may be
32 possible. Significant binding over that of appropriate control fragments
indicates that
9
CA 02239259 1998-07-31
1 the fragment has MAR activity. Control fragments could include DNA sequences
2 which have been shown to not bind the nuclear matrix by previous assays, or
could
3 be prokaryotic or prokaryotic cloning plasmid derived sequences which by
nature do
4 not bind the nuclear matrix.
In an alternative method, MAR fragments can be obtained by the isolation of
6 nuclear matrices followed by digestion with one or more restriction enzymes.
For
7 instance, nuclear matrices equivalent to 20 Azso units of nuclei are
incubated in 1 ml
8 of binding/digestion buffer (as described above) with 1000 units of
restriction
9 enzymes) for 3 hours at 37 degrees C (Hall et al. (1991)). The endogenous
DNA
fragments which remain attached to the nuclear matrices are recovered (for
example
11 by centrifugation at 2000 x g for 10 minutes, washing once with
digestion/binding
12 buffer, treatment of the pellet fraction with Rnase A at 200 ,ug/ml
followed by
13 proteinase K at 500,ug/ml (Hall et al., (1991 )). The resulting DNA
fragments are then
14 extracted with phenol/chloroform and precipitated with ethanol, and then
cloned into
a plasmid cloning vector using standard techniques (Sambrook et al., (1989)).
16 Variations on this method are possible and can be found in, for example,
Mirkovitch
17 et al. (1984) and Nomura et al. (1997).
18 In the exemplified case, MAR Bx7 was isolated from the 5' flanking region
19 (approximately the 2.2kb region upstream from the transcription initiation
site) of the
Triticum aestivum variety Glenlea Bx7 storage protein gene. The DNA sequence
of
21 this MAR is depicted in SEQ ID NO: 1. MAR Bx7 contains features typical of
MARs,
22 being AT rich (63%), and containing motifs homologous to the DNA
topoisomerase II
23 consensus sequence (position 709), T-box (position 119) and ATATTT motif
24 (position 437). As shown in Table 1, MAR Bx7 (EM820 fragment) had about 5.9
times the nuclear matrix binding activity of a prokaryotic cloning vector
control
26 sequence, and about 2.3 times the nuclear matrix binding activity of the
known
27 maize Adh1 gene MAR.
28 Given the variability exhibited in MAR sequences, it will be appreciated
that if
29 any sequence changes were made to the exemplified MAR sequence, the
sequence
would remain functionally identical to the exemplified MAR as long as the
sequence
31 still bound the nuclear matrix in an experiment designed to test the
ability of a DNA
32 sequence to bind the nuclear matrix (for example by either of the above
assays).
CA 02239259 1998-07-31
1 Changes to the exemplified MAR sequence, including, for example, insertions,
2 deletions or base changes, may be effected through the use of known
techniques,
3 such as the use of commercially available mutagenesis kits from Stratagene,
La
4 Jolla, CA, USA, or Clontech, Palo Alto, CA, USA). Naturally occurring
sequence
variations may also be identified. Therefore, all variants of MAR Bx7 which
exhibit
6 greater nuclear matrix binding activity than a prokaryotic cloning vector
control
7 fragment under equivalent assay conditions shall be understood to fall
within the
8 scope and spirit of the invention.
g Example 2 herein provides methods for using MAR Bx7 to identify and isolate
other MARs from the 5' flanking region of endosperm-specific storage protein
genes
11 of monocotyledonous plants. Analysis of sequence homology between MAR Bx7
12 and the known MARs from cereal crops, being the maize- and rice-derived
MARs
13 reported by Avramova et al. (1993) and Nomura et al. (1997) determined that
these
14 MARs have at most 60% and 68% homology, respectively, to MAR Bx7 over the
region of greatest similarity. This relatively low level of homology is not
surprising,
16 as the known rice and maize MARs are not obtained from the 5' flanking
regions of
17 endosperm-specific storage protein genes. As further discussed in Example 2
18 herein, the inventors have analysed the sequences of 5' flanking regions of
various
19 endosperm-specific storage protein genes of wheat or Aegilops tauschii, a
close
relative of wheat. Levels of homology between 86-99% were observed over
regions
21 of approximately 100-450 base pairs. Given this degree of sequence homology
22 (greater than about 80%), it is expected, based on the observed properties
of MAR
23 Bx7, that these 5' flanking regions of endosperm-specific storage protein
genes of
24 wheat or Aegilops tauschii contain sequences sufficiently similar to MAR
Bx7 that
they would have similar functional properties, and would bind the nuclear
matrix and
26 function as MARs.
27 As noted above, Aegilops tauschii is closely related to wheat. Bread wheat
28 (Triticum aestivum L.) is a hexaploid made up of three genomes, designated
29 genomes A, B and D. Each genome was contributed by a diploid progenitor
species. Candidate diploid species include T. uartu, T. monococcum or T.
31 boeoticum for the A genome, Aegilops speltoides for the B genome, and T.
tauschii
32 (also known as Aegiiops squarrosa or Aegiiops tauschii~ for the D genome
(Mujeeb-
11
CA 02239259 1998-07-31
1 Kazi (1993)). There are also tetraploid species containing two of the above
2 genomes. These include Triticum turgidum and Triticum durum (genomes A and
B).
3 All of these species are therefore inter-related and, as such, any MAR found
on the
4 B genome of T. aestivum would also be found in the B genome of diploid and
tetraploid species carrying that genome. The same principle would apply to the
6 other genomes.
7 There is also a high degree of homology between the genomes of other
8 monocotyledonous plant species and Triticum aestivum. This homology is known
as
9 gene synteny and allows genes from easily manipulated cereal species having
comparatively small genomes, such as rye or rice, to be used to clone genes
from
11 wheat. Synteny has been observed between wheat, barley and oats (Hermann,
12 G.G. (1996)); wheat and rye (Langridge, P., et al. (1998)); and wheat,
rice, and
13 maize (Ahn, S., et al. (1993)). Due to gene synteny, there is a high
probability that
14 the 5' flanking regions of endosperm specific storage protein genes of
barley, oats,
rye, rice, and maize would contain MARs. Wheat, barley and rye are members of
16 the family Triticeae.
17 Using the methods disclosed in Examples 1 and 2 herein, fragments of the 5'
18 flanking regions of endosperm-specific storage protein genes of
monocotyledonous
19 plant species such as wheat, barley, oats, rye, rice and maize can be
identified,
isolated, and tested for nuclear matrix binding activity and their effect on
the
21 expression of coding sequences in plants. Those sequences which have at
least
22 80% homology to MAR Bx7 are particularly preferred candidates form
investigation.
23 Any such sequences which exhibit nuclear matrix binding activity, and which
do not
24 have a deleterious effect when placed adjacent to a heterologous promoter,
shall be
understood to be MARs within the scope and spirit of the invention.
26 As discussed above, MARs are identified by their ability to bind the
nuclear
27 matrix, and it is the very property of nuclear matrix binding activity that
defines a
28 MAR, rather than any other feature or property. A helpful general
discussion of
29 MARs is provided in Spiker et al. (1996). As noted therein, work with MAR
sequences is still in its early stages, particularly in plant systems, and
much remains
31 to be learned about the mechanism by which MARs affect transgene
expression.
32 Nevertheless, a number of studies have shown that MARs (animal, yeast,
soybean
12
CA 02239259 1998-07-31
1 and tobacco MARs) are useful for increasing transgene expression in plants
(Allen
2 et al. (1996); Allen et al.(1993); Mlynarova et al. (1995); Breyne et
al.(1992); and
3 Schoffl et al. (1993)). Decreased silencing of transgenes through the use of
MARs
4 has also been demonstrated (Ulker et al. (1997)). These studies have
examined the
utility of MARs in less than ideal conditions. For instance Mlynarova et al.
(1995)
6 achieved increased expression of the GUS (~3-glucuronidase) reporter gene in
7 tobacco plants using a chick lysozyme MAR. As discussed earlier, as
different
8 MARs do not have extensive homology, nor are there strictly conserved DNA
9 elements, MARs are species specific and differ in their ability to bind the
nuclear
matrix. It is therefore to be expected that MARs will have greatest utility in
11 increasing transgene expression in organisms closely related to the source
of the
12 MAR sequence. This is borne out in the results reported by Spiker et al.
(1996) of
13 their earlier work comparing expression of the GUS reporter gene in tobacco
using
14 either a heterologous MAR derived from yeast, or a native tobacco MAR. Use
of the
yeast (heterologous) MAR resulted in a 12-fold increase in transgene
expression,
16 whereas use of the tobacco (homologous) MAR resulted in a 60-fold increase
in
17 expression of the transgene. As such, it is expected that while MARs of the
present
18 invention, obtained from the 5' flanking region endosperm-specific storage
protein
19 genes of monocotyledonous plants, may be used in any plant species, they
will be
particularly effective in increasing transgene expression in the economically
21 important monocotyledonous cereal species.
22 To employ MARs of the invention to increase or stabilize transgene
23 expression in plants, recombinant nucleic acid molecules are made wherein
an
24 expression cassette or cassettes, each comprising a promoter, coding region
and
terminator (polyadenylation site), is cloned in such a way that it is adjacent
to or
26 preferably flanked by an MAR of the invention. In a preferred embodiment,
the
27 resulting recombinant nucleic acid molecule is cloned into a high copy
number
28 plasmid or a binary plasmid for use in Agrobacterium. Suitable high copy
number
29 cloning plasmids such as pUCl9 or pBluescript are well known in the art and
are
available commercially from such sources as Life Technologies (Gaithersburg,
MD,
31 USA) and Stratagene (La Jolla, CA, USA). Agrobacterium binary plasmids
include
32 pBINl9 and pB1101 and can also be obtained commercially from such sources
as
13
CA 02239259 1998-07-31
1 the American Type Culture Collection (10801 University Boulevard, Manassas,
2 Virginia, 20110-2209, USA) and Clontech (Palo Alto, CA, USA). Methods for
the
3 construction of such constructs are known in the art and are described in
commonly
4 used laboratory manuals such as Sambrook et al. (1989).
The constructs containing the gene of interest are then stably inserted into
6 the genome of a plant using known genetic transformation protocols
including,
7 without limitation, incorporation into protoplasts using polyethylene glycol
("PEG") or
8 electroporation (Datta et al. (1990)), using Agrobacterium as a delivery
system
9 (Tingay et al. (1997)), or bombardment using a device such as described in
U.S.
Patent No. 5,179,022. Using these methods, fertile regenerated plants can be
11 produced containing the gene of interest adjacent to or flanked by an MAR
of the
12 invention. Such plants will exhibit, on average, increased expression and
stability of
13 the gene of interest.
14 The invention is further illustrated by the following non-limiting
examples.
16 EXAMPLE 1
17 Isolation of a MAR adjacent to the Bx7 storage protein
18 gene from Triticum aestivum variety Glenlea
19 Primers for nested polymerase chain reaction ("PCR") amplification were
synthesised by Life Technologies (Gaithersberg, MD, USA). Their sequence was
21 compiled from the coding region of the wheat storage protein gene Bx7*
(Genbank
22 Accession No. M22209) (Anderson et al. (1989)) and from sequence in the
23 untranslated upstream region of the related Bxl7gene from T. aestivum
(wheat)
24 L86-69 (derived from cv. Olympic x cv. Gabo) (Reddy et al. (1993)). These
were
used to amplify the region upstream of Bx7 in T. aestivum cv. Glenlea by
26 polymerase chain reaction. The primer sequences were: (outer primers)
27 GAGCTCTCCCATCCAATTG (SEQ ID NO: 2) and AGAAGCTTGGCCTGGATAGT
28 (SEQ ID NO: 3); and (inner primers) GGGTCGATGGTATCAATCC (SEO ID NO: 4)
29 and GGCCTGGATAGTATGACCC (SEO ID NO: 5). The first round of PCR used
200 ng purified 'Glenlea' DNA as template and Taq DNA polymerase in 35 cycles
of
31 30 sec at 92°C (denature), 30 sec at 52°C (anneal) and 2.5
min at 72°C (extend) in a
32 thermocycler (Thermolyne Temptronic) using the outer nested primer pair.
The
14
CA 02239259 1998-07-31
1 second round of PCR was identical to this, but used 0.2 pl of the first
round reaction
2 product and the inner nested primers. The resulting 2.2 kb DNA fragment from
the
3 second round of PCR was purified by ethanol precipitation and cloned into
the
4 EcoRV site of pBluescript SK (obtained from Stratagene, La Jolla, CA, USA)
using
TA-cloning (Zhou et al. (1995)).
6 The cloned DNA insert was sequenced using the commercial sequencing
7 service of the National Research Council of Canada, Plant Biotechnology
Institute
8 (Saskatoon, SK, Canada). The sequence was compared to the Bxl7 sequence and
9 to the Bx7 sequence. The plasmid carrying the 2.2 kb MAR insert, called pBx7-
2.2,
was digested separately with EcoRV & Mscl and with Mscl & Smal. The resulting
11 fragments, 0.8 and 0.9 kb respectively, were individually subcloned into
pBluescript
12 SK using standard practice (Sambrook et al., (1989) and called pEM820
(containing
13 MAR Bx7) and pMSm900, respectively.
14 To prove that the cloned sequence functions as a MAR, nuclei were isolated
from 7-day etiolated 'Glenlea' seedlings using the procedure described by
16 Steinmuller & Appel(1986) as modified by Cockerill & Garrard (1986).
'Glenlea'
17 wheat was grown in the dark on damp vermiculite for 5 days. Etiolated
seedlings
18 were harvested, 100 g ground to a powder in liquid nitrogen and the powder
19 resuspended in 250 ml of isolation buffer (20 mM Tris.HCl pH 7.8, 250 mM
sucrose,
5 mM MgCl2, 5 mM KCI, 40% (v/v) glycerol, 0.25% (v/v) Triton X-100, 0.1 %
(v/v)
21 ,u-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride ("PMSF"), 1 ,ug/ml
leupeptin
22 (Boehringer) and 1 ,ug/ml aprotinin (Boehringer)). The suspension was
warmed to
23 10°C by stirring, and then filtered through nylon mesh (0.1 mm) and
centrifuged at
24 0°C, 10,000 g for 15 minutes. All subsequent procedures were
performed on ice
using chilled buffers. The pellet was carefully resuspended in 150 ml
isolation buffer,
26 washed and centrifuged as before but at 6000 x g. The pellet was washed
again
27 with 100 ml isolation buffer and re-centrifuged as above. The final pellet
was
28 resuspended in a minimal volume (approximately 5 ml) of 30 mM sodium
phosphate
29 pH 7.8, 1.5 M sucrose, 5 mM MgCl2, 1 mM PMSF, and layered onto a 2.5 ml
cushion
of the same buffer with 2.3 M sucrose. The sample was centrifuged at 24000 rpm
in
31 a Beckman SW27Ti rotor for 30 minutes at 4°C.
32 The supernatant was removed completely and the pellet resuspended in 2 ml
CA 02239259 1998-07-31
1 digestion buffer (10 mM Tris.HCl pH 7.8, 250 mM sucrose, 10 mM NaCI, 3 mM
2 MgCl2, 1 mM PMSF) washed and re-centrifuged (750 g, 10 minutes, 4°C).
The
3 pellet was resuspended in 2 ml digestion buffer supplemented with 1 mM CaCl2
and
4 0.1 mg/ml Dnase I (Pharmacia) and incubated at 23°C for 1 hour. After
centrifugation (as above), pellets were resuspended in 5 ml digestion buffer
and
6 mixed with an equal volume of ice-cold 4 M NaCI, 10 mM EDTA, 1 mM PMSF, 20
7 mM Tris.HCl, pH 7.8. After 10 minutes on ice, the sample was centrifuged at
1500
8 g, 15 minutes at 4°C, and the pellet re-extracted twice with ice-cold
2 M NaCI, 10
9 mM EDTA, 10 mM Tris.HCl pH 7.4, 1 mM PMSF, 0.25 mg/ml BSA (Sigma Fraction
V) and centrifugation at 4500 g, 15 minutes, 4°C. The pellet (of
nuclear matrix) was
11 washed with digestion buffer supplemented with 0.25 mglml bovine serum
albumin
12 ("BSA"). The final pellet was resuspended in the same buffer (1 ml)
combined with
13 an equal volume of glycerol and stored at -20°C.
14 Binding assays were carried out using a modified procedure of Hall et al.
(1991 ). Crude minipreps of clones to be assayed for binding were digested
with
16 Xbal/Xhol (pEM820 and pMSm900). A positive control, the MAR associated with
17 the Adh-1 gene of maize cloned into the BamHl/Hindlll site of pUCl9, was
kindly
18 provided by Z. Avramova, Purdue University, IN, USA. This construct,
referred to as
19 pBH1.3, was digested with BamHl/Hindlll for the binding experiment.
Digested
plasmids were labelled with [a32P]dCTP using the Klenow fragment (Gibco/BRL)
to
21 end-fill the digests, and incubated with nuclear matrices corresponding to
1.0 A 2so
22 unit of nuclei (as characterized on a spectrophotometer as per Hall et al.,
(1991 )), for
23 one hour in the presence of 20 ~g unlabelled competitor DNA (pUCl9) in a
total
24 volume of 100 ,ul binding buffer (20 mM Tris.HCl pH 7.4, 20 mM KCI, 70 mM
NaCI, 5
mM EDTA, 1 mM dithiothreitol, 1 mM PMSF). After incubation, the suspension was
26 centrifuged and the supernatant transferred to a fresh tube. The pellet was
washed
27 once with binding buffer and resuspended in extraction buffer (20 mM
Tris.HCl pH
28 7.4, 5 mM EDTA, 1 % (w/v) sodium dodecyl sulphate 100 ,ul (SDS) and 5 ,ug
sheared
29 salmon sperm DNA (Boehringer)). This was vortexed briefly and heated to
80°C for
15 minutes. An equal volume of phenol was added to the sample which was again
31 vortexed and heated to 80°C briefly. The sample was then centrifuged
at 13,000 x
32 g, at room temperature in a microfuge. The aqueous phase was aspirated and
to it
16
CA 02239259 1998-07-31
1 was added 0.1 volume 1 M NaCI. The DNA was precipitated in ethanol (using
2 standard techniques (Sambrook et al., (1989)). The following was loaded onto
a 1
3 Tris/acetate/edta agarose gel (Sambrook et al., (1989)): the entire final
pellet of DNA
4 recovered from the nuclear matrices; 10 ~I of the unbound DNA (contained in
the
nuclear matrix supernatant) and 1 ,ul of the labelled DNA (used in the binding
6 experiment). These were separated for 1 hour at 65 V in TAE. The gel was
fixed by
7 immersion in 1 % (w/v) hexadecyltrimethylammonium bromide, 50 mM sodium
8 acetate pH 5.5 for 1 hr. The gel was dried between paper towels and
9 autoradiographed using Biomax film (Kodak). The resulting autoradiogram was
placed in a Bio-Rad (Hercules, CA, USA) visual densitometer and the area under
the
11 peaks corresponding to the bands was integrated using a Bio-Rad integrator.
12 The results of the assay showed that 26.4% of the amount of sequence
13 pEM820 in the assay bound to the nuclear matrix and was found in the pellet
14 fraction as compared to 10.3% for pMSm900 and 11.4% for the previously
described
(Avramova and Bennetzen, (1993)) maize Adh-I MAR (Table 1 ). The prokaryotic
16 derived plasmid cloning vector control sequences did not bind the matrix
(less than
17 5%) and were found exclusively in the supernatant. This shows that the
sequence
18 of the invention does act as a MAR and binds the matrix more strongly (more
than
19 twice) than the previously described maize MAR, making for superior
utility.
For utility in enhancing and stabilizing expression for any transgene, it must
21 first be proven that MAR Bx7 does not have a deleterious effect when placed
22 adjacent to a heterologous promoter. As MAR Bx7 is derived from a promoter
which
23 is active only in the cereal endosperm, it was necessary to prove that MAR
Bx7,
24 when placed adjacent to a constitutive promoter, did not alter the normal
expression
pattern of the promoter by making it endosperm specific. To prove this, a
series of
26 transient expression experiments was conducted. Plasmid pACT1 d (Ray Wu,
27 Cornell University, NY, USA) carries the rice actin promoter fused to the
uidA gene
28 (encoding the ~i-glucuronidase ("GUS") protein) and the nopaline synthase
("NOS")
29 polyadenylation signal. Two further plasmids were constructed for transient
expression experiments. The actin::GUS::NOS cassette from pAct1 d was modified
31 by flanking it with either the EcoRV-Mscl (EM) (for plasmid pEM.Act) or the
32 Mscl-Smal (MSm) fragment (for plasmid pMSm.Act) of pBx7-2.2. This was done
by
17
CA 02239259 1998-07-31
1 cloning the MAR fragments (EM or MSm) in tandem into the Pvull and EcoRV
sites
2 of pSP72 (obtained from Promega, Madison, WI, USA) and then inserting the
3 actin::GUS::NOS cassette into the Pstl/Xbal sites between the MARs. For
4 transformation, plasmids were grown in E. coli DHSa and purified using a
Maxi-Prep
kit (Promega). Plasmids were coated onto tungsten particles (1 ,um, BioRad,
6 Hercules, CA, USA) using the method described by Knudsen and Muller ((1991
).
7 Tissue for bombardment was embryos and leaves. Embryos were dissected from
8 surface-sterilized wheat seeds ('Glenlea') harvested 15 to 25 days after
anthesis.
9 These were placed on solid medium (Knudsen and Muller, (1991 ); Donovan and
Lee
(1977)); the composition of this medium is: Murashige and Skoog standard
("MS")
11 medium (Murashige and Skoog (1962)) supplemented with 4.038 g/I casein
12 hydrolysate (BDH), 3% (w/v) sucrose, 100 mg/ml inositol, 0.4 mg/ml thiamine
and
13 1 % agarose (Gibco/BRL) pH 5.8), and bombarded with plasmid-coated tungsten
14 particles using a biolistic particle gun (Sanford et al. (1987)). The
embryos were
incubated in the dark for 24 hours before being assayed for GUS activity by
the
16 colorimetric GUS assay (Klein et al. (1988)). Leaves were cut into 5 mm
strips,
17 surface-sterilized with ethanol, and transferred to solid medium for
bombardment
18 followed by GUS assay.
19 The results were that both the plasmid with the constitutive rice actin
promoter only, and the plasmid with the rice actin promoter adjacent to MAR
Bx7
21 were active in both embryos and leaves (Table 2). This proves that MAR Bx7
is
22 independent from the promoter from which it was derived (which is not
active in
23 embryos or leaves) and therefore is not deleterious to the expression
pattern of
24 other promoters when placed adjacent to them to enhance expression and
stability.
26 EXAMPLE 2
27 Use of MAR Bx7 to identify and isolate other MARS from the 5' flanking
28 regions of endosperm-specific storage protein genes
29 of monocotyledonous plants
As discussed previously, other MAR sequences from cereal crops (maize and
31 rice) have been reported (Avramova et al., (1993); Nomura et al. (1997)).
However,
32 these MARs differ significantly from MAR Bx7. The computer software program
18
CA 02239259 1998-07-31
1 Align Plus (Scientific and Educational Software) was used to compare the
maize and
2 rice sequences to MAR Bx7. The maize sequence is identified as Genbank
3 Accession number X00581 and the rice sequence is identified as Genbank
4 Accession number X95271. In their entirety, these sequences were,
respectively,
2% and 1 % homologous to MAR Bx7. However, the maize sequence had a region
6 of 495 base pairs that was 60% homologous to a 527 base pair region of MAR
Bx7
7 and the rice MAR has a 225 base pair sequence which is 68% homologous to a
208
8 base pair region of MAR Bx7. The region of MAR Bx7 having homology to the
9 maize sequence is not the same region which is homologous to the rice
sequence
(Table 3). This illustrates the differences between MAR sequences even in
related
11 plant species. MAR Bx7 has been shown to be a stronger MAR than the maize
12 sequence (Table 1 ).
13 Using MAR Bx7 to search other known DNA sequences (using the BLAST
14 algorithm of Altschul et al. (1990)), a number of sequences with stretches
of over
100 base pairs with greater than 80% homology are found. These sequences
(Table
16 4) are all of endosperm specific storage protein genes from wheat or close
relatives
17 of wheat (Aegilops tauschir), where enough sequence data in the promoter
region is
18 present to match that of MAR Bx7. Given the high degree of homology of
these
19 promoter regions to MAR Bx7, it is likely that the other sequences contain
regions
capable of binding the nuclear matrix. Any sequence containing a stretch of
over
21 100 base pairs with 80% or greater homology with MAR Bx7 and capable of
binding
22 to the nuclear matrix would be considered to be functionally equivalent to
MAR Bx7.
23 The primary source of such sequences would be the 5' flanking region of
endosperm
24 specific storage protein genes from wheat or its close relatives. These
could be
cloned using methods generally known in the art including, for example,
generating
26 DNA primers for the polymerase chain reaction (PCR) using sequences in the
27 Genbank database where many promoter sequences for storage protein genes
are
28 catalogued (for example accession numbers in Table 4) or using the sequence
of
29 MAR Bx7; isolation of a cDNA clone for a storage protein gene (using
standard
cDNA library techniques as in Sambrook et al. (1989) for example) and using
31 promoter walking (for example using a commercially available kit such as
Promoter
32 Finder from Clontech, Palo Alto, CA, USA); or probing a genomic library
using as a
19
CA 02239259 1998-07-31
1 probe a DNA sequence derived from the coding region or promoter of a storage
2 protein gene using moderate stringency washing techniques designed to
identify
3 sequences having at least 80% homology to the probe (for example first
washing in
4 2 x SSPE, 0.1 % SDS at room temperature for 10 minutes followed by washing
in 1 x
SSPE, 0.1 % SDS at 65 degrees C for 15 minutes (manufacturers protocol for
6 moderate stringency washing for Hybond N+ membranes (Amersham Pharmacia,
7 Baie D'Urfe, PQ, Canada). Other membrane types may require different
conditions
8 however a high stringency wash should be omitted). These methods are not
9 exclusive and any method which allows the cloning of a promoter for a gene
could
be used.
11
12 EXAMPLE 3
13 Use of MAR Bx7 to enhance transgene expression
14 and stability in a monocotyledonous plant
A plasmid such as pMAR.Act1 d.MAR is constructed so that the MAR Bx7
16 (EM820) sequence flanks both sides of the ~i-glucuronidase expression
cassette
17 (rice actin promoter-GUS gene-NOS polyadenylation site - the expression
cassette
18 contained in plasmid pACT1 d described in Example 1 herein ).
Alternatively, a
19 single MAR sequence could be used to flank one side of the expression
cassette
only. A construct with the expression cassette but lacking the MAR sequences)
is
21 used as a control.
22 The plasmids are transformed into E. coli as described in Sambrook et al.
23 (1989), and purified using a Maxi-Prep kit (Promega, Madison, WI, USA).
Plasmids
24 are coated onto tungsten or gold particles (BioRad, Hercules, CA, USA)
using the
method described by Knudsen and Muller (1991 ).
26 Immature wheat embryos (1.0 to 1.5mm in length) are pre-cultured and
27 transformed with either the plasmid containing the ~i-glucuronidase
expression
28 cassette flanked by the MAR, or the control plasmid, using a method such as
that
29 described in US Patent No. 5,610,042 or US Patent No. 5,631,152.
The resulting transformed plants are characterized as to their expression
31 levels using, for example, the fluorometric assay for ~i-glucuronidase as
described by
32 Jefferson (1987). Leaf tissue from each of the plants is ground in
extraction buffer
CA 02239259 1998-07-31
1 (50mM NaP04 pH 7.0 buffer, lOmM ~3-mercaptoethanol, lOmM disodium EDTA,
2 pH8.0, 0.1 % sodium dodecyl sulphate and 0.1 % Triton X-100). An aliquot of
1 to 10
3 ,ul is added to 50 ,ul of extraction buffer plus 1 mM MUG (methyl
umbeliferone
4 glucuronide) which is pre-warmed to 37 degrees C. The mixture is incubated
at 37
degrees C for 30 to 60 minutes and then stopped by the addition of 25 ,ul of 1
M
6 sodium carbonate. The mixture is then quantified numerically using a
fluorometer.
7 The protein content of the plant extract is determined using commercially
available
8 protein assays such as those sold by Bio-Rad (Hercules, CA, USA). In this
way, the
9 values for the level of gene expression per mg protein can be determined for
each
plant. The expression for the plants containing the MAR sequence is compared
to
11 the plants lacking the sequence, and those plants exhibiting increased or
stabilized
12 gene expression relative to the control plants are selected.
13 The transformed plants are also crossed to non-transformed plants. The
14 progeny are then analysed for expression as described above. The series of
crossing is done over more than one generation. In this way, the degree of
silencing
16 of gene expression for the non-MAR containing construct can be compared to
the
17 MAR containing construct.
18
19
21
CA 02239259 1998-07-31
1 Table 1. Results of binding assay with the two subcloned fragments of the
Bx7
2 2.2kb promoter fragment (EM820 and Msm900) and the Adhl MAR positive
control.
3 Each fragment has its own internal negative control vector fragment. Data is
4 presented for the area under the peaks corresponding to the bands on the
autoradiogram after densitometric analysis. Area units are assigned by the
6 integrator.
7 Area undervector Adhl vector EM820 vector Msm900
8 peak control- fragment control- fragment control- fragment
Adhl EM820 (MAR Bx7) MSm900
9 supernatant59975328 78028861 7465160 3351453 60897560 1859769
pellet 2213018 8917389 64511520 17037888 57563920 5936224
11 % in pellet3.7 11.4 4.5 26.4 3.1 10.3
12
13
14
Table
2.
Results
of
transient
GUS
expression
in
tissues
transformed
with
pActld
and
MAR-flanked
actin::GUS::NOS
constructs.
16 Construct Tissue
Embryo Endosperm Callus Leaf
17 pAct 1 d +++ ++ N p +
18 pEM.Act +++ ++ +++ +
19 pMSm.Act +++ ++ ND +
Unshot _ -
_
21
22 ND
= not
determined
23 +++
= 20%
of
tissue
had
>10
spots
per
tissue
piece
and
<20%
had
no
spots
24 ++
= approximately
50%
had
no
spots
per
tissue
piece
and
<20%
had
>10
spots
+
= at
teat
one
spot
per
construct
per
tissue
26 - = no spots
27
22
CA 02239259 1998-07-31
1 Table 3. Homology of MAR Bx7 with other disclosed cereal species MAR
2 regions.
3 Accession Number and regionRegion of Homology in % homology
MAR
4 of homology (base pairs) Bx7 (base pairs)
X00581 ( 169-664) 6-553 60
6 X95271 ( 18-243) 599-807 68
7
8
9 Table
4.
BLAST
similarity
search
using
MAR
Bx7
as
query
sequence.
All
accessions
are
wheat
or
Aegilops
endosperm
specific
storage
protein
gene
11 promoter
regions.
12 Accession Length of Homologous Region (Base % Homology
pairs)
13 Number
14 X13927 343
gg
X13927 458 g2
16 M22208 174 94
17 X03042 104
86
18
19
23
CA 02239259 1998-07-31
1 REFERENCES
2
3 Ahn, S., et al. Mol. Gen. Genet., 241, 483-490 (1993).
4 Allen et al., Plant Celt, 5, 603-613, (1993).
Allen et al., Plant Cell, 8, 899-913, (1996).
6 Altschul et al., J. Mol. Biol., 215, 403-410, (1990).
7 Anderson, O.D., Greene, F.C., Theor. Appl. Genet., 77, 689-700, (1989).
8 Avramova and Bennetzen, PI. Mol. Biol., 22, 1135-1143, (1993).
9 Breyne et al., Plant Cell, 4, 463-471, (1992).
Breyne et al., Transgenic Res., 3, 195-202, (1994).
11 Cockerill & Garrard, Cell, 4, 273-382, (1986).
12 Datta et al., Biotechnology, 8, 736-740, (1990).
13 Donovan and Lee, Plant Sci. Lett., 9, 107-113, (1977).
14 Jefferson, PI. Mol. Biol. Reporter, 5, 387-405, (1987).
Hall et al. PNAS, USA, 88, 9320-9324, (1991).
16 Hermann, G.G., et al., Symp. Soc. Exp. Biol., 50, 25-30, (1996).
17 Klein et al., PNAS, USA, 85, 4305-4309, (1988).
18 Knudsen and Muller, Planta, 185, 330-336, (1991 ).
19 Langridge, P., et al. Mol. Gen. Genet., 257, 568-575, (1998).
Mirkovitch et al., Cell, 39, 223-232, (1984).
21 Mlynarova et al., Plant Cell, 7, 599-609, (1995).
22 Mujeeb-Kazi, A. In Biodiversity and Wheat Improvement, A. B. Damania
(editor).
23 1993. A Wiley-Sayce Publication, pp. 95-102.
24 Murashige and Skoog, Physiol. Plant., 15, 474-497, (1962).
Nomura et al., Plant Cell Physiol., 38, 1060-1068, (1997).
26 Peach and Velten, Plant Mol. Biol., 17, 49-60 (1991 ).
27 Reddy, P., Appels, R., Theor. Appl. Genet., 85, 616-624, (1993).
28 Sambrook et al., Molecular Cloning, Cold Spring Harbor Press, (1989).
29 Sanford et al., Particulate Sci. Technol., 5, 27-37, (1987).
Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74, 563-568, (1977).
31 Schoffl et al., Transgenic Res. 2, 93-100, (1993).
32 Spiker et al., Plant Physiol., 110, 15-21, (1996).
24
CA 02239259 1998-07-31
1 Steinmialler & Appel, Plant Molecular Biology, 7, 87-94 (1986).
2 Tingay et al., Plant J., 11, 1369-1376, (1997).
3 Ulker et al., Plant Physiol. 114, 306, (1997).
4 Van der Geest, Plant J., 6, 413-423, (1994).
Zhou et al., Biotechniques, 19, 34-35, (1995).
6 All publications mentioned in this specification are indicative of the level
of
7 skill in the art to which this invention pertains. To the extent that they
are consistent
8 herewith, all publications are herein incorporated by reference to the same
extent as
9 if each individual publication was specifically and individually indicated
to be
incorporated by reference.
11 Although the foregoing invention has been described in some detail by way
of
12 illustration and example, for purposes of clarity and understanding it will
be
13 understood that certain changes and modifications may be made without
departing
14 from the scope or spirit of the invention as defined by the following
claims.
CA 02239259 1999-06-28
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jordan, Mark C.
Rampitsch, Christof
Cloutier, Marie S. J.
(ii) TITLE OF INVENTION: Matrix Attachment Regions
(iii) NUMBER OF SEQUENCES: 5
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: McKay-Caret' and Company
(B) STREET: 10155 102nd Street
(C) CITY: Edmonton
(D) STATE: Alberta
(E) COUNTRY: Canada
(F) ZIP: T5J 4G8
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,239,259
(B) FILING DATE: 31-JUL-1998
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McKay-Caret', Mary Jane
(C) REFERENCE/DOCKET NUMBER: 28002CA0
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (780) 424-0222
(B) TELEFAX: (780) 424-0290
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 819 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
1
CA 02239259 1999-06-28
(A) ORGANISM: Triticum aestivum
(B) STRAIN: Glenlea
(xi) SEQUENCE DESCRIPTION:
SEQ ID NO:1:
ATCGACTTGA TATTATGGAT ATTTATGTATTTCTCTACAAATTTGATCAA ACTTTAAAAG60
GTTTAACTTC TCAAAAAAAA AAACTAATACACCTTATATTTTAAAACAGA GAGAGTAGTT120
TTTTTTTACC CAAACCCCAA CTGCCCAGCAAAGCGCGCTCAACTCTTCTA GTCTAAATAA180
CTAGCATCCA CTAACACATT TCTCCCGACATGCAAGCACGTCACCTATGA AAATGCCCAC240
CTCAATATGC AACCATGCAT AGAAGAAAGCTCACCTCAGCATGCAAACAT GCAGCATAAT300
TTCCATTTTA CTTGGCTATT TATGTTTGATAAATATTTCACAAATATACA ATAATCAAAA360
ACAATAAATT ATATGTGTTT TTAGTTTTAGTTCTCATATCCAAATATACA TGTTTCATAC420
AACCAAATCT CATTTAAATA TATTGTAAAATATTCCGGCAACAACTTGTG GGGGCCTTAA480
ATATATTGTA AAATATTCCG GCAACAACTTGTGGGGTACATCTAGTTACA GTGGAATATT540
AGTGATGGCG TGACCAAGCG ATAAGGCCAACGAGAGAAGAAGTGCGTCGT CTATGGAGGC600
CAGGGAAAGA CAATGGACAT GCAAAGAGGTAGGGGCAGGGAAGAAACACT TGGAGATCAT660
AGAAGAACAT AAGAGGTTAA ACATAGGAGGGCATAATGGACAATTAAATC TACATTAATT720
GAACTCATTT GGGAAGTAAA CAAAATCCATATTCTGGTGTAAATCAAACT ATTTGACGCG780
GATTTACTAA GATCCTATGT TAATTTTAGACATGACTGG 819
(2) INFORMATION FOR SEQ ID
N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pai rs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: singl e
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other
nucleic acid
(A) DESCRIPTION: /desc = "PCR
primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
2
CA 02239259 1999-06-28
GAGCTCTCCC ATCCAATTG 19
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
AGAAGCTTGG CCTGGATAGT 20
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GGGTCGATGG TATCAATCC 19
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
3
CA 02239259 1999-06-28
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GGCCTGGATA GTATGACCC 19
4
